Download Suppressor analysis of the protein kinase Elm1p, an enzyme

Retrospective Theses and Dissertations
1997
Suppressor analysis of the protein kinase Elm1p, an
enzyme involved in the regulation of yeast
morphology
Nicholas Patrick Edgington
Iowa State University
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Edgington, Nicholas Patrick, "Suppressor analysis of the protein kinase Elm1p, an enzyme involved in the regulation of yeast
morphology " (1997). Retrospective Theses and Dissertations. Paper 11979.
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Suppressor analysis of the protein kinase Elmlp^ an enzyme
involved in the regulation of yeast morphology
Nicholas Patrick Edgington
A dissertation submitted to the graduate faculty
in partial fulfUlment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Molecular, Cellular, and Developmental Biology
Major Professor: Alan Myers
Iowa State University
Ames, Iowa
1997
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iii
TABLE OF CONTENTS
ABSTRACT
J
v
CHAPTER 1. INTRODUCTION...
Filamentous Growth in S. cerevisiae
Molecular Characterization of Filamentous Growth
Dissertation organizatioiL
1
1
19
28
CHAPTER 2. DIFFERENTIAL EFFECTS OF CAMP-DEPENDENT
PROTEIN KINASE ACTIVITY ON SACCHAROMYCES CEREVISIAE
FILAMENTOUS GROWTH DEPENDING ON GENETIC AND
NUTRITIONAL SIGNALS
Abstract.
Introduction
Materials and Methods
Results
Discussioru
Acknowledgements
References
30
30
31
36
43
61
75
76
CHAPTER 3. CONTROL OF SACCHAROMYCES CEREVISIAE
FILAMENTOUS GROWTH BY A PROTEIN KINASE PATHWAY
INCLUDING SEL2P AND CDC28
Abstract
Introduction
Results
Discussion
Material and Methods
Acknowledgements
References
82
83
88
110
117
128
128
CHAPTER 4. ANALYSIS OF SEL2/HSL1 REVEALS A LINK BETWEEN
CYTOKINESIS AND CELL CYCLE CONTROL IN SACCHAROMYCES
CEREVISIAE
Abstract.
Introduction
Material and Methods
Results
Discussion
Acknowledgements
References
133
134
137
143
160
171
172
iv
i
CHAPTERS. GENERAL CONCLUSIONS
180
APPENDIX. IDENTinCATION OF ELMS
183
REFERENCES
201
ACKNOWLEDGMENTS
215
V
ABSTRACT
Saccharomyces cerevisiae is an ascomycete that has several
developmental options, with concomitant morphological changes, that
depend on cell t3^e and nutrient status. In response to these signals,
vegetative Saccharomyces cerevisiae cells asstime one of two distinct
morphologies, the yeast form or the filamentous form. Molecules atfecting
this morphogenetic response were identified by analyzing transposoninduced or spontaneous suppressor mutations that restore yeast form growth
to a strain that constitutively exhibits filamentous form characteristics owing
to mutation of the protein kinase Elmlp. One transposon-tagged mutation
was isolated and is allelic to the negative regulatory subtmit Bcylp, thus
activation of cAMP-dependent protein kinase (PKA) by deletion of Bcylp
suppressed the constitutive filamentoiis growtii phenotype of elml strains.
The suppression is cell-type dependent and occurs on minimal media. PKA,
therefore, has differential effects on cell morphology depending on the
presence or absence of the al/a2 repressor coded for by the MAT locus, and
on the nutritional environment.
A second screen for spontaneous extragenic suppressors resulted in
the isolation of ten mutant strains that define three gene loci, termed SELl,
SEL2, SEL3. SEL2 has been cloned and sequence aruilysis indicates that the
gene product encodes an S. cerevisiae homolog of the serine/threonine
vi
protein kinase niml* of S. pombe. Evidence from this study suggests a
characterized protein kinase cascade tiiat impacts upon activity of the cyclindependent kinase Cdc28p, participates in regulation of filamentous
growth.
Deletion of 5WEI, a homologue of S. pombe weeV, also restored yeast form
growth to elml strains. Swelp is functionally analogous to the weel* kinase
in that it phosphorylates Cdc28p. Sel2p, based on its similarity to the nimV
kinase, is predicted to inhibit Swelp. Together, these data suggest regulation
of Cdc28p by phosphorylation determines whether the pseudohyphal growth
characteristics induced by elml mutations can occur. Sel2p is localized to the
bud neck during the budded phase of the cell cycle. Overexpression of Sel2p
results in a growth arrest late in mitosis that is suppressed by mutations in
Clb2p, and an allele of CDC28, suggesting that Sel2p degradation may be
required for the telophase to G1 cell cycle transition.
1
CHAPTER 1. INTRODUCTION
Filamentous Growth in S. cerevisiae
Knowledge of the fundamental mechanisms that dictate cell shape and
polarity is a critical aspect of imderstanding how cells respond appropriately
to their environment. The budding yeast S. cerevisiae has proven to be a
very useful genetically tractable eukaryotic model system in which to study
the regulation of morphology. Although S. cerevisiae appears to be a
morphologically simple unicellular organism, yeast cells require complex
regulatory hierarchies to integrate cell wall growth, osmotic stability, and
mating functions, with cell division and nutrient availability.
S. cerevisiae cells can exist in different developmental states that
depend on cell-type and nutritional status. Haploid cells in their basal state
grow as ellipsoidal cells. On solid rich medium, haploids at the bottom of
colonies can alter their cell mode and enter another developmental state
known as invasive growth (116). Haploid cells undergoing this
differentiation alter their bud site selection, slightly elongate, and require
many of the same elements used in the mating MAP kinase cascade. Diploid
yeast cells can enter an alternative growth mode when there are adequate
sources of carbon, but limiting amounts of nitrogen. These cells become
elongated, switch their budding pattern from bipolar to unipolar, delay the
cell cycle in the G2 phase, remain attached after c5rtokinesis, and penetrate
2
into solid growth medium (16,48, 73). This mode of growth is known
collectively as pseudohyphal growth. Together, these two cell-type specific
developmental states, invasive and pseudohyphal growth, are referred to as
filamentous form growth.
Nutrient regulation
Nutrient source and availability regulate all of the developmental
options, as well as cell size and division in 5. cerevisiae. Mammalian cells
have evolved such that nutrient availability does not regulate cell division,
and thus have evolved mechanisms that either restrict cell division in the
presence of plentiful nutrients, or that make cell division dependent on other
conditions, such as growth foctor availability. Therefore, in addition to their
role as basic building blocks of metabolism, nutrients such as carbon, sulfur,
phosphate, and nitrogen sources are analogous to mammalian growth factors
in regulating S. cerevisiae cell division and differentiation (131). S. cerevisiae
cell populations wiU continue to mitotically divide as long as adequate
nutrients are available. Under conditions of carbon source depletion on rich
medium, or specific starvation for carbon, nitrogen, sulfur, or phosphorus,
cells arrest in a metabolically inactive state known as Go or stationary phase
with uiu-eplicated DNA (85,138).
During a defined period of growth of cells in G1 phase, a minimal
critical cell size is required before newly bom daughter cells can initiate a new
3
round of division. The threshold of critical cell size is depaident on the
quality of nutrients available, such that poor carbon or nitrogen sources result
in smaller size requirements before commitment to the next round of cell
division (60, 90). Thus nutrient source quality also regulates cell size in S.
cerevisiae. Under specific conditions of nitrogen starvation and in the
presence of a poor nonfermentable carbon source such as acetate, diploid cells
undergo meiosis (54,101). Nutrients appear to regulate meiosis at two
different stages of differentiation (79).
Nutrient source and availability is also an important determinant for
the initiation of pseudohyphal and invasive growtii. Brown and Hough
foimd that polyploid brewing S. cerevisiae cells grown in continuous culture
imder conditions of low ammonitun sulfate, and normal levels of glucose,
became elongated, and that this process was reversible by the replacement of
ammoniimi sul^te (16). Phosphate and glucose limitation resulted in wildtype ovoid shaped cells, however limitation of other poor nitrogen sources
such as methionine and asparagine resulted in elongated cells (16). Haploid
invasive growth occurs on rich mediimi underneath mature colonies (116),
but the initiating nutritive conditions are as yet imdefined. Presimiably,
nutrient depletion in conjunction with oxygen deprivation, are initiating
signals. Conflicting reports of whether haploid yeast cells are capable of
pseudohyphal growth in nitrogen-limited medium have been reported, and
perhaps this reflects variances in strains (48,141).
4
Oxygen availability may also play a role in pseudohyphal growth, since
oxygen limitation in conjunction with nitrogen limitation was more effective
in producing elongated chains of poljrploid brewing yeast cells in liquid
cultiure (74). It is interesting that oxygen limitation is important for
pseudohyphal development in liquid cultures, since cell cultures in wellaerated nitrogen-limited liquid do not promote pseudoh3rphal growth (48).
Thus the requirement for a solid medium for pseudoh3rphal growth to occur,
may simply reflect the requirement of oxygen limitation at the bottom of
mature colonies, fa. siunmary, nutritive type and availability regulate nearly
all aspects of S. cerevisiae life, including cell size, cell division, and
developmental options, and it is the interplay between these external signals
with genetically programmed signal transduction cascades that allow yeast to
monitor, adapt, and survive in hostile environmental conditions.
Bud-site selection components
S. cerevisiae cells divide in an asymmetric manner, such that the
daughter cell that is produced is much smaller than the mother cell. Because
of this asymmetry, a specific location must be chosen where directed growth
will occur to produce a mating structure or a new bud. Two factors, extrinsic
and intrinsic, regulate where polarized growth will occur on the cell surface
of S. cerevisiae cells (33). Extrinsic factors are the nutritional conditions, and
the haploid response to the secretion of a mating peptide from a haploid cell
5
of the opposite mating type. Intrinsic factors are genetically programmed, and
depend on the mating type of the cells.
The intrinsic bud-site selection of haploid cells is the axial pattern, in
which the mother and daughter cells initiate new buds proximally to each
other (17,21,33,93,137). A chitin rich circular bud scar marks the position
where a bud was produced on the mother cell, and a birth scar is produced on
the daughter cell at the same site of division (115). Thus haploid axial
division results in new bud-sites being chosen that are juxtaposed to these
physical markers. The diploid pattern of bud-site selection is bipolar, in
which the mother and daughter form a new bud at either the distal or
proximal pole. Based on the different phenotypes of classes of bud-site
selection mutations, it appears that the programs that define eodal or bipolar
bud-site selection appear to be independent of one another, since specific
mutations exist that interfere with one but not the other pathway. It is
postulated that in axial budding, a labile marker is present at the previous site
of budding, and thus nutrioit conditions that delay bud formation allow the
marker to be lost, and ttie next site is chosen randomly (22, 94). hi diploids
however, the bipolar signal remains, and therefore appears to be permanently
marked, regardless of the growth rate.
Detailed analysis of the molecular basis of bud-site selection has
revealed four classes of bud-site selection mutations. One class is comprised
of proteins of a GTPase module, Budlp/Rsrlp (Ras-related GTPase), Bud2p
6
(GTPase-activating protein or GAP), Bud5p (GDP-GTP exchange factor or
GEF), and their mutation results in randomized budding in both haploid and
diploid cells (9,18,19,100,109, 111, 119). Budlp has specific interactions with
cell polarity components which are necessary for bud emergence, thus Budlp
links upstream cell type-specific bud-site selection pathways with downstream
bud emergence and polarized growth components (108).
The remaining three classes represent mutations that affect only
haploid or diploid bud-site selection. Mutations in the proteins Bud3p,
Bud4p, Axllp, Axl2p/Budl0p, and the neck ring-associated septins, exhibit
defects in haploid-sp>ecific axial bud-site selection, which results in a bipolar
budding pattern (19,20,42,44,50,118,123). The third class represents a large
nimiber of mutations that affect only the diploid bipolar pattern, resulting in
randomized budding, and have little or no effect on haploid axial budding.
This class contains proteins that play roles in lipid metabolism (Sur4p, Fenlp,
Rvsl61p, Rvsl67p), actin filament organization and/or morphogenesis
(Actlp, Spa2p, Pea2p, Aip3p/Bud6p, End3p, Rvsl61p, Rvsl67p, Bnilp, Sac6p,
Srv2p, Slalp, Sla2p), secretion/endocytosis (Sec3p, Sec4p, Sec9p, End3p), and
one other gene which has not yet been cloned (BUD7) (2,8,10,34,41,127,134,
143, 144). Many of the mutations listed above have other cellular roles in
addition to affecting bipolar budding, and thus in certain cases, it is only
specific alleles that have budding defects. The fourth class consists of two
genes with specific bipolar defects, but do not result in random patterns when
7
disrupted. Mutation of BUDS in diploid strains results in a budding pattern in
which buds are formed only at the proximal division site pole,^ and BUD9
mutations resiilt in budding only from the distal pole (144).
A model for the haploid axial organization of bud-site selection
suggests that proteins that regulate the axial pattern, BudSp, Bud4p, Axllp,
and Axl2p, function to regulate the activation, assembly or localization of
Bud5p, which then activates Budlp by guanine nucleotide exchange of GDP
for GTP (17). Presumably, these proteins function only in haploids due to the
diploid-specific repression of expression, activity, or localization. Sequence
analysis of BudSp, and Bud4p have shown no significant homologies to any
other proteins in database searches, however immimolocalization or direct
imaging with green fluorescent protein (GFP) fusions, have demonstrated
that BudSp and Bud4p are localized to the mother'daughter neck region, at
the onset of mitosis as a double ring structure (20,50,118,123). The
localization of BudSp and Bud4p, are weakly interdependent on each other,
and also require the previous localization of the septin proteins, Cdc3p,
CdclOp, Cdcllp, Cdcl2p, to the bud neck (20,123). Axl2p is a novel type I
integral membrane protein with a single transmembrane-spanning domain,
and remains at the bud neck during the entire budded period of growth, and
transiently after cytokinesis and cell abscission (50,118). Bud3p and Bud4p
may serve as division site markers that influence the localization of Axl2p in
the subsequent rotmd of division. The role Axllp in axial selection is not
8
clear, however it is regulated transcriptionally by the diploid-spedfic al/a2
repressor, and therefore is expressed only in haploids, in contrast to 6ud3p,
and Bud4p (20,44,123). Thus, Axllp may play a role in the haploid-spedfic
activation of BudSp and Bud4p. Presimiably, it is the combined interactions
of all of the components of axial bud-site selection that then activate Budlp.
The regulation of the diploid bipolar budding pattern appears to
involve the actin cytoskeleton, membrane organization, and bud-site
selection specific molecules. It may be that the location of these bud-site
selection components is critical for their function, and this localization is
dependent on proper cytoskeletal and membrane organization. These
components may act as spatial cues in marking the proximal and distal poles
of the cell. These landmarks would then serve to stimulate Budlp, resiilting
in bud-site selection, emergence and growth. Zahner et al, provides a model
to explain the observed defects in a number of bipolar budding defective
mutations, in which one spatial cue is located at the tip of the nascent bud
and another at the bud neck region (144). As the daughter enlarges, the
proximal division site cue at the bud neck is partitioned between the mother
and daughter cell, and may be mediated by Bud9p, and many other
components tfiat are required for polarized growth. The placement of the
distal pole landmark appears to be independent of polarized growth
components, but requires the activity of BudSp (143,144). In siurunary, S.
cerevisiae cell division exhibits asymmetric polarized growth to produce a
9
daughter ceU. The location in the cell membrane where bud emergence will
occur is defined by external nutritive and genetically programmed intrinsic
factors.
Filamentous form growth in 5. cerevisiae also results in the alteration
of bud-site selection. Invasive growth of haploid cells results in a change in
the bud-site selection from an axial to a bipolar pattern (116). In addition,
diploid cells undergoing pseudohyphal growth switch their budding pattern
from a bipolar pattern to a imipolar distal budding pattern, in which each
new bud is formed opposite of the birth pole (48, 73). These differences in
bud-site selection have allowed the use of a genetic screen to clone two
proteins that regulate bud-site selection, 6ud4p and Axl2p (50,123). The
differential budding pattern of filamentous
versus yeast form cells is not
required to allow filamentous growth to occur. Instead, filamentous cells
with defects in bud-site selection genes still form elongated cells and penetrate
the solid agar media at wild-type frequencies (103). A rationale for the
alteration in bud-site selection in filamentotis differentiation is that tmipolar
budding allows sessile yeast cells to place daughter cells away from the
nutrient-deprived colony during subsequent cell divisions, and thus forage
for adequate nutrients (48). Although great progress has been made in the
molecular analysis of bud-site selection, the mechanisms by which bud-site
selection components are regulated in filamentous
have not been investigated.
growth of S. cerevisiae
10
Cell polarity and morphogenesis
Once a bud-site has been selected as a region for polarized growth,
components responsible for bud emergence and growth assemble at the
incipient bud site. These components then serve to act as, or initiate the
formation of a scaffold upon which cytoskeletal elements can form, direct
polarized secretion, and deliver other signaling proteins (17, 21, 25,32, 33, 72,
93). As the bud increases in size, growth components localize to the motherdaughter neck region during cytokinesis. Molecular analysis of this process
has identified a large nimiber of proteins that play roles in. cytoskeletal
organization and vesicle secretion, and are essential for polarized growth (4,
25,137).
Deletion of the rho-related small GTPase Cdc42p, or Cdc24p, the GEF
activator of Cdc42p, results in depolarized actin and isometric cell expansion
(1,9,59). This suggests that Cdc42p and Cdc24p have essential functions in
the generation of cell polarity, but not cell growth. The activated GTP-bound
form of Budlp protein can specifically interact with Cdc24p, and thus connects
the bud-site selection pathway to polarized growth components (108). Cdc24p
and Cdc42p are part of a multisubunit complex that includes a src homology
(SH3) domain-containing protein, Bemlp (23,110). Bemlp physically
interacts with Cdc24p, Budlp, and Ste20p, a mating pathway MAPKKK, and is
11
located at the tip of the developing bud, suggesting that Bemlp is important
in mediating protein-protein interactions and signaling (29,110,126,145).
Another important protein that is involved in the organization and
signaling of polarized growth is the budding yeast formin, Bnilp (37, 70,89,
144). Biulp is located at the tip of mating projections and buds, and physically
interacts with profilin, actin, Cdc42p, Aip3p, Rholp, RhoSp, and Rho4p (37,
58, 70). Cells with mutations in Bnilp have sjmthetic lethal interactions with
a mutation in the septin Cdcl2p, and have abnormal bud neck morphologies
(89). Therefore, Bnilp may serve as the downstream lii\k between Cdc42p
activation and cytoskeletal organization for bud emergence. The ras-related
small GTPase Rho proteins appear to be important for continued growth after
bud emergence, since mutations in RHO genes results in cells that arrest with
very small buds (92, 99,129,142). The physical interaction of Rholp, Rho3p,
and Rho4p with Bnilp suggest a mechanism for the coordination of bud
emergence with bud growth in S. cerevisiae.
The budding yeast septins also play an important role in bud-site
selection, bud morphogenesis, sporulation, and cytokinesis (30,42, 89,144).
The septins are evolutionarily conserved in fungi, invertebrates, amphibians,
and mammals, and in budding yeast are represented by the genes CDC3,
CDCIO, CDCll, CDC12 (27,89,112,122). They form, or are part of, a 10 nm
filament system that is present at the incipient bud site and during bud
growth at the bud neck region. Late in cell division, the septins form a double
12
ring structure, such that one ring is located in the mother, and the other in
the dau^ter (43, 65). Deletion of specific septins is lethal, and mutations in
septins result in abnormal septum formation and chitin deposition,
h3rperpolarized growth, randomized budding, and defects in cytokinesis (89).
Genetic interactions with the septins and components of polarized growth,
Afrlp, Spa2p, Bnilp, Ste20p, Cla4p, and Qis4p, the regulatory subunit of
chitin synthase m, suggest that the septins may act as a scaffold upon which
morphogenesis components assemble to restrict growth to the bud, and later
in the cell cycle, to complete C3rtokinesis and cell scission (29,42,71,89).
Studies with Latnmculin-A, a potent inhibitor of actin assembly, have shown
that localization of the septins to the bud site is independent of the actin
cytoskeleton, thus it appears that septin localization to the bud neck is
mediated by other factors (5). Although a preponderance of genetic data
suggests that the septins are a critical aspect of cellular morphogenesis, the
specific molecular interactions of the septins in many of these activities, such
as c3rtokinesis, are not yet defined.
Potential insight into the role of the septins in cytokinesis may be
gained by the study of Ste20p , Cla4p, and a recently described Cla4p
homologue SKMl, which are budding yeast PAK-related serine/threonine
kinases that can physically bind to Cdc42p via a conserved GTPase protein
binding motif, and share an essential function during cytokinesis (29, 57, 96,
98). Mutations in Ste20p and Cla4p result in proper formation of the septin
13
ring, but the ring appears to localize to the bud tip rather than the neck
region, resulting in abnormal cell morphologies (29). The misplacement of
the septins may be due to inappropriate growth on the mother side of the
neck ring, thus Ste20p and Qa4p may restrict Cdc42p activity and cell growth
to tile daughter side of the neck ring to facilitate c3^okinesis and septum
formation. In addition to the PAK-related kinases, mutations in three other
kinases, Ycklp, Yck2p, and Elmlp, and several phosphatase subunits, Cdc55p,
Tpd3p, Pph21p, Pph22p, Fpzlp, and Fpz2p, also affect cellular morphogenesis,
resulting in some cases in aberrant actin and chitin distribution, and defects
in cytokinesis and nuclear migration, suggesting that a ntmiber of
phosphorylation-dependent signal transduction cascades play an important
role in cell morphogenesis (12,38,53,78,117,135)
In summary, budding yeast morphogenesis can be ordered in a series of
steps which are regulated in a temporal and spatial manner to dictate the
organization of components required for cellular remodeling. First, intrinsic
and extrinsic cues define asymmetric growth by regulating bud-site selection
proteins, which in turn directs the assembly of bud emergence components.
Reorganization of the cytoskeleton, and the subsequent vectorial delivery of
the secretion apparatus, in addition to the activation of the bud growth
components to tiie incipient bud site, is apparently mediated by the bud
emergence components. Late in the cell division, growth components are
14
redirected to the mother-daughter bud neck, by an unknown mechanism to
complete cytokinesis and cell scission.
A defining characteristic of filamentous cells is an elongated shape that
is a result of hyperpolarized deposition of cell membrane and wall growth
components at the bud tip (73). Thus, it may not be surprising that proteins
responsible for polarized growth may also be required for filamentous growth
to occur. Accordingly, proteins involved in cell polarity, Spa2p, Srv2p, Bnilp,
Cdc42p, Ste20p, and tropomyosin, were found to be required for
pseudohyphal growth to occur in lutrogen-limited media (86,102,103).
Septins may also play a role in filamentous growth since specific mutant
alleles of CDC12 result in enhanced filamentous growth (13).
Cell cycle control and morphogenesis
Temporal and spatial coordination of nuclear division and cellular
morphogenesis is essential for the successful production of viable progenitor
(mother) and progeny (daughter) yeast cells. Interruption of this coordination
would residt in nonviable progeny. For example, morphogenesis in the
absence of nuclear division or segregation may result in daughter cells which
lack a nucleus. Alternatively, DNA replication in the absence of
morphogenesis would result in multinuclear mother cells. One mechaiusm
to insure this coordination, is to initiate both processes concomitantly
utilizing a common upstream signaling component.
15
Iti S. cereoisiae, Cdc28p, the master cell cycle dependent regulatory
kinase, is the common signaling component (91,113). Cdc28p catalytic
activity is dependent on positive regulatory subimits, known as cyclins, that
vary in abimdance during cell cycle progression (46,49,104,114,128,140).
Presumably, it is the temporally regulated differential association of Cdc28p
with cyclins that confer substrate specificity to Cdc28p, and allow Cdc28p to
execute the multiple functions reqtiired for cell division. Two conditions,
adequate nutrients and cell size or mass accumulation, are monitored in G1
cells, and are required to be above a defined threshold in order for a new
round of cell division to occur. The point of commitment for initiating a
new round of division is known as Start (51). At Start, Cdc28p associates with
the G1 cyclins, Clnlp, Cln2p, and Cln3p (114), to initiate bud emergence,
spindle pole duplication, and DNA replication (28). The mitotic cyclins or
Clbs, appear later in the cell cycle during S phase and G2 phase to perform
functions such as spindle pole body separation and elongation, nuclear
division and separation (46,66,67,105-107,128).
Early in Gl, a prebud site is selected, where bud formation components
are assembled. Actin cables and patches become directed to the prebud site
(64), presimiably, to direct the vectorial delivery of vesicles and other
components necessary for bud growth. The actin rearrangement occurs after
Start, before bud emergence, and depends on Cdc28p catalytic activity (82,84).
Thus, incubation of a cdc28 temperature-sensitive mutant strain at the
16
restrictive temperature results in depolarized actin distribution. The
Cdc28p/Clnl, 2p-dependent acounulation of actin at the prebud site does not
require new protein synthesis, suggesting that Cdc28p may be acting via direct
phosphorylation of a component of polarized growth (84). Supporting the
h3^othesis that Cdc28p regulates polarized growth directly, S3mthetic lethal
interactions were observed in strains containing deletions for Clnlp and
Cln2p, and mutations in a component of bud-site selection, Bud2p (11, 30).
These data suggest that perhaps a Clnlp or QriZp substrate is necessary to
reduce the concentration or location of GTP-bound Budlp in order for bud
emergence to occur. Thus, in the absence of Qnlp and Cln2p, Bud2p GAP
activity becomes essential to reduce the concentration of GTP-bound Budlp.
Actin remains at tiie tip of the growing bud, imtil the bud is about
thirty percent as large as the mother cell. Bud growth then switches from
apically directed to isotropic or generalized growth. Actin localization
becomes disuse and circumferential during isotropic growth. As c)^okinesis
occurs, actin localization is redirected to the mother-daughter neck region
(64). Overexpression of Qn2p, or deletion of the mitotic cyclin Qb2p, results
in apically dominated growth and elongated cells with actin staining
concentrated at the bud tip. Clb2p and Clblp overexpression results in
delocalized actin staining, that is dependent on Cdc28p activity (84).
Inactivation of the Cdc28p/Clb complex near the end of mitosis is required to
allow actin to polarize at the neck of the mother and daughter cells. A
17
possible mediator of Qb2p-dependent apical/isotropic growth switch may be
the protein Naplp (61,62). A Xenopus Mapl homologue specifically binds to,
and is phosphorylated in vitro by p34°'*^/cyclin B complexes, but not
p34a/c2/cyciin A complexes (61). Yeast strains deleted for Naplp, and Qbs 1,3,
and 4, exhibit an elongated cell morphology reminiscent of wild type yeast
filamentous form growth, suggesting that Naplp may be involved in the
Clb2p-dependent apical/isotropic growth switch of cells during mitosis (62).
In addition to Cdc28p-dependent activity in initiating bud emergence, a
second mechanism exists to ensure coordinated nuclear division and bud
morphogenesis, bi response to C3rtoskeletal depolarization or a defect in bud
emergence, a morphogenesis checkpoint pathway is activated that delays
nuclear division during the G2 phase of the cell cycle until the cytoskeleton
can be repolarized (82,83,125). Yeast cells lacking this checkpoint have
nuclear division uncoupled to bud growth, and therefore become binucleate
in response to a bud defect. The morphogenesis checkpoint was found to be
dependent on phosphorylation of the T5rr-19 residue of Cdc28p (83), and
specifically inhibits Cdc28p/G2 cyclin complexes (14). The phosphorylation of
Cdc28p is performed by Swelp, a tyrosine kinase, that is evolutionarily
conserved in many organisms (3,14,81). Presumably, Swelp is activated by
the checkpoint pathway, and delays nuclear division until the cytoskeleton
can repolarize. The dual specificity phosphatase Mihlp (120) can remove the
T3n:-19 phosphate, and thus may play a role in downregulating the checkpoint
18
pathway during adaptation or normal cell division. Collectively, two
mechanisms exist to ensure the coordination of nuclear division with the
morphological progression of bud emergence and bud growth. First, Cdc28p
in conjunction with G1 and G2 phase cyclins temporally coordinates nuclear
division with actin localization and deposition of cell growth materials.
Secondly, the bud morphogenesis checkpoint establishes a link to signal
cytoskeletal defects to the cell cycle machinery.
Cell cycle regulation appears to play a significant role in filamentous
form growth. Kron and Fink have demonstrated that pseudoh3^hal yeast
cells exhibit a G2 delay in the cell cycle (73). S. cerevisiae yeast cells divide
asymmetrically, producing daughter cells which are smaller in size than the
mother cell. This asjmimetry results in a delay in the G1 phase of the
daughters cell cycle while it reaches a critical size before passing Start (52).
The mother cell has already reached critical size, and therefore initiates a new
roimd of cell division immediately. Pseudohyphal cells, however, exhibit an
alternate form of growth, such that the mother cell pauses in G2, while the
daughter continues to grow. At the time that cytokinesis and cell separation
occur, the daughter reaches critical size and continues into G2, and thus the
mother and daughter initiate new roimds of cell division s3mchronously (72,
73). Thus, filamentous form cells utilize a G2 cell-size checkpoint, as opposed
to the yeast form G1 cell-size control mechanism. Filamentous form yeast
cells also have an altered apical/isotropic switch. During the G2 phase of the
19
cell cycle, bud growth is isotropic, and actin staining is circumferential in the
mother and daughter cell (64, 84). In contrast, filamentous form cells
continue to exhibit apically dominated cell growth, and exhibit actin staining
at the tip of the bud during the G2 phase of the cell cycle (73). These data
suggest that the mitotic delay and hyperpolarized cell growth of filamentous
cells may be the result of high G1 cyclin activity or reduced G2 cyclin activity.
In support of this hypothesis, Clnlp appears to be partially stabilized in wildt37pe cells under conditions of pseudoh3^hal growtii (7). In addition,
overexpression of Clnlp or Cln2p, or deletion of Qblp or Qb2p, results in
enhanced pseudohyphal growth imder conditions of nitrogen-limitation (72).
Collectively, these data suggest that the modification of the cell cycle may be a
critical prerequisite for filamentous growth.
Molecular Characterization of Filamentous Growth
Nutrient regulation
The molecul2ir analysis of pseudohjrphal growth has been a fruitful
area of research in the last several years. Although elongated S. cerevisiae
strains were noted to be capable of forming pseudohyphae under conditions
of limited nitrogen as early as 1965 (16), Gimeno, et. al., 1992 (48), was the first
to characterize the phenomenon in detail. The authors foimd ttiat a
mutation in the gene SHR3, which leads to reduced uptake of many amino
20
adds, enhanced pseudohyphal growth in nitrogen-limited minimal media.
In addition the authors tested the effect of a dominant mutation of RASZ'^^,
which encodes one of die two RAS genes in S. cerevisiae, and found that this
mutation enhanced pseudohyphal growth in diploids on low nitrogen media.
Both the shr3 mutation, which starves the cells of amino adds by preventing
amino add permeases from reaching the plasma membrane (88), and the
RASZ""'^^ mutation, which is very sensitive to conditions of nitrogen
starvation, implied that the signed for pseudohyphal growth in diploids may
be the lack of appropriate nitrogen sources. In budding yeast, Ras genes have
been shown to activate adenylate cydase. Adenylate cyclase converts ATP to
cAMP which then releases the inhibitory subimits of protein kinase A (PBCA),
and results in actived catalj^c subimits (40,133,139). Thus it appears that the
stimulation of pseudoh3rphal growth by the activated RAST^^ mutation is a
result of high PKA activity. In support of this hypothesis, overexpression of
the phosphodiesterase gene PDE2 results in the complete inhibition of the
stimulatory effects of the RAS2"^^^ mutation on pseudoh3^hal growth (136).
The signal tremsduction of RAS2 appears to bifurcate however, as it has been
demonstrated that RAS2"^ can also stimulate the GTPase Cdc42p, resulting
in activation of a transcriptional reporter responsive to filamentous growth
induction (102). The nutrient signal for haploid invasive growth has not
been characterized, however since this differentiation occurs imder mature
21
colonies, it is likely that the cells are experi^dng some type of nutrient
deprivation.
Genetic screens for enhancement of filamentation due to gene
overexpression
The pseudoh3rphal development screen
Involvement of the cell cycle and filamentous growth was first
suggested by the isolation of a potential transcription factor. Gimeno and
Fink performed a screen to isolate genes that in high-copy would enhance
pseudoh5rphal growth on nitrogen-limiting media, which led to the isolation
of several PHD gaies (47). PHDl was found to be homologous to several cellcycle regulated transcription factors in a region containing SWI4- and MPBllike DNA binding motifs. Overexpression of PHDl results in vigorous
filamentous growth regardless of the media composition (47). Deletion of
PHDl did not prevent pseudohjrphal growth in diploids suggesting that there
may be redundancy in this developmental pathway, or that PHDl
overexpression leads to enhanced pseudohyphal growth nonspedfically
through inappropriate DNA binding (47). A second gene, PHD2, was foimd to
be allelic to MSNl/MSSlO/FUPl, a transcription factor previously implicated
in carbon and iron metabolism, and mating functions (35, 36, 75).
22
Mammalian enhancers of filamentation
Screens utilizing a H. sapiens cDNA library to screen for high-copy
enhancers of pseudohyphal growth in S. cerevisiae have been performed to
identify mammalian genes important for generating or maintaining cell
polarity or morphogenesis (31, 63, 76). The underlying assumption is that the
basic mechanisms of c3rtoskeletal regulation will be conserved between higher
and lower eukaryotes. In addition, the identification of these mammalian
Actors may also result in insight into the mechanisms of yeast cell polarized
growth. Overexpression of both himian (hsRBPT) and yeast genes (RBPT) that
compose part of the RNA polymerase II subunit, enhanced pseudohyphal
development in diploids on lutrogen-limited media (63). The subcomplex
comprising these genes are also involved in allowing yeast to withstand long
periods of stress and nutrient deprivation (24). These data suggest that there
may be a connection between a core element of the yeast transcriptional
complex, and changes in cell growth control mediated by nitrogen limitation.
N-terminally truncated HEFl overexpression results in enhanced
pseudohyphal growth, and cell elongation in diploids on minimal mediiun
in S. cerevisiae (76). HEFl interacts with a number of mammalian proteins
implicated in C3rtoskeletal regulation, and contains several coi\served SH2 and
SH3 domains (76). Intriguingly, the effects of truncated HEFl overexpression
occurred only in diploids, suggesting that perhaps a diploid-spedfic factor was
necessary for this effect. Kir, a Ras-related G-protein, was identified in a
23
similar screen for high-copy enhancement of cell elongation in S. cerevisiae
(31). High-copy expression of Kir resulted in constitutive cell elongation,
regardless of nutrient conditions, and was dependent on Stellp, Ste7p, and
SteZOp (31). Thus Kir overexpression on minimal mediimi is similar to
RAS2""'" overexpression under conditions of nitrogen-limitation, and
apparently interacts with tiie same downstream components. The studies
above support the evolutionary conservation of proteins involved in cell
polarized growth in budding yeast and mammalian cells, and substantiate the
role of f(as proteins, signaling proteins contaiiung conserved protein-protein
interaction domains, and nutrient availability, in regulating cellular
morphogenesis.
Genetic screens for inhibition of filamentation due to recessive mutations
Mating/Filamentous MAPK pathway
Components of the mating MAP kinase pathway, STEll (MEKK), STE7
(MEK), STE12 (transcription factor), and STE20 (PAK kinase homologue) (6,
55, 80,124), were foimd to be necessary for pseudohyphal development in
diploids (86), Although the gene products are required for mating functions
in haploids, it had been previously observed that they were also expressed in
diploids (97). The requirement for these gene products in pseudohyphal
growth provided a rationale for their expression pattern in diploids. The
24
same MAP kinase elements required for pseudoh3rphal growth on low
nitrogen medium are also required for invasive growth of haploids on rich
media (116), suggesting that elements of one MAP kinase pathway can play
dual developmental roles, mating and invasive growth, in haploid cells.
The fact that both the invasive growth and the mating pathway share
elements of a single MAP kinase cascade, raises questions of how specificity is
achieved. This suggests that there must be components of these pathways
that do not overlap, and thus provide specificity for a single response
pathway. One specificity determinant is the manner of activation of Ste20p.
During haploid mating, the protein kinase Ste20p appears to be stimulated by
the free Py subunits of G proteins that link pheromone-receptor binding with
downstream mating pathway processes (77). Ste20p phosphorylates Stellp,
which in turn phosphorylates the MAP kinases Fus3p, and Ksslp, which then
directly or indirectly activate Stel2p transcriptional activity to induce
transcription of gene products that contain pheromone response elements
(PREs). ti the filamentous growth pathway, Ste20p still phosphorylates
Stellp, however it is activated or localized by Cdc42p instead of the Py
subimits. Another difference between the mating pathway and the
filamentous growth pathway is that Fus3p and Ksslp are not required for
pseudohyphal growth (86), and have differential effects on invasive growth
(116).
25
Ksslp promotes and Fus3p inhibits haploid invasive growth. Two
nuclear proteins, Diglp/Kstlp and Dig2p/Rst2p, have been identified as
physically interacting with Ksslp and Fus3p, and appear to negatively regulate
invasive growth (26,130). The Dig proteins physically interact with, and are
predicted to repress Stel2p transcriptional activation of genes containing
filamentous response elements (FREs) under conditions of adequate
nutrients. During invasive growth, Ksslp may phosphorylate and release
Diglp and Dig2p, resulting in Stel2p transcriptional activation of genes
responsible for filamentous growth (26). Stel2p was foimd to have a DNA
binding specificity that was different hrom ttie previously identified PRE
sequence due to interactions with Teclp, a protein not involved in the
mating pathway. During filamentous growth, Stel2p associates with Teclp to
bind and transcriptionally activate FRE-containing genes (95). Accordingly,
tecl or stel2 homozygous deletion significantly inhibits pseudohyphal
growth in diploids on low nitrogen solid mediimi (45, 95). In simimary, the
utilization of common elements in two distinct developmental pathways
relies on combinatorial control of downstream effectors to provide specific
outputs in response to differing external factors, namely nutrients amd mating
pheromone.
Other genetic screens for recessive mutations
Mosch and Fink performed transposon mutagenesis to identify factors
that were required for aspects of pseudohyphal growth in S. cerevisiae (103).
26
Mutations were classified according to which aspects of filamentous growth
were lacking such as cell elongation, cell polarity, and agcir penetration.
Mutations were identified in transcription factors (Stel2p, Teclp, Cdc39p), a
protein phosphatase (Cdc55p), proteins witti roles in morphogenesis and
diploid bud-site selection (Srv2p, Tpmlp, Spa2p, Bnilp, Pea2p, BudSp), a
protein involved in intron turnover (Dbrlp), and several imcharacterized
proteins (Whi3p, Nablp, DfgSp, DfglOp, Dfgl6p) (103). The data indicate that
filamentous growth may require a large number of proteins in order to occur,
and that these signaling pathways are genetically separable.
Many standard 5. cerevisiae laboratory strains are unable to imdergo
filamentous growth. This may be due to a strong selective pressure directly
against unusual or elongated cells, or indirectly by procedures such as replica
plating in the artificial environment of the research laboratory. The gene
FLOS was identified in a search for recessive mutations which allowed a
strain incapable of pseudohyphal growth, S288C, to become competent (87).
The diploid Belgism strain Z1278b can undergo pseudohyphal growth on low
nitrogen medium, and when £1278b haploids are crossed to haploid S288C
strains, the resulting diploid is competent to undergo pseudohyphal growth
(48, 87). This suggests that 'wild-type' S288C contains recessive mutations in
genes that are required for pseudohyphal growth to occur. Although other
mutations in S288C were also present that inhibited pseudohyphal growth,
backcrosses to Z1278b, resulted in a single Mendelian recessive factor present
27
in S288C that prevented pseudoh3rphal growth. This allowed the cloning of
FLOS by complementation of the pseudohyphal growth pheno^rpe hy
introduction of a plasmid library made from the strain £1278b, which
contained a functional copy of FLOS (87). FloSp is a putative transcriptional
activator of Flolp^ a cell wall-associated protein involved in flocculation (68).
The exact nature of the requirement of FloSp for filamentous growth, or its
relationship to other proteins required for filamentous growth, is not clear.
Genetic screen for constitutive filamentation due to recessive mutations
Our lab has taken another approach to identify gene products that are
involved in the regulation of filamentous growth. We have performed a
genetic screen to identify recessive mutations which result in constitutive cell
elongation on rich mediimi, thus the genetic screen is predicted to identify
proteins that negatively regulate filamentous growth. Twenty-six abnormally
elongated strains defined 14 ELM (elongated morphology) genes, three of
which, CDC28 (CDK), CDC12 (septin), GRRl (G1 cydin degradation), were
previously identified gene loci (13). All of the mutant strains exhibit many if
not all aspects of pseudohyphal growth, including a G2 delay. ELMl was
cloned and foimd to encode a serine/threonine protein kinase homologue
(12), which exhibits serine/threonine kinase activity in vitro, on general
phosphoacceptor substrates (69). Elmlp is predicted to act downstream or
28
parallel to Ste20p, Stellp, and Ste7p^ since elml ste double mutant strains
remain elongated (69). The elucidation of downstream signaling components
or substrates of Elmlp may provide valuable insights into the mechanisms of
cell polarity and morphogenesis in S. cerevisiae and other fungi.
Dissertation oiganization
This dissertation is formatted to include submitted papers. Chapter 1
contains a general introduction and a literature review that provides an
overview of the significant aspects of filamentous growth in S. cerevisiae.
Chapter 2 consists of the characterization of a suppressor of elml mutant
strains, and has been submitted to the journal Genetics. Chapter 3 consists of
a submitted paper to the journal Genes and Development, detailing the
characterization and genetic interactions of SELl and CDC28, two gene lod
that regulate filamentous growth. Chapter 4 consists of a submitted paper to
the journal Molecular and Cellular Biology concerning the cellular
localization of Sel2p to the bud neck, and genetic interactions with the G2
cyclin, Clb2p. General conclusions are presented in Chapter 5, and the
Appendix contains the details of the cloning of ELMS, a novel putative
membrane protein that is evolutionarily conserved firom fungi to mammals.
References for Chapter 1 and Chapter 5 are listed after the Appendix. The
authors of the submitted papers in Chapters 2 and 4, consist of Dr. Alan
Myers, my major advisor, and myself, and thus I performed all of the
29
experimental procedures, with intellectual contributions and suggestior\s by
Dr. Myers. I wrote the papers, which were subsequently edited by Dr. Myers,
bi Chapter 3,1 perfomed all experiments relating to SEL2, and Dr. M.
Blacketer and T. Bierwagen performed all experiments relating to CDC28.
The paper was written collaboratively, and subsequently edited by Dr. Myers.
Two suppressor screens of elml mutant cell elongation, have resulted
in the characterization of the differential effects of protein kinase A activity
on filamentous growth, and the isolation of several spontaneous extragenic
suppresors of elml strair\s. One of these is a unique serine/threonine kinase,
Sel2p, which appears to play a role in filamentous growth and the execution
of cytokinesis perhaps by regulating Cdc28p complexes at the bud neck.
30
CHAPTER 2. DIFFERENTIAL EFFECTS OF CAMP-DEPENDENT
PROTEIN KINASE ACTIVITY ON SACCHAROMYCES
CEREVISLAE FILAMENTOUS GROWTH DEPENDING ON
GENETIC AND NUTRITIONAL SIGNALS
A paper submitted to Genetics
Nicholas P. Edgington and Alan M. Myers
Abstract
In response to combined environmental and genetic signals vegetative
Saccharomyces cerevisiae cells assume one of two distinct morphologies, the
yeast form or the filamentous form. Molecules affecting this morphogenetic
response were identified by analyzing suppressor mutations that restore yeast
form growth to a strain that constitutively exhibits filamentous form
characteristics owing to mutation of the protein kinase Elmlp. Activation of
cAMP-dependent protein kinase (PKA) by deletion of the negative regulatory
subimit Bcylp suppressed the constitutive filamentous growth phenotj^e of
elml strains. Suppression was media-specific, and occurred only in MATa or
MATa haploids, not in MATa/MATa strains, hi contrast, in MATa/MATa
cells bcyl enhanced the filamentous
growth phenotype of elml strains. PKA
activation also enhanced or inhibited the filamentous growth response of
otherwise wild type strains, again with cell-type specificity. PKA, therefore,
has differential effects on cell morphology depending on the presence or
31
absence of the al/a2 repressor coded for by the MAT locus, and on the
nutritional environment. Mutations eliminating the protein kinase Snflp
also suppressed filamentous growdi both in elml mutants and otherwise
wild type strains. Thus, a variety of parameters influences the cell's decision
of whether or not to &cecute the fUam^tous growth response.
Introduction
The ascomycete Saccharomyces cerevisiae can exist in two different
morphologic states during vegetative growth, termed the yeast form and the
filamentous form (GIMENO et al. 1992; KRON and Gow 1995; ROBERTS and FINK
1994; WITTENBERG and REED 1996), and thus provides a model system for
investigating mechanisms that regulate progranuned changes in cell shape.
The environmental signals that stimulate filamentous growth are complex.
In the phenomenon referred to as pseudohyphal growth (GIMENO et al. 1992)
the filamentous form occurs if the nitrogen source in the meditmi is limited
and an adequate carbon source is present. In addition, pseudohyphal growth
occurs only when cells are in contact with an agar surface, not during growth
in liquid media, and occurs in diploid but not haploid cells. Another t3^e of
filamentous growth phenomenon, termed invasive growth, occurs beneath
the surface of mature colonies on rich agar media and is haploid specific
(ROBERTS and FINK 1994), Although the filamentous forms that occur in
invasive growth and pseudoh3rphal growth are very similar, apparently they
32
arise in response to distinct combinations of regulatory factors including
genetic criteria (the genotype of the MAT locus), external conditions (nutrient
avcdlability and surfoce contacts), and the activities of specific signal
transduction molecules. The complexity of these morphogenetic responses is
reflected in the fact that strains of different genetic backgrounds differ in their
abilities to adopt the filamentous form. For example, pseudoh3^hal growth
has been reported in certain haploid strains (WRIGHT et al. 1993), and invasive
growth occurs in some diploid strains (AM., unpublished results).
The mechanism(s) by which cells sense appropriate environmental
signals, and in response cause the global changes in cellular architectiure and
physiology that constitute the filamentous growth form, axe as yet not
defined. Several molecules with defined signaling functions, however, are
known to stimulate or repress filamentous form differentiation in the
pseudohjrphal and invasive growth responses. Components of the MAP
kinase cascade that also function in the pheromone response pathway
(HERSKOWTTZ 1995) transmit a positive signal for both the pseudohyphal and
invasive growth responses (LJU et al. 1993; ROBERTS and FINK 1994); these
include Stellp and Ste7p (MEKK and MEK homologs, respectively), together
with the upstream activator kinase Ste20p and the downstream transcription
factor Stel2p. The connection between the MAP kinase cascade and the
Stel2p-mediated transcriptional response pertaining to morphology changes
is complex. The MAP kinase components active in the pheromone response
33
pathway, Fiis3p and Ksslp, are not required for pseudohyphal growth (Liu et
al. 1993) but are involved in invasive growth (ROBERTS and FINK 1994). Stel2p
interacts with the transcriptional regulator Teclp to mediate its
morphological effects (GAVIRIAS et al. 1996; MADHANI and FINK 1997). In
addition^ potential negative regulators of Stellp action in invasive growth,
Diglp and Dig2p, were identified irutially by their ability to form a complex
with Ksslp (COOK et al. 1996). Teclp, Diglp and Dig2p seemingly function
specifically in the morphologic differentiation pathway(s) and are not
involved in pheromone response. Elimination of Ste7p, Stellp, or Stel2p
does not entirely prevent pseudoh3rphal growth (Liu et al. 1993), suggesting
the existence of an alternate means of signaling this response.
The S. cerevisiae Ras pathway influences filamentous form
differentiation. The activated allele
stimulates the rate and
frequency of filamentous form differentiation in cells exposed to the
pseudohyphal growth regime (GIMENO et al. 1992). This stimulation is blocked
by deletion of STE20 and reduced by elimination of STE7, STEll or STE12,
indicating the 5. cerevisiae Ras proteins act through a MAP kinase cascade as
Ras proteins are known to do in other organisms (MOscH et al. 1996).
Signaling between Ras2 and the STE MAP kinase cascade requires the Rho
family G protein Cdc42p (MOscH et al. 1996). The action of Ras2 on the STE
MAP kinase cascade is sp>ecific for its function in filamentous form
differentiation and does not cause the pathway to activate genes transcribed as
t
I
34
part of the mating response (MOSCH et al. 1996). The mammalian ras-related
protein Kir also induces filamentous
growth in 5. cerevisiae, again dependent
on the STE7/STE11 MAP kinase pathway (DORDM et al. 1996)
The activity of Ras in filamentous growth may not be explained
entirely by its stimulation of the STE MAP kinase cascade. Even though
strains lacking STE7, STEll, or STE12 were reduced in the frequency
of filamentous form cells, the mutant RAS allele still caused a 5-fold increase
in the percentage of pseudohyphal cells in ste mutants as compared to strains
containing oiUy the wild type allele RAS2 (MOSCH and FINK 1997; MOSCH et al.
1996). Mutants lacking components of the STE MAP kinase pathway but
containing RASl^"^" displayed a response quantitatively similar to the fully
wild type control strain (MoscH et al. 1996). Furthermore, overexpression of
cAMP phosphodiesterase, which presumably reduces cAMP concentration,
attenuated the ability of RAS2^'"^ to stimulate filamentous form growth
(WARD et al. 1995). This result suggests Ras affects filamentous form growth
through its other known function in S. cerevisiae, namely stimulation of
cAMP-dependent protein kinase (PKA) by activation of ad^ylate cyclase
(BROACH and DESCHENES 1990). Consistent with this hypothesis, a molecule
that functions downstream of Ras in the regulation of PKA, namely Srv2p,
was foxmd to be required for specific aspects of pseudohyphal growth (MOSCH
and FINK 1997). The function of Ras in control of morphologic differentiation,
therefore, may involve psurtidpation in at least two signal trarwduction
35
pathways, namely the STE MAP kinase cascade and the PKA pathway. The
current study provides direct evidence that PKA is a regulator of filamentous
form differentiation.
S. cerevisiae mutations are known that cause cells to adopt multiple
aspects of filamentous form growth independent of ploidy, nutritional
environment, or surface contacts, bi addition, in the heterozygous state these
mutations potentiate the pseudoh3rphal growth response in otherwise wild
type diploid strains (BLACKETER et al. 1993; BLACKETER et al. 1994; BLACKETER et
al. 1995). Some of these mutations are deletions, suggesting the affected genes
code for molecules required for repression of the filamentous form in
inappropriate growth conditions. One such gene, ElMl, codes for a serinethreonine protein kinase termed Elmlp (BLACKETER et al. 1993; KOEHLER and
MYERS 1997). As a means of identifying other factors involved in the
regulation of filamentous form growth the current study isolated second-site
suppressor mutations that restore yeast form growth to elml mutant strains.
The results indicate that PKA activity affects filamentous form growth when
differentiation is stimulated either by elml mutations, or by the invasive
growth or pseudohyphal growth regimes in otherwise wild t5^e cells. The
effects of PKA activity differ significantly dependent on the genotype of the
MAT locus and the growth medium. Specifically, PBCA activity inhibits
filamentous form growth in haploid cells, but stimulates morphologic
differentiation in diploids in a manner similar to the effects of RAS2^''". The
36
data indicate that Elmlp and PKA activity are components of a complex
signaling mechanism that has differential effects on morphologic
development depending on die particular combination of signal inputs to
which the cell is exposed.
Materials and Methods
Strain backgrounds, media and genetic methods
Strains in the D273-10B/A1 genetic background are derived from this
strain obtained from A. Tzagoloff (Colimibia University). Auxotrophic
markers were obtained by outcrossing the parent to strain W303-1A (WALUS
et al. 1989), and subsequently backcrossing meiotic progeny at least five times
to D273-10B/A1. Strains in the Z backgrotmd (GRENSON et al. 1966) were
obtained from C. Styles (Whitehead b\stitute) (Liu et al, 1993; ROBERTS and
FINK 1994).
The following media were used: YPD (1% yeast extract, 2% peptone, 2%
glucose); SD (2% glucose, 0.17% yeast nitrogen base without amino adds and
ammonia, 0.5 % ammonium stdfate, supplemented as required with
histidine, leucine, methionine, and uracil at 20 mg/ml each); and SLAD, the
nitrogen limitation medium described previously (GIMENO eta/. 1992) except
that histidine was omitted. SLAD and SD media differ only by ammonium
sulfate content. Solid media for yeast contained 2% agar. Yeast strains were
37
cultured at 30°C unless specified otherwise. Standard methods were used for
genetic manipulations (ROSE et al. 1990).
Strain construction and plasmids
Yeast strains are listed in Table 1. DNA manipulations, nucleotide
sequence analysis, and 5. cerevisiae transformation were performed according
to standard me^ods (AUSUBEL et al. 1989). Haploid strains containing null
mutations in the BCYl locus were obtained by replacement of the wild t3rpe
gene with disruption fragments excised from plasmids pbq/l::LElI2 or
pba/l::URA3 (TODA et al. 1987b). Diploids lacking functional BCYl genes were
obtained by mating a bq/l::URA3 haploid to a bq/l::LEU2 haploid of the
opposite mating type. Congenic BCYl control strains were obtained by
transforming the same parent strains with pRS315 {LEU2) or pRS316 (URA3)
(SIKORSKI and HIETER 1989). A null mutation in the BUDS locus was obtained
by replacing the wild tjrpe gene with bud3::URA3 contained on a fragment
excised from plasmid pBUD3D<Q (CHANT et al. 1995). The bcyl budS double
mutant strain NEY2105 was obtained by replacement of ttie BUD3 locus
followed by replacement of the BCYl locus. A null mutation in the SNFl
locus was obtained by replacing the wild type gene with snfl-15::LEU2
contained on a fragment excised from plasmid pJH80 (HUBBARD et al. 1992).
The MAT A gene was introduced to form pseudodiploid strains as part of the
YCpSO-based plasmid pMATa-URA3 (ASTELL et al. 1981). TPKl was
38
Table 1. S. ceremsiae strains
Strain
Genotjrpe
Source
D271-10B/A1 genetic background:
122D63b
MATa ura3 cdcl2-122
(BLACKETER et al. 1995)
123D65d
MATa ura3 elm5-l
(BLACKETER et al. 1995)
aDU
MATa ura3 metS
(BLACKETER et al. 1995)
NEY2157
MATa. Ieu2 ura3
(BLACKETER et al. 1995)
NEY2159
MATa leu2 ura3 his3 met6
(BLACKETER et al. 1995)
NEDN6d
MATa leu2 ura3 met6 elml-1
(BLACKETER et al. 1995)
104E)62d
MATa ura3 met6 elml-1
(BLACKETER et al. 1995)
NEY1489
MATa Ieu2ura3 met6 elml-1
(BLACKETER et al. 1995)
NEY1667
MATa leu2 ura3 metS sel4::mTn3 elml-1
Transformation of NEY1489
NEY1908
MATa/MATa Ieu2/leu2 ura3/ura3
NEY1489 [pRS316l x
met6/met6 elml-l/elml-1 [pRS316,
NEDN6d [pB{S315]
pRS315]
NEY1917 MATa/MATa leu2/leu2 ura3/ura3
met6/MET6 his3/HIS3 [pRS316, pRS315]
NEY2157[pRS316]x
NEY2159 [pRS315]
NEY1932 MATa leu2 ura3 met6 elml-1 bcyl::LEU2
Transformation of NEDN6d
NEY1933 MATaleu2 ura3 metS elml-1
Transformation of NEY1489
bq/l::URA3
39
Table 1. (continued)
Strain
Genot5rpe
NEY1907 MAT^/MATa leul /leul uraS/uraS
Source
NEY1932xNEY1933
met6/met6 elml-l/elml-1
bcyl::URA3/bq/l::LEU2
NEY1934 MATa leu2 ura3 bcyl::URA3
Transformation of NEY2157
NEY1935 MATa leu2 ura3 his3 bcyl::LEU2
Transformation of NEY2159
NEY1916 MATa/MATa Ieu2/leu2 ura3fura3
NEY1934 X NEY1935
met6/MBT6 his3/HlS3
bcyl::URA3/bq/l::LEU2
NEY2099 MATa ura3 elmS-l bqfl::URA3
Transformation of 123D65d
NEY2103 MATa leu2 ura3 metS elml-1 bq/l::LEU2
Successive transformations
[pMATa-URA3]
NEY2105 MATa leu2 ura3 met6 bcyl::URA3
bud3::LEU2 elml-1
of NEDN6d
Successive transformations
of NEDN6d
NEY2123 MATa ura3 cdcl2-122 bcyl::URA3
Transformation of 122D63b
NEY2239
MATa/MATa Ieu2/leu2 ura3lura3
Transformation of NEY1489
met6/met6 elml'l/elml-1
X NEDN6d
snfl::LEU2/SNFl
NEY3000
MATa/MATa Ieu2/leu2 ura3/ura3
Transformation of NEY2157
met6/MET6 his3/HIS3 snfl::LEU2/SNFl
XNEY2159
40
Table 1. (continued)
Strain
Genotype
Source
NEY3001
MATa. Ieu2 ura3 met6 elml-1 SNFl
Progeny of NEY2239
NEY3002
MATa. Ieu2 ura3 met6 elml-1 snfl::LEU2
Progeny of NEY2239
£ background:
L5487
MATa leu2 ura3
C. Styles
L5684
MATa. Ieu2 ura3
C. Styles
NEY1888
MATa/MATa Ieu2/leu2 ura3fura3
L5684[pRS3151xL5487
[pRS316,pRS315]
[pRS316]
NEY1930
MATa leu2 ura3 bcyl::LEU2
Transformation of L5487
NrEY1931
MATaleu2ura3 bcyl::URA3
Transformation of L5684
NEY1922
MATa/MATa Ieu2/leu2 ura3/ura3
NEY193G X NEY1931
bq/l::URA3/ bcyl::LEUl
introduced as part of the multiple copy plasmid YEpTPKl (TODA ei al. 1987a),
and the activated allele RAS2^^^ was introduced as part of the centromeric
plasmid pl045-564 (WILSON et al. 1993).
All gene replacements were confirmed by DNA gel blot hybridization
analysis (data not shown). All strains containing bq/1 mutations were
analyzed as soon as possible after collection from the transformation in which
the mutation was introduced. Haploids in which BCYl had been deleted by
41
gene replacement were patched from the transformation plates onto selective
SD medium, grown 1-2 days, then spread onto the appropriate medium for
analysis. Diploids lacking bofl were obtained by mixing haploid cells from
the initial SD patches on YFD medium, incubating overnight, spreading on
selective SD medium for single colonies, growing 1 day, patching the diploids
on selective SD mediimi, then applying diploid cells to the appropriate
mediimi for analysis. Included in this analysis was testing the
bq/l::URA3/bcyl::LElI2 diploids for heat shock sensitivity and requirement
for glucose as the carbon source. These controls ensured that suppressor
mutations had not arisen during construction of the diploids. Strains lacking
a functional SNFl allele also were analyzed as soon as possible after their
construction, similar to the bcyl strains.
Generation and cloning of sel4:nnTn3
Transposon mutagenesis was performed as described (CHUN and GOEBL
1996) using methods described previously (HOEKSTRA et al. 1991). The
transposon-mutagenized plasmid library provided by K. Chun (Indiana
University) was digested with Notl, and used to transform elml-1 strain
NEY1489 to uracil prototrophy. Small colonies were visualized using an
inverted microscope, and those containing rotmd cells were collected. The
original transformant NEY1667 was mated to elml-1 tester strain NEDN6d,
and the resultant diploid displayed elongated morphology. Tetrads derived
42
from this diploid invariably produced two uradl-requiring strains (SEL4) with
elongated morphology and contained two spores that failed to germinate.
Thus, suppression of cell elongation in NEY1667 resulted from a single
recessive mutation that also prevented spore germination. NEY1667 also was
mated to die BLMl strain NEY2159. Tetrads from this diploid produced spore
clones in which the cells exhibited elongated morphology, indicating the
suppressor mutation in strain NEY1667 is extragenic, as opposed to a
reversion within the ELMl locus. This mutation is termed sel4::mTn3.
The mutation sel4::mTn3 was cloned as follows. m-Tn3-(URA3) was
detected in genomic DNA of strain NEY1667 by DNA gel blot hybrization
using as a probe linearized plasmid pBLUESCRIPT-SK+ (Stratagene), which
contains the E. coli gene bla also present within the transposon. A single
hybridizing Clal fragment of approximately 9 kb was detected. Genomic DNA
from NEY1667 was digested with Clal and size fractionated by velocity
sedimentation in a sucrose gradient (AUSUBEL et al. 1989). A plasmid library
from the fraction containing the 9 kb fragment was constructed in plasmid
pBC-SK+ (Stratagene), which is marked with a chloramphenicol resistance
gene and lacks bla. Transformants containing m-Tn3-(URA3) were selected
by plating in the presence of both chloramphenicol and ampidlliti. DNA gel
blot hybridization and restriction mapping confirmed that m-Tn3-(URA3)
was present in the recovered plasmid pDLl.
43
The sequence of a segment of the S. cerevisiae genomic DNA present
in pDLl was determined vising universal primers, and fovmd to be identical to
a region of the BCYl locus. These data indicated sel4::mTn3 is a complex
rearrangement in the region containing the BCYl locus. Modification of the
BCYl locus in this mutation was solely responsible for the observed
phenotypes, however, because introduction of plasmid pBCYl containing the
wild tjrpe allele (TODA et cd. 1987b) into NEY1667 {elml-l sel4:nnTn3)
complemented all phenotypes including suppression of cell elongation.
Thus, a mutation in the BCYl locus was responsible for suppression of
elml-1.
Results
Identification of hcyl as a media-specific suppressor of e/ml-induced
filamentous growth
Strain NEY1489 exhibits constitutive cell elongation owing to a
recessive mutation in the ELMl locus (Figure 1A,B). A modified Tn3
transposon marked with URA3, m-Tn3-(liRA3), was integrated into the
genome of this strain at random locations (CHUN and GOEBL 1996).
Approximately 2000 luracil'independent transformants growing on selective
minimal mediimi were scored for cell morphology by visual observation. A
single strain, NEY1667, was identified in which the cell morphology was
44
uniformly roimd owing to a single recessive, extrageiuc suppressor mutation.
A 9 kb Clal fragment containing m-Tn3-(lIRA3) was isolated from total
genomic DNA of strain NEY1667 by selection in E. coli for the ampidllinresistance gene hla contained within the modified Tn3 transposon (HOEKSTRA
et al. 1991). This fragment was used to replace the corresponding
chromosomal region of the elml-1 strain NEY1489, and all uracil
prototrophic transformants displayed the round cell morphology typical of
the original suppressed strain NEY1667 (data not shown). Thus the cloned
fragment contains a mutation, termed sel4:mTn3, that suppresses the
morphologic abnormality of elml~l straiits.
The mutation sel4::mTn3 was found to be allelic with BCYl, which
codes for the negative regulatory subimit of PKA (TODA et al. 1987b). The
nucleotide sequence of the chromosomal region adjacent to m-Tn3-(IilLA3)
in the cloned fragment indicated the insertion had occurred within BCYl.
The sel4::mTn3 mutant strain NEY1667 displayed several phenotypes
characteristic of bq/1 mutants including sensitivity to heat shock, ser\sitivity
to nitrogen starvation, and requirement for glucose as the carbon source (data
not shown). As with bcyl spores, sel4::mTn3 spores were germination
deficient, because tetrads from crosses between NEY1667 and the ura3 tester
strain aDU invariably contained two viable uradl-requiring spores and two
inviable spores. Introduction of a plasmid containing BCYl into NEY1667
suppressed all of these growth defects, and also restored the cell elongation
phenotype typical of eltnl-1 mutant strains (data not shown).
Directed deletions of BCYl constructed by gene replacemoit suppressed
the cell elongation phenot3rpe of the elml-1 strain NEY1489. The wild type
BCYl allele in strains carrying either elml-l or the deletion allele
elml::URA3 was replaced by the insertion allele hcyl::LEU2 or bq/l::URA3,
and transformants were grown on minimal selective medium. All of the
elml bofl double mutant strains exhibited round cell morphology identical to
that of the original elml-1 sel4::mTtt3 strain NEY1667 (Figure 1C,D and data
not shown). Thus, elml mutations are unable to cause abnormally elongated
cell morphology in the presence of bcyl mutations, prestmiably owing to
hyperactivity of PKA resulting from loss of its negative regulatory subtmit.
The appearance of bcyl cells was the same in elml and ELMl genetic
backgrounds (Figure 1D,E); in both instances the cells were more climipy than
wild t5rpe strains and seemingly were more spherical than the ovoid shaped
wild type cells. Another phenotype attributable to elml mutations is a high
frequency of non-axial bud-site selection, which is evident from the
prevalence of extended branched chains of cells in haploid elml mutants
(Figure 1B,C). The bq/1 mutation also suppressed this phenotype, causing
restoration of the axial budding pattern tjrpical of haploid cells (i.e. mother
46
FIGURE 1. - Suppression of e/ml-induced cell elongation by bofl. (A) Strain
NEY2157 (ELMl BCYl). (B) Strain NEY1489 (elml-1 BCYl). Cells
shown in panels A and 6 are from cultures grown to early
exponential phase in YPD liquid medium. (C) Strain NEY1489
(elml-l BCYl). (D) Strain NEY1933 {elml-1 bcylr.URAS). (E) Strain
NEY1934 (ELMl bcyl::URA3). Cells shown in panels C-E are from
cultures grown for 48 hours on SD solid medium. (F) Strain
NEY1933 (elml-1 bqfl::URA3) from a culture grown for 18 hours
on YPD solid medium.
47
and daughter cells both bud adjacent to the previous site of cytokinesis)
(CHANT and HERSKOWITZ 1991; FREIFELDER 1960) (Figure ID).
The suppressor phenot}^ described above was observed in cells grown
in SD synthetic minimal media. Jn contrast, elntl bcyl double mutants
grown in YPD rich medium displayed the elongated morphology typical of
elml mutant strains (Figure 1D,F). Transferring the elongated elml bq/1 cells
from YPD to SD plates resulted in reappearance of the suppressed phenot5rpe
(data not shown), ruling out die possibility that genetic change was
responsible for the observed loss of suppression. Thus, suppression of elml
by bq/1 is media-spedfic.
Activation of RAS2 or PKA suppresses cell elongation in elml
mutants: The effect of bcyl mutations on the morphology of elml strains is
likely to be mediated by activation of PKA. To test this h3^othesis two
alternative means of PKA activation were tested for ttie ability to suppress cell
elongation in elml-1 mutant strains. A centromeric plasmid carrying the
activated allele
(WILSON et al. 1993), or a 2 jun circle-based plcismid
carr5dng the gene TPKl coding for the PKA catalytic subimit (TODA et al. 1987a)
were introduced into ttie elml-1 strain NEY1489. In both instances the
elongated morphology phenotype was partially suppressed, but not to the
same imiform extent as occurred when &cyl mutations were present (Figure
2). These data indicate activation of PKA suppresses the effects of elml-1 on
cell morphology. Less uniform suppression by
or TPKl as
48
FIGURE 2. - Activation of RAS2 or PKA suppresses dml-induced cell
elongation. Strain NEY1489
uraS) was traxisformed with
one of the following URAS-containing plasmids. Cells shown are
from cultures grown for 18 hours on selective SD solid medium.
(A) pRS316 (emptyjplasmid). (B) pl045-564 (centromeric plasmid
containing
) (C) "V^Ep^P^CI (high copy episomal plasmid
carrying ^d type TPKl).
compared to bq/1 mutations may reflect the degree to which PKA activity is
increased by each condition.
Interaction of the MAT and BCYl loci in suppression of elml
Filamentous form differentiation is known to vary dependent on
whether cells are haploid or diploid (GIMENO et al. 1992; ROBERTS and FINK
1994). Thus, bcyl mutations were tested for di^erential effects on cell
morphology in elml mutant haploid and diploid strains. Congenic haploid,
diploid, and AlATa/MATa pseudodiploid strains carrying both bcyl and elml
mutations were grown in SD mediimi. Neither the elml/elml diploid nor
the elml pseudodiploid were suppressed by bq/1 (Figure 3A-C; Figure 4A), in
49
FIGURE 3. - Effects of bcyl on MAT^/MATa elml mutants. (A) Strain NEY1908
iMATi/MATaelml-l/elml-l). (B) Strain NEY1907 (MATa/MATa
elml-l/elml-1 bcyl::URA3/bcyl::LEU2). (C) Strain NEY2103
{MATa elml'l hcyl::LEU2) transformed with pMATi-URA3).
Cells shown in panels A-C are from cultures grown for 18 hours on
selective SD mediiim. Panel C can be compared to Figure ID to
observe the effect of the episomal MAT locus on the ability of fecyl
to suppress elml in a haploid backgrotmd. p) Strain NEY1908. (E)
Strain NEY1907. Cells shown in panels D and E are of the same
genotype as those shown in panels A and B, respectively. In panels
D and E cells shown are from cultures grown on YPD plates for 18
hours.
50
contrast to MATi. elml bcyl strains (Figtire ID) or congenic MATa strains
(data not shown). Mitotic segregants of the pseudodiploid strain that had lost
the plasmid-bome MATA locus, when grown in minimal medium,
invariably displayed die round cell morphology t3rpical of elml bq/l strains
(data not shown). Thus, the differential suppression of elml by bq/l in
mutant haploid and diploid strains results specifically firom the genot3rpe of
the MAT locus, not from an effect of cell's ploidy.
The bcyl mutation had some effect on the morphology of homozygous
elml diploids grown on rich medium. This was not a suppression effect; to
the contrary, in these conditions bcyl seemed to normalize the elongated cell
morphology. In this D273-10B/A1 genetic background elml homozygous
diploids grown on YPD plates frequently exhibit irregular cell shapes
(Figure 3D). When homozygous bcyl mutations were present the length to
width ratio of the double mutant cells became much more uniform, and cell
shape was more homogenous (Figure 3E). Thus bcyl suppresses the
morphologic effects of elml in haploid strains but has a different effect in
MATA/MATa strair\s that results in a noticeably more uniform cell shape.
Interaction of the bud emergence pattern and BCYl in suppression of elml
The genotype of the MAT locus is known to affect cell polarization
such that specific bud site selection patterns occur in haploids and diploids.
51
raising the possibility that the differential effects of fecyl on cell elongation in
haploid and diploid cells is explained by their distinct budding patterns.
According to this hypothesis the strict axial budding pattern in MATa or
MATa cells is required for suppression of elml by bq/l, whereas the bipolar
budding pattern of MATa/MATa cells does not allow suppression. The
direct effect of the bcyl mutation may be to restore the default axial budding
pattern to haploid elml cells, and this in turn may prevent cell elongation.
To test this hypothesis MATa strains were constructed that budded in
the bipolar pattern instead of the normal strict axial pattern. To accomplish
this BUDS was deleted in haploid cells so that the budding pattern would be
converted from axial to bipolar (CHANT and HERSKOWTTZ 1991) independent of
the effects of elml. The effects of fccyl in a budS elml haploid were observed,
and suppression of the cell elongation phenotype was again foimd to occiur.
In this instance the bipolar budding pattern was retained and the uniform
round cell morphology still was evident (Figure 4). Thus, bcyl suppresses the
elongated cell morphology phenotype in elml haploid strains whether the
budding pattern is axial or bipolar, suggesting that the effects of the MAT
locus on cell shape are independent of its regulation of bud site selection.
52
J
r
t
0
J
N
m
FIGURE 4. - Effects of budding pattern on suppression of elml by bq/1. (A)
Strain NEY2103 (MATaelml-1 bq/l::LEll2) transformed with
pMAT3L'URA3. (B) Strain NEY2105 (MATa elml-1 b<^l::URA3
bud3::LEU2). Cells are from cultures grown on selective SD
medium for 18 hours.
Cell type-specific ejects of hcyl on filamentous growth
Loss of ELMl function is thought to cause the same morphogenetic
differentiation effects as those that occur in wild type cells imdergoing
pseudohyphal or invasive growth (BLACKETER et al. 1993; BLACKETER et al.
1995). Thus, the effects of bcyl mutations on filamentous growth in
otherwise wild type haploids and diploids were tested. Homozygous
deletions of BCYl were constructed in the D273-10B/A1 and S genetic
backgrotmds. The rate and extent of filament formation in response to
nitrogen limitation in the 2 genetic backgroimd was significantly greater in
bcyl/bq/1 mutants than in the diploid wild type control strain (Figure 5A,B)/
similar to the stimulatory effects observed previously for the activated allele
53
iS
FIGURE 5. - Effects of bq/1 mutations on pseudohyphal growth of diploid
strains in response to nitrogen limitation. (A) NEY1888
(MATsi/MATaBCYl/BCYl) (2 genetic background). (B) NEY1922
(MATa^/MATa bcyl::URA3/bqfl::LEU2) (2 genetic background). (C)
NEY1917 (MATA/MATaBCYl/BCYl) P273-10B/A1 genetic
background). (D) NEY1916 {MATA/MATabcyl::URA3/bcyl::LEU2)
(D273-10B/A1 genetic background). Colonies shown in panels A-D
were grown for 2 days on nitrogen-limited SLAD medium. (E)
NEY1916 iMATA/MATabq/l::URA3/bafl::LEU2) (D273-10B/A1
genetic background). (F) NEY1922 {MATa/MATa
bcyl::URA3/bq/l::LEU2) (Z genetic background). Colonies shown
in panels E-F were grown for 2 days on standard nitrogen
concentration SD plates.
54
RASI^'^9 (GIMENO et al. 1992). The wild type D273-10B/A1 diploid strain was
unable to undergo filamentous growth in response to nitrogen limitation;
this observation is consistent with previous reports (BLACKETER et al. 1993;
BLACKETER et al. 1995), although separately maintained D273-10B/A1 strains
were found to be competent for pseudohj^hal growth (Liu et al. 1996).
Introduction of homozygous bcyl mutations into the pseudoh3^hal-defident
D273-10B/A1 diploid backgroimd resulted in rapid and uniform filamentous
growth when the cells were plated on the nitrogen-limiting medium SLAD
(Figure 5C,D). Filamentous growth in these strains is dependent on the
nitrogen limitation signal, because on SD mediiun with normal ammonitun
ion concentration the cells display the tjrpical yeast form morphology (Figure
5E,F). Thus, in parallel with the observed ejects on elml diploids, bq/1
mutations enhance filamentous growth in wild type diploid strains
imdergoing pseudohyphal differentiation.
Mutations in bofl were adso tested for effects on the ability of otherwise
wild type haploid cells to undergo filamentous differentiation in response to
the invasive growth regime (ROBERTS and FINK 1994). Wild type and fecyl
mutant cells in tt\e D273-10B/A1 backgroimd were grown on YPD plates for 4
days, after which cells were washed off of the surface of the plates and those
remaining beneath the agar surface were visualized with an inverted
microscope. Wild type cells exhibited the filamentous form previously
described (ROBERTS and FINK 1994) (Figure 6A). The bcyl mutants also
55
FIGURE 6. - Effects of bcyl mutations on filamentous growth of haploid strains
in response to the invasive growth regime. (A) Strain NEY2159
transformed with pRS316 (MATa BCYl). (B) Strain NEY1935
(MATa bcylr.URAS). Colonies were grown for 4 days on YPD
plates, then cells were washed off the surface of the plate. The cells
shown remained under the surfoce of tiie agar mediimi after
washing.
penetrated the agar, however, the cells remained roimd (Figure 6B). Similar
results were obtained in the 1 genetic backgroimd (data not shown). Thus,
again in parallel with their effects on elml mutant strains, bqfl mutations
suppressed cell elongation in haploids undergoing filamentous form
differentiation.
Effects of snfl mutations on filamentous foim differentiation
Mutations in the SNFl gene were tested for the ability to suppress elml
mutations and to atfect filamentous growth in otherwise wild type strains.
These experiments were undertaken because snfl mutations cause a battery of
phenotypes similar to those caused by bq/1, but seemingly do so
independently of PKA function (HARDY et al. 1994; HUBBARD el al. 1992;
THOMPSON-JAEGER et al. 1991). One copy of SNFl was deleted in an elml-l
homozygous diploid, and meiotic progeny clones of the resultant strain
NEY2239 were collected. The elml-1 snfl::LEU2 double mutant progeny grew
significantly more slowly on the spore germination plates than did the elml
single mutants (data not shown). After growth on rich YPD plates for 2 days it
was evident that snfL suppressed cell elongation in the elml mutant strair«
(Figure 7A,B). This suppression is similar regarding morphologic form to the
effects of bqfl, but is distinct in that it occurs in rich medium. The effects of
elml on bud site selection were not suppressed by snfl, however, as bipolar
budding was frequently evident in the double mutant haploid strains (Figure
7C). Again, this is distinct from the effects of bq/l, which include restoration
of the axial budding pattern to elml mutant strains (Figure 1C,D).
SNFl also was deleted in otherwise wild type strains, which were then
examined for the ability to undergo the filamentous growth response. Tetrad
analysis was performed on the snfl/SNFl diploid NEY3000, and progeny
clones were tested for invasive growth under YPD medium as described
(BLACKETER et al. 1993). The snfl mutation cosegregated with the phenotype
of significantly reduced invasive growth (Figure 7D). The snfl cells
remaining under the surface of the agar medium were spherical in shape, as
opposed to the elongated cell morphology of SNFl sibling clones (Figure7E,F).
7. - Effects of snfl on cell elongation. (A) Strain NEY3001 (AlATa elml1 SNFl), (B) Strain NEY3002 (MATae/ml-l snfl::LEU2). Cells
shown are from cultures grown on YPD solid medium for 2 days.
(C) The same strains as ttiose shown in panel A or B, selected to
illustrate the bipolar budding pattern of haploid elml cells
(NEY3001, left section) and haploid elml snfl double mutant cells
(NEY3002, center and right sections). (D) bivasive growth assay.
Tetrads derived from the SNTl/snfl::LBU2 Ieu2/leu2 diploid
NEY3000 were subjected to the invasive growth assay. All colonies
that exhibited wild type levels of invasive growth were leucine
auxotrophs and thus contained SNFl, whereas all colonies that
exhibited reduced invasive growth were leucine prototrophs and
thus carried snfl::LEU2. (E) A SNFl segregant from the tetrad
analysis depicted in panel D. Cells shown remained beneath the
surface of the YPD agar medium after removal of unburied cells by
washing the plate imder rtmrung tap water. (F) A $nfl::LEU2
segregant from the tefrad analysis of panel D undergoing invasive
growth as described for panel E.
58
59
Thus, snfl resembles bq/1 in its ability to prevent cell elongation during
invasive growth of haploids.
Effects of bofl mutations on cell elongation induced by other elm
mutations: ELMl is one of fourteen genes identified previously in a screen
for mutations that constitutively cause cell elongation (BLACKETER et al 1995).
Loss of function mutations in the BCYl locus were generated in strains
containing various elm mutations, to test whether the effects of PKA
activation on cell elongation were specific to elml mutations or were more
general. Suppression of cell elongation by bqfl::URA3 was obvious in most
instances; two examples are shown in Figure 8. ELMS is identical to HSL7 (N.
Edgington., unpublished results) (MA ef al. 1996) and codes for a putative
membrane protein. Disruption of BCYl in an elmS-l strain had the same
effects on cell morphology as were observed in the elml-1 strain (Figure
8A,B). Likewise, inactivation of BCYl in the presence of cdcl2-122, an allele of
the septin gene CDC12 that causes constitutive filamentous growth
(BLACKETER et al. 1995), also suppressed the cell elongation phenotype (Figure
8C,D). As in the bq/1 elml double mutant, suppression of cdcl2-122 by bofl
was specific for growth in sjmthetic medium and did not occur in the complex
medium YPD (Figure 8E,F). Thus, PKA is able to suppress haploid cell
elongation when excessive polarized growth is induced by a variety of
different mutations.
60
FIGURE 8. - Effects of bofl on cell elongation induced by elm5-l or cdcl2~122.
(A) Strain 123D65d transformed with pRS316 (MATa elm5-l). (B)
Strain NEY2099 {MATaelmS-l bcyl::URA3). (C) Strain 122D63b
transformed with pRS316 (MATa cdcll-lll). P) Strain NEY2123
(MATa cdcl2-122 bqfl::UF^3). Cells shown in panels A-D are
from cultures grown for 48 hours on SD solid medium. (E) Strain
122D63b. (F) Strain NEY2123. Cells shown in panels E-F are of the
same genotype as those shown in panels C-D; in panels E-F the
cells shown are from cultures grown for 48 hours on YPD solid
medium.
61
Discussion
Control of filamentous
growth by combinatorial action of multiple signaling
pathwa3rs
The spectrum of morphologic and physiological changes that entail
filamentous growth is induced in wild type cells by at least two seemingly
distinct growth regimes, and mutations in certain genes cause the same
battery of changes to occur constitutively. Four different protein kinase
pathways have been implicated in control of this response, namely the PICA,
Snflp, Elmlp, and STE MAP kinase patiiways. Although the possibility exists
that some of these pathways may be interacting directly in hierarchical
fashion, this situation is highly imlikely for all of them. These observations,
along with consideration of the complex signals required for filamentous
growth (e.g. low nitrogen, high carbon source, sur^ce contacts, genotype of
the MAT locus), suggests a model proposing that multiple independent
signaling pathways impinge on a common regulatory mechaxusm, which in
turn controls the decision to execute the filamentous growth response
(Figtire 9A). The hypothetical common mechanism may integrate
information about various aspects of the cell's growth state and environment,
such that filamentous growth will occur only when an appropriate set of
combined conditions are met. Most of the signaling pathways identified as
regulators of filamentous growth are known to be involved also in other
62
A.
Snflp
PKA
Bmlp
STE
MARK
c.
PKA
^
Elmlp
Bim
I
i
FF
YF
PKA
D.
MAPK
PKA
a1/a2-{
I
Ste12p/
Teclp
C|P
YF
YF
FF
FF
FIGURE 9. - Models for control of filamentous
growth. (A) Multiple signal
transduction pathways are proposed to act in a combinatorial
manner to dictate the morphologic form. Each of the kinase
pathways indicated may respond to a specific component of a
filamentous growth sigiud. (B) RAS is proposed to activate two
pathways, the STE MAP kin^ cascade, and the PKA pathway,
either of which can stimulate pseudohyphal growth in diploid
cells. (C) Model for haploid-spedfic suppression of filamentous
growth by PKA. PKA activity is propos^ to provide a negative
signal that prevents filamentous growth. This negative signal may
require a haploid-spedfic factor, that is not expressed in when
al/a2 is present. (D) Model for diploid-spedfic stimulation of
filamentous growth by PKA. In addition to the repressive function
proposed in panel C, PKA activity may stimulate a second pathway
that provides a positive signal for filamentous growth. The fact
that PKA has a stimulatory effect only in diploid cells may be
explained by requirement of a diploid-spedfic factor in the positive
signaling pathway.
63
cellular processes such as the pheromone response, release &om glucose
repression, response to nutrient withdrawal or depletion, or sponilation. By
integrating the specific combination of information provided by various
signaling pathways, cells can be directed towards specific developmental
output responses such as mating, sponilation, stationary phase, filamentous
growth, or meiosis. How this integration is accomplished is not known,
however, possible scenarios are 1) combinations of phosphorylation/
dephosphorylation events on certain key Actors, each regulated by a different
input pathway, and/or 2) different input pathways regulating specific
transcriptional responses so that the combination of factors produced in any
given set of multiple conditions determines the developmental state.
The fact that PKA activation suppresses the morphologic abnormalities
resulting from elml mutations during growth in rich medium but not
synthetic medium is likely to be a manifestation of this proposed
combinatorial control process. The bq/1 mutation presumably activates PBCA
to its maximal level regardless of the nutrient supply. Thus, if the level of
PKA activity were the sole determinant of morphology, then bcyl should
suppress elml regardless of the growth medium. To explain the observation
that suppression is media-specific we suggest that a second signal, generated
in response to the nutritional environment, interacts with the PKA signal.
The nature of this second signal during growth in synthetic medium is
proposed to interact with PBCA such that cell elongation is prevented.
64
however, during growth in YPD a qualitatively or quantitatively different
signal may interact with the PKA pathway in such a way as to allow
filamentous
growth.
For certain kinase pathways functioning in control of filamentous
growth evidence exists supporting the hypothesis that they act in parallel.
Snflp is a positive regulator of the appropriate response to nutrient
limitation, whereas PKA activity inhibits this response (HARDY et al. 1994;
HUBBARD et al 1992; THOMPSON-JAEGER et al. 1991). In conditions where PKA is
absent from the cell Snflp still exhibits a positive regulatory function in the
starvation response, indicating that Snflp functions independently of PKA
(HARDY et al. 1994; HUBBARD et al. 1992; THOMPSON-JAEGER et al. 1991). The
suggestion that Elmlp acts in a pathway independent of PKA comes from the
fact that multiple copy plasmids bearing ELMl partially rescue the
temperature sensitive phenotype of mutations affecting adenylate cyclase,
which inactivate the PKA pattiway Q. GARRETT, submitted). These data are
consistent with Elmlp functioning downstream of PBCA or in a parallel
pathway. Similarly, this study shows PKA activity suppresses the effects of
elml mutations, consistent with Elmlp functioning upstream of PKA or in a
separate pathway. Taken together these considerations support the model
that Elmlp and PKA influence filamentous growth through distinct effectors.
Finally, elml mutations cause the occurrence of filamentous
growth
characteristics even when the STE MAP kinase cascade is disrupted by
65
deletion of STE7, STEll, or STE20 (A.M-, unpublished results). Therefore,
Elmlp must function either downstream of the MAP kinase cascade or
through a distinct effector mechanism.
Dual roles of RAS2 in control of pseudoh3rplial growth in diploid cells
Activation of RAS2 was known previously to stimulate filamentous
growth in diploid S. cerevisiae cells (GIMENO et al. 1992), however, the effector
pathway involved was not clearly identified. Mutations reducing cAMP
levels attenuated the stimulation of filamentous growth by activated RAS,
implicating the PKA pathway as the effector (WARD et al. 1995). On the other
hand mutations in various STE genes had the same effect, implicating a MAP
kinase cascade as the effector by which RAS stimulates filamentous growth
(MOSCH et al. 1996). The feet that the transcriptional reporter gene
FG(TyA)::lacZ, which normally is expressed only in filamentous growth
conditions, was induced more substantially by activated RAS2 than by a bq/1
mutation suggested that PKA might not be a significant effector of
filamentous growth and that the actual effector pathway comprises a MAP
kinase cascade (MOSCH et al. 1996).
The data presented here provide direct evidence that PKA activity
stimulates filamentous growth in diploids and that the effects of RAS
signaling in this process are not mediated entirely by activation of a MAP
kinase cascade. The fact that PKA activity stimulates filamentous growth but
66
does not substantially activate the pseudohj^hal-spedfic transcriptional
reporter FG(TyA)::lacZ may be explained by a role independent of the
transcriptional response controlled by Stel2p/Teclp (Figure 9B). This
hypothesis is consistent with the fact that activated RAS2 caused a 5-fold
increase in the frequency of pseudohyphal form cells in nitrogen-limited
cultures even in the absence of Stellp, Ste7p, or Stel2p (MOSCH et al. 1996).
Taken together the available data indicate that activation of RAS stimulates
two different effector pathways, PKA and a MAP kinase cascade, and that both
of these provide positive signals for the filamentous growth response in
diploid cells.
Opposing loies of PKA in haploid invasive growth and diploid pseudohjrphal
growth
In contrast to its function as an activator of filamentous growth in
diploid cells, PKA activity is a repressor of filamentous growth in haploid
cells. In elml mutant cells PKA activity also blocked filamentous form
growth in haploids but not in diploids, and in this instance the differential
effects could be attributed directly to the presence or absence of al/a2. These
results are consistent with a model suggesting the action of PKA as a repressor
of filamentous growth is opposed by the action of al/a2 (Figure 9C). Possibly
a factor required for suppression of filamentous growth by PKA activity is
repressed at the transcriptional level by al/o2. This model suggests that
67
Elmlp activity represses transition to the filamentous growth state, such that
recessive mutations cause constitutive filamentous growth in the absence of
any inducing signal. H3^eractivation of PKA is proposed to provide a second
repression signal that compensates for the loss of Elmlp. Similarly,
application of the normal invasive growth signal to haploid cells can not
overcome the repressive effect of hyperactive PKA.
An apparent paradox arises considering the fact that in one instance
(i.e. MATa./MATa cells) PKA stimulates filamentous growth, whereas in
other conditions (i.e. cells lacking al/a2) PKA represses filamentous growth.
The suggestion that al/ot2 eliminates the ability of PKA to repress
filamentous growth is not sufficient to explain how PKA also could stimulate
the response. To account for these observations we speculate that PKA
activity can stimulate two different pathways, each of which is able to affect
the decision of whether to undergo filamentous growth (Figure ID). One of
these routes may be the repression pathway suggested above, which is active
only in haploid cells because it requires a haploid-spedfic protein. The second
pathway suggested is one that stimulates filamentous growth, and this route
may be active only in MATa/MATa cells because it requires a certain
diploid-spedfic protein. Some precedence for a single protein kinase pathway
being utilized differentially in control of filamentous
growth is provided by
considering the involvement of the MAP kinases Fus3p and Ksslp in
invasive growth. Both of these enzymes are known to function directly
68
downstream of Ste7p in the pheromone response pathway (HERSKOVVITZ 1995).
Neither FUS3 nor KSSl is necessary for pseudohyphal growth of diploids (Liu
et al. 1993), whereas the two MAP kinases play opposing roles in invasive
growth of haploids, one stimulatory and the other inhibitory (ROBERTS and
FINK 1994). Thus the STE MAP kinase pathway is similar to the PKA patiiway
in that it can be utilized either to repress or stimulate filamentous growth.
The transcription factor al/a2 was known previously as a regulator of
filamentous form differentiation, because MATn/MATa strains undergo
pseudoh5^hal growth, whereas this does not occur in MATa/MATa or MATa
/MATa diploids, or MATa or MATa haploids (GIMENO et cd. 1992). A possible
explanation for this effect is that the bipolar budding pattern conditioned by
al/a2 is required for filamentous growth. This study reveals a more direct
role of al/a2 as a determinant of cell shape, in addition to its known roles in
regulation of cell polarity (i.e budding pattern) and developmental potential.
In conditions where PKA is hyperactive and a filamentous growth stimulus
is applied, cells containing al/a2 are elongated whereas cells lacking this
regulator remain round. The effects of al/o2 on cell shape do not result
indirectly from its regulation of bud site selection, because an alternative
means of providing the bipolar budding pattern (i.e. deletion of BUD3 in a
haploid strain) did not allow cell elongation in these conditions (Figure 4).
The models presented in Figure 9 provide one possible mechanism by which
al/a2 could regulate cell shape.
69
Possible roles of PKA activity in regulation of filamentous growth
Considering that PKA is known to regulate multiple responses to
nutrient withdraw!, we expect that this regulatory molecule also serves to
recognize external nutrient conditions during the invasive growth response.
Here we speculate about two potential downstream roles of PKA that could
explain its ability to influence filamentous growth. One possibility is that
PiCA acts directly at the site of polarized growth to influoice the cellular
architecture responsible for determining cell shape. Assembly of the actin
cytoskeleton in a specific manner is known to be required for polarized cell
growth, regardless of the circumstances by which that growth is stimulated
(DRUBIN 1996). A potential coimection between the actin C3rtoskeleton and
PKA is provided by the adenylyl cyclase-assodated protein Srv2p, which in
addition to adenylyl cyclase is able to bind both to actin monomers and the
actin binding protein Abplp (FREEMAN et al. 1995; GERST et al. 1991; LILA and
DRUBIN 1997). These observations raise the possibility that actin assembly,
through the action of Srv2p, causes cAMP levels and hence PKA activity to
increase in areas of polarized growth. Altered PBCA activity, as it occiurs in
bcyl mutants, or possibly in response to filamentous growth signals
involving RAS2, might influence the cell polarization process and thus affect
development of filamentous cell shape. Consistent with this speculation is
the observation that Srv2p is necessary for certain aspects of filamentous
70
growth to occur in response to the normal nutrient limitation signal (M6SCH
and FINK 1997).
Another mechanism by which PKA might regulate filamentous
growth is through regulation of cyclin gene expression. Cyclins are regulatory
subimits of the cyclin-dependent kinase that contains the catal3rtic subimit
CDC28 and is responsible for control of cell division cycle progression
(NASMYTH 1996). CDC28 activity is known to be an upstream regulator of
cellular polarization; in the absence of CDC28 activity in the G1 phase of the
cell cycle actin fails to localize to the site of future bud growth, whereas in
conditions of excess G1 CDC28 activity cells are h5rperpolarized and buds
assume elongated shapes (LEW and REED 1993). Specific cell division cycle
alterations occur in pseudoh3^hal form cells (KRON et aL 1994), suggesting a
role of CDC28 in control of filamentous growth. Further evidence consistent
with this hypothesis is that mutations affecting phosphorylation of CDC28 are
able to suppress the effects of elml (N.E., in preparation), and a specific
mutation in CDC28 causes constitutive filamentous growth in the absence of
the normal signal (AM., in preparation).
PKA is known to repress expression of cyclins in both a transcriptional
and post-transcriptional manner during adjustment of cell size when the
carbon source is changed (BARONI et al 1994; TOIOWA et cd. 1994). Thus we
speculate that PBCA activity may be involved in modification of CDC28
function in response to some aspect of a filamentous growth signal, and that
71
this alteration of CDC28 may in turn mediate downstream events in the
filamentous growth response. Such a mechanism cotild be consistent with
the observation that PKA activity has different effects on filamentous growth
depending on whether al/oc2 is present, because changes in cyclin gene
expression are known to cause distinct phenot3^pes in haploids and diploids.
Deletion of SWI4, a transcription foctor involved in Gl-spedfic expression of
the cyclin genes CLNl and CLN2, is viable in haploids but is lethal in diploids
(OGAS et al. 1991). Furthermore, deleting cydins of the PCLl family, which
partially overlap in function with CLNl and CLN2, caused abnormal cell
elongation in diploids but not in haploids (MEASDAY et al. 1997).
A role of Snflp in regulation of filamentous growth
The observation that deletion of Snflp represses cell elongation during
invasive growth may indicate a specific signal input that is integrated as part
of the process controlling the filamentous growth response. Alternatively,
Snflp inactivation could function to regulate cell morphology through a
downstream effector shared by the PKA pathway. The latter hypothesis stems
from the knowledge that inactivation of Snflp causes abnormalities in the
response to starvation that are very similar to the effects of PKA
hyperactivation (HARDY et al. 1994; HUBBARD et al 1992; THOMPSON-JAEGER et al.
1991). Distinct differences were observed, however, between the effects of fecyl
and snfl in suppression of the morphological defect induced by elml.
72
Specifically, PKA activation suppressed the elml phenotype only during
growth in S3aithetic SD medium, whereas snfl action was observed during
growth in SD or in rich YPD medium. Furthermore, snfl did not suppress
the bud site selection defect of elml strains, whereas bq/1 did suppress this
pheno^^. We suggest, therefore, that the effectors through which Snflp and
PKA regulate filamentoiis growth are distinct.
Owing to these specificities we suggest that Snflp acts as a sensor of a
certain aspect of the filcunentous growth signal, most likely the presence of
glucose, and PKA is sensing a different component of the signal. Snflp was
initially described as regulator of the cell's response to the absence of glucose
from the growth medium (JOHNSTON and CARLSON 1992). The ability of Snflp
to sense carbon source availability is well suited to transmitting part of the
pseudohyphal growth signal, which requires not only that nitrogen be limited
but also that the an adequate carbon source be present. Further evidence
suggesting a connection between glucose sensing and cell morphology control
is provided by analysis of mutations in the gene GRRl. Grrlp affects glucose
repression and glucose-induced gene expression, and is known as well to
affect cell morphology such that deletion of GRRl results in abnormally
elongated cells similar in shape to filamentous form cells (BLACKETER et al.
1994; BLACKETER et al. 1995; FUCK and JOHNSTON 1991; OZCAN and JOHNSTON 1995;
VALUER and CARLSON 1991). In addition to its role in carbon source responses,
Grrlp has been identified as a factor that regulates the abimdance of G1 cyclins
73
by controlling their degradation (BARRAL et al. 1995). This latter observation is
consistent with the suggestion that CDC28 activity is a downstream effector
that integrates information from various signaling pathways to elicit the
filamentous growth response. The data presented here suggest that Snflp
participates in one of these signaling pathways.
PKA as a regulator of morphologic difierentiation in other fungi
The involvement of PKA in control of morphologic ditferentiation is
not vmique to S. cerevisiae, but instead is tjrpical in the fungal kingdom.
Exogenous provision of cAMP to the dimorphic zygomycete Mucor spp.
constrains growth of haploid strains to the yeast form morphology, regardless
of atmospheric conditions that would normally induce transition to the
hyphal form (ORLOWSKI 1991). In addition, the endogenous level of cAMP
phosphodiesterase increases during the dimorphic transformation of Mucor
rouxii (CANTORE et al. 1983), consistent with the hypothesis that reduced
cAMP, and hence reduced PKA activity, stimulates transition to the hj^hal
form.
One of the morphogenetic transitions that occurs as part of the
infectious life cycle of the rice blast fungus Magnaporthe grisea, namely
appressorium formation, also is regulated by cAMP levels and PKA.
Exogenous cAMP promotes formation of appressoria in this organism in
laboratory growth conditions where this morphological transformation
74
normally does not occur. In addition, deletion of the M. grisea gene coding
for the PKA catalytic subuxut blocked appressorium formation (MITCHELL and
DEAN 1995). Thus PKA activity promotes morphologic development in the
rice blast fungus. A MAP kinase pathway also is involved in this
morphogenetic transition. The M. grisea gene PMKl codes for a structural
and functional homolog of the Ksslp and Fus3p components of the S.
cerevisiae MAP kinase cascade involved in mating. Deletion of PMKl
prevented appressorium formation in M. grisea (Xu and HAMER 1996).
cAMP also controls morphogenetic development in the com smut
basidiomycete Ustilago maydis, with obvious parallels to the effects in S.
cerevisiae described in this study. Haploid U. maydis cells exhibit a budding
yeast form morphology, whereas mating of haploids carrying different alleles
at both the a and h gene loci resvilts in transition to the filamentous form
(GILLISSEN et al. 1992). Mutations that inactivate adenylate cyclase result in
constitutive Mamentous growth (GOLD et al. 1994). This phenotype was
suppressed by addition of exogenous cAMP or mutation of the gene uacl,
which codes for the regulatory subunit of PKA and thus is analogous to S.
cerevisiae BCYl; prestmied hyperactivation of PBCA by the ubcl mutation also
attenuated filamentous growth of ottierwise wild t3^e cells (GOLD et al. 1994).
PKA activation in U. maydis also affected bud site selection and cell
separation, specific phenotypes also foxmd in this study to be associated with
the bcyl mutation in S. cerevisiae.
75
A final example of the involvement of cAMP and PKA in morphologic
development is provided by studies in which the PKA regulatory subunit of
the filamentous ascomycete Neurospora crassa was eliminated by mutation of
the gene mcb (BRUNO et cd. 1996). This mutation caused loss of polarized
growth and abnormal septum placement, suggesting a regulatory role for
PKA in organization of the cortical actin cytoskeleton. Collectively these data
reveal a common theme in fungal growth and development in which PKA
activity and MAP kinase cascades regulate cellular morphogenesis in
response to external and genetic signals. Investigations into the differential
regulation of cell polarization by PKA in S. cerevisiae, therefore, may provide
unique insights into the regulation of cell shape not only in budding yeast,
but also in medically and agriculturally important pathogenic fungi.
Acknowledgments
We thank K. Chim for providing the transposon mutagenesis library, M.
Wigler, J. Chant, J. Carmon, M. Lorenz, and M. Carlson for providing
plasmids, and K. Olsen for technical assistance. This investigation was
supported by award no. MCB-9319028 from the National Science Foimdation.
This is journal paper No. J-
of the Iowa Agriculture and Home
Economics Experiment Station, Ames, Iowa, Project No. 3197, supported by
Hatch Act and State of Iowa Funds.
76
References
ASTELL, C R., L. AHLSTROM-JONASSON, M. SMITH, K. TATCHELL, K. A. NASMYTH et
al., 1981 The sequence of the DNAs coding for the mating type lod of
Saccharomyces cerevisiae. Cell 27: 15-23.
AusuBEL, F. M., R. BRENT, R. E. KINGSTON, D. D. MOORE, J. A. SMITH et al., 1989
Current Protocols in Molecular Biology. John Wiley and Sons, New York.
BARONI, M. D., P. MONTI and L. ALBERGHINA, 1994 Repression of growth-
regulated G1 cyclin expression by cyclic AMP in budding yeast. Nature 371:
339-342.
BARRAL, Y., S. JENTSCH and C. MANN, 1995 Gi cydin turnover and nutrient
uptake are controlled by a common pathway in yeast. Genes Dev. 9:399409.
BLACKETER, M. J., C. M. KOEHLER, S. G. COATS, A. M. MYERS and P. MADAULE,
1993 Genetic control of dimorphism in S. cerevisiae: Involvement of a the
novel protein kinase homologue Elmlp and protein phosphatase 2A. Mol.
Cell. Biol. 13:5567-5581.
BLACKETER, M. J., P. MADAULE and A. M. MYERS, 1994 The S. cerevisiae
mutation elm^l facilitates pseudohjrphal differentiation and interacts
with a deficiency in phosphoribosylpyrophosphate synthase activity to
cause constitutive pseudohyphal growth. Mol. Cell. Biol. 14:4671-4681.
BLACKETER, M. J., P. MADAULE and A. M. MYERS, 1995 Genetic analysis of
morphologic differentiation in S. cerevisiae. Genetics 140: 1259-1275.
BROACH, J. R. and R. J. DESCHENES, 1990 The function of RAS genes in
Saccharomyces cerevisiae. Adv. Cancer Res. 54: 79-140.
BRUNO, K. S., R. ARAMAYO, P. F. MINKE, R. L. METZENBERG and M. PLAMANN, 1996
Loss of growth polarity and mislocalization of septa in a Neurospora
mutant altered in the regulatory subimit of cAMP-dependent protein
kinase. EMBO J. 21:5772-5782.
CANTORE, M. L., M. A. GALVAGNO andS. PASSERSON, 1983 cAMP levels and in
situ measurement of adenylate cyclase and cAMP phosphodiesterase
activities during yeast-to-hyphae transition in the dimorphic fungus
Mucor rouxii. Cell Biol. Int. Rep. 7:947-954.
77
CHANT, J. and I. HERSKOWTTZ, 1991 Genetic control of bud site selection in yeast
by a set of gene products that constitute a morphogenetic pathway. C^ 65:
1203-1212.
CHANT, J., M. MISCHKE, E. MITCHELL, I. ITEBSKOWRRZ and J. R. PRINGLE, 1995 Role
of 6ud3p in producing the axial budding pattern of yeast. J. Cell Biol. 129:
767-778.
CHUN, K. T. andM. G. GOEBL, 1996 The identification of transposon-tagged
mutations in essential genes that affect cell morphology in Saccharomyces
cerevisiae. Genetics 142: 39-50.
COOK, J. G., L. BARDWELL, S. J. KRON and J. TTIORNER, 1996 Two novel targets of
the MAP kinase Kssl are negative regulators of invasive growth in the
yeast Saccharomyces cerevisiae. Genes Dev. 10: 2831-2848.
DORIN, D., L. COHEN, K. DEL VILLAR, P. POULLET, R. MOHR et al., 1996 Kir, a
novel Ras-family G-protein, induces pseudoh)^hal growth in
Saccharomyces cerevisiae. Oncogene 11: 2267-2271.
DRUBIN, D. G., 1996 Origins of cell polarity. Cell 84:335-44.
FLICK, J. S. and M. JOHNSTON, 1991 GRRl of Saccharomyces cerevisiae is
required for glucose repression and encodes a protein with leudne-rich
repeats. Mol. Cell. Biol. 11:5101-5112.
FREEMAN, N. L., Z. CHEN, J. HORENSTEIN, A. WEBER and J. FIELD, 1995 An actin
monomer binding activity localizes to the carboxyl-tenninal half of the
Saccharomyces cerevisiae cydase-assodated protein. J. Biol. Chem. 270:
5680-5685.
FREIFELDER, D., 1960 Bud formation in Saccharomyces cerevisiae. J. Bacteriol.
80:567-568.
GAVIRIAS, V., A. ANDRLVNAPOLOUS, C. J. GIMENO and W. E. TIMBERLAKE, 1996
Saccharomyces cerevisiae TECl is required for pseudoh3^hal growth.
Molec. Microbiol. 19:1255-1263.
GERST, J. E., K. FERGUSON, A. VOJTEK, M. WIGLER and J. FIELD, 1991 CAP is a
bifunctional component of the Saccharomyces cerevisiae adenylyl cydase
complex. Mol. Cell. Biol. 11:1248-1257.
78
GILLISSEN, B., J. BERGEMANN, C. SandMANN, B. SCHROEER, M. BOLKER et al., 1992
A two-component regulatory s3rstem for self/non-self recognition in
Ustilago maydis. Cell 68:647-657.
GIMENO, C. J., P.O. LJUNGDAHL, C. A. STYLES and G. R. FINK, 1992 Unipolar cell
divisions in die yeast S. cerevisiae lead to filamentous growtii: regulation
by starvation and RAS. Cell 68:1077-1090.
GOLD, S. E., G. A. DUNCAN, K. J. BARRET and J. W. KRONSTAD, 1994 cAMP
regulates morphogenesis in the pathogenic fungus Ustilago maydis. Genes
Dev. 8:2805-2816.
GRENSON, M., M. MOUSSET, J. M.
and J. BECHET, 1966 Multiplicity of the
amino add permeases in 5. cerevisiae. 1. Evidence for a specific arginine
transporter. Biochim. Biophys. Acta 127:325-338.
HARDY, T. A., D. HUANG and P. J. ROACH, 1994 Interactions between
cAMP-dependent and SNFl protein kinases in the control of glycogen
accumulation in Saccharomyces cerevisiae. J. Biol. Chem. 269: 27907-27913.
HERSKOWTTZ, I., 1995 MAP kinase pathways in yeast: For mating and more.
CeU 80:187-197.
HOEKSTRA, M. F., H. S. SEIFERT, J. NICKOLOFF and F. HEFFRON, 1991 Shuttle
mutagenesis; bacterial transposons for genetic manipulations in yeast.
Methods Enzymol. 194:329-342.
HUBBARD, E. J. A., X. YANG and M. CARLSON, 1992 Relationship of the
cAMP-dependent protein kinase pathway to the SNFl protein kinase and
invertase expression in Saccharomyces cerevisiae. Genetics 130: 71-80.
JOHNSTON, M. andM. CARLSON, 1992 Regulation of carbon and phosphate
utilization, pp. 193-281 in The Molecular and Cellular Biology of the Yeast
Saccharomyces. Gene Expression, edited by E. W. Jones, J. R. Pringle and J.
R. Broach. Cold Spring Harbor Press, Plainview, NY.
KOEHLER, C. M. and A. M. MYERS, 1997 Serine-threonine protein kinase
activity of Elmlp, a regulator of morphologic differentiation in
Saccharomyces cerevisiae. FEBS Lett., in press.
KRON, S. J. andN. A. R Gow, 1995 Budding yeast morphogenesis; signaling,
cytoskeleton and cell cycle. Curr. Opin. Cell Biol. 7:845-855.
79
KRON, S. J., C. A. STYLES and G. R. FINK, 1994 S3aninetric cell division in
pseudohyphae of tiie yeast Saccharomyces cerevisiae. Mol. BioL Cell 5:
1003-1022.
LEW, D. J. and S. L REED, 1993 Morphogenesis in the yeast cell cycle:
Regulation by Cdc28 and cyclins. J. Cell. BioL 120:1305-1320.
LILA, T. andD. G. DRUBIN, 1997 Evidence for physical and functional
interactions among two Saccharomyces cerevisiae SH3 domain proteins,
an adenylyl qrdase-assodated protein and the actin C3rtoskeleton. Mol.
Biol. Cell 8:367-385.
LIU, H., C. A. STYLES and G. R. FINK, 1993 Elements of the yeast pheromone
response pathway required for filamentous growtti of diploids. Science
262:1741-1744.
LIU, H., C. A. STYLES and G. R. FINK, 1996 Saccharomyces cerevisiae S288C has a
mutation in FLOS, a gene required for filamentous growth. Genetics 144:
967-978.
MA, X.-J., Q. Lu and M. GRUNSTEIN, 1996 A search for proteins that interact
genetically with histone H3 and H4 amino termini uncovers novel
regiilators of the Swel kinase in Saccharomyces cerevisiae. Genes Dev. 10:
1327-1340.
MADHANI, H. D. and G. R. FINK, 1997 Combinatorial control required for the
specificity of yeast MAPK signaling. Science 275:1314-1316.
MEASDAY, V., L. MOORE, R. RETNAKARAN, J. LEE, M. DONOVIEL et al., 1997 A
family of cyclin-like proteins that interact with the Pho85 cyclin-dependent
kinase. MoL Cell. Biol. 17:1212-1223.
MITCHELL, T. K. and R. A. DEAN, 1995 The cAMP-dependent protein kinase
catalytic subimit is required for appressorium formation and pathogenesis
by the rice blast fungus Magnaporthe grisea. Plant Cell 7:1869-1878.
MOSCH, H.-U. and G. R. FINK, 1997 Dissection of filamentous
growth by
transposon mutagenesis in Saccharomyces cerevisiae. Genetics 145:
671-684.
80
MOSCH, H.-UV R. L, ROBERTS and G. R. FINK, 1996 Ras2 signals via the
Cdc42/Ste20/mitogen-activated piotein kinase module to induce
filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci.
USA 93:5352-5356.
NASMYTH, K., 1996 Viewpoint putting the cell cycle in order. Science 274:
1643-5.
OGAS, J., B. J. ANDREWS and I. HERSKOWITZ, 1991 Transcriptional activation of
CLNl,CLN2, and a putative new G1 cyclin {HCS26) by SWI4, a positive
regulator of Gl-spedfic transcription. Cell 66:1015-1026.
ORLOWSKI, M., 1991 Mucor dimorphism. Microbiol. Etev. 55: 234-258.
OzcAN, S. and M. JOHNSTON, 1995 Three different regulatory mechanisms
enable yeast hexose transporter (HXT) genes to be induced by different
levels of glucose. Mol. Cell. Biol. 15:1564-1572.
ROBERTS, R. L. and G. R. FINK, 1994 Elements of a single MAP kinase cascade
in Saccharomyces cerevisiae mediate the two developmental programs in
the same cell type: Mating and invasive growth. Genes Dev. 8:2974-2985.
ROSE, M. D., F. WINSTON and P. HEITER, 1990 Methods in Yeast Genetics. A
Laboratory Course Manual. Cold Spring Harbor Laboratory Press,
Plainview, NY.
SncoRSKi, R. S. and P. HDETER, 1989 A system of
shuttle vectors and yeast host
strains designed for efBdent manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122: 19-27.
THOMPSON-JAEGER, S., J. FRANCOIS, J. P. GAUGHRAN and K. TATCHELL, 1991
Deletion of SNFl afiiects the nutrient response of yeast and resembles
mutations which activate the adenylate cyclase pathway. Genetics 129:
697-706.
TODA, T., S. CAMERON, P. SASS and M. WIGLER, 1987a Three different genes in
the yeast Saccharomyces cerevisiae encode the catalytic subunits of the
cAMP-dependent protein kinase. Cell 50:277-287.
TODA, T., S. CAMERON, P. SASS, M. ZOLLER, J. D. SCOTT et al., 1987b Qoning and
characterization of BCYl, a locus encoding a regulatory subunit of the
cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol.
CeU. Biol. 7:1371-1377.
81
TOKIWA, G., M. TYERS, T. VOLPE and B. FUTCHER, 1994 fohibition of G1 cyclin
activity by the Ras/cAMP pathway in yeast. Matuie 371:342-345.
VALUER, L. G. and M. CARLSON, 1991 New SNF genes, GALll and GRRl affect
SUC2 repression in Saccharomyces ceremsiae. Genetics 129: 675-684.
WALUS, J. W., G. CHREBET, G. BRODSKY, M. ROLFE and R. ROTHSTEIN, 1989 A
h3rper-iecombination mutation in S. cerevisiae identifies a novel
eukaryotic topoisomerase. Cell 58:409-419.
WARD, M. P., C. J. GIMENO, G. R. FINK and S. GARRETT, 1995 SOK2 may regulate
cyclic AMP-dependent protein Idnase-stimulated growth and
pseudohyphal development by repressing transcription. Mol. Cell. Biol. 15:
6854-6863.
WILSON, B. A., M. KHALIL, F. TAMANOI and J. F. CANNON, 1993 New activated
RAS2 mutations identified in Saccharomyces cerevisiae. Oncogene 8:
3441-5.
WITTENBERG, C. and S. I. REED, 1996 Plugging it iru signaling circuits and the
yeast cell cycle. Curr. Opin. Cell Biol. 8:223-230.
WRIGHT, R. M., T. REPINE and J. E. REPINE, 1993 Reversible pseudohjqphal
growth in haploid Saccharomyces cerevisiae is an aerobic process. Curr.
Genet. 23:388-391.
Xu, J.-R. and J. E. HAMER, 1996 MAP kinase and cAMP signaling regulate
infections structure formation and pathogenic growth in the rice blast
fungus Magnaporthe grisea. Genes Dev. 10:2696-2706.
82
CHAPTER 3. CONTROL OF SACCHAROMYCES CEREVISIAE
FILAMENTOUS GROWTH BY A PROTEIN KINASE PATHWAY
INCLUDING SEL2P AND CDC28
A paper submitted to Genes and Development
Nicholas P. Edgington, Melissa J. Blacketer, Trade A. Bierwagen
and Alan M. Myers
Abstract
The ascomycete Saccharomyces cerevisiae exhibits alternative
vegetative growth states referred to as the yeast form and the filamentous
form, and switches between the two morphologies depending on specific
environmental signals. To identify molecules involved in control of
morphologic difierentiation this study characterized mutant S. cerevisiae
strains that exhibit filamentous growth in the absence of the normally
required external signal. A specific amino add substitution in the
cyclin-dependent protein kinase CDC28 was found to cause constitutive
execution of most filamentous growth characteristics. These effects indude
specifically modified cell polarity characteristics in addition to the defined
shape and division cyde alterations typical of the filamentous form.
Constitutive filamentous growth induced by mutation of the protein kinase
Elmlp was prevented by modification of CDC28 such that it could not be
phosphorylated on t3^osine residue 19. Mutations aHecting the conserved
Sel2p-Swelp protein cascade that mediates this phosphorylation of CDC28
83
either prevented or enhanced filamentous growth. The data suggest that an
enviroiunentally-responsive signaling mechanism specifically modulates
CDC28 function, which in turn exerts global ejects that cause filamentous
form growtii. The modulation of CDC28 function is likely to affect the
specificity of its interactions with cydins.
Introduction
The ascomycete Saccharomyces cerevisiae is able to adopt one of two
distinct morphologic forms during vegetative growth, referred to as the yeast
form and the filamentous form (Gimeno et al. 1992). Two distinct signaling
regimes induce the filamentous form in wild type cells. In the phenomenon
known as pseudohyphal growth diploid cells become filamentous when
grown on solid agar medium containing a rich carbon source but limited for
ammonia (Gimeno et al. 1992). Haploid cells also become filamentous, but in
response to a different signal. This phenomenon, known as invasive growth,
occurs under the surfoce of mature colonies grown on rich agar medium
(Roberts and Fink 1994). The genetic tractability of S. cerevisiae provides a
facile model system for analysis of the molecular control mechanisms that
mediate this morphologic differentiation response.
The filamentous forms that occur during invasive or pseudohyphal
growth are similar (Gimeno et al. 1992; Kron et al. 1994; Roberts and Fink
1994). Filamentous form cells are significantly elongated compared to the
yeast fonn, and cell separation after cytokinesis is delayed. The establishment
of cell polarity is specifically altered in the filamentous form, such that cell
siirface expansion, i.e. bud growth, predominantly occurs at the pole opposite
the previous cytokinesis site, ti contrast, in the yeast form bud growth occurs
much more frequently adjacent to the to previous site of cell division
(Friefelder 1960). Filamentous form cells are able to grow invasively beneath
the surface of agar media, whereas yeast form cells grow exclusively on the
surface of agar plates.
Yeast form and pseudohyphal form cells also differ from each other in
their control of the cell division cycle. In the yeast form new daughter cells
(i.e. cells that have not budded previously) grow by isotropic surface
expansion during the G1 phase of the cell cycle; after reaching critical size the
Start checkpoint is passed (Pringle and Hartwell 1981). After Start the cells are
committed to executing the remaining series of events in the cell division
cycle in a specific order, and because of this commitment, mitosis of the
mother cell in the yeast form can occur independent of daughter cell size
(Pringle and Hartwell 1981). The pseudohyphal form differs in that newly
formed daughter cells form buds shortly after cytokinesis, without a G1
growth phase, and mitosis seemingly is restricted dependent on growth of the
bud to a critical size (Kron et al. 1994). These observations suggest the major
control of cell cycle progression occiurs during G2 in filamentous form cells
and during G1 in yeast form cells. Despite the complexity of this process.
85
there is a simple visual indication of the ditference in cell cycle progression,
which is that buds emerge simultaneously in mother-daughter pairs in the
filamentous form. The yeast form differs in that the mother cell usually
forms a bud before the daughter cell, indicative of faster passage of Start.
The apparent differences in cell division control suggest that the cyclin
dependent protein kinase CDC28 is involved in filamentous growth. CDC28
is a central regulatory factor in determining whether 5. cerevisiae cells pass
several different cell cycle control points, including Start, the transition
between G2 phase and entry into mitosis, and the completion of mitosis after
chromosome separation is complete in telophase (for reviews see Nasmyth
1993; Futcher 1996; Wittenberg and Reed 1996). To date no evidence is
available to indicate that the function of CDC28 is altered in response to
nutritional conditions, although a mechanism such as this is a possible
explanation for the differences in cell cycle progression between yeast and
filamentous form cells.
At least two different mechanisms must be considered as means of
modulating CDC28 function for the purpose of morphologic differentiation.
Although expression of CDC28, the catalytic subtmit of the protein kinase
complex, is constant throughout the cell cycle, it performs distinct functions
at the various transition points owing to association with different activating
regulatory subtmits termed cyclins (Futcher 1996). Eleven different cyclins are
known to activate CDC28, and their expression varies specifically through the
86
cell qrde. Thus, altered cyclin expression or activity in response to the
environmental growth regime could specifically modulate CDC28 activity.
Another possibility is regulatory phosphorylation of the catal3^c subunit. In
organisms including Schizosaccharomyces pombe, Drosophila melanogaster,
and humans, a protein kinase first identified in S. pombe as weel is known to
phosphorylate the CDC28 counterpart on a conserved tjnrosine residue and
prevent its activity (Russell and Nurse 1987; Nurse 1990). Subsequent
removal of ttiis phosphate group reactivates the cyclin-dependent kinase.
This phosphorylation mechanism is also a potential means of regulating
CDC28 activity in response to an environmental signal so that filamentous
growth characteristics result.
As a means of identifying molecules involved in the control of
filamentous growth, mutant strains were analyzed that constitutively exhibit
elongated cell morphology and other specific characteristics independent of
the growth regime that normally is required for morphologic differentiation
in wild type cells (Blacketer et al. 1993; Blacketer et al. 1994; Blacketer et al.
1995). One of the genes identified by this screen, ELMl, codes for a serinethreonine protein kinase termed Elmlp (Koehler and Myers 1997). Deletion
of ELMl causes constitutive filamentous growth, independent of growth
medium, surface contact, or cell ploidy (Blacketer et al. 1993), suggesting
Elmlp functions as a downstream regulator of morphologic differentiation.
The targets of Elmlp that may be responsible for the changes in cellular
87
growth are not known. Another gene identified by mutations that cause
constitutive filamentous growth characteristics is ELM7 (Blacketer et al. 1995).
The product of ELM7 was suggested to play a central role in this process
because of its extensive genetic interactions with many other mutations
affecting cell morphology (Blacketer et al. 1995).
Through further characterization of ELM? and ELMl, this study
describes two lines of evidence indicating CDC28 function is a controlling
factor in the cell's decision to grow vegetatively in the filamentotis form or
the yeast form. First, a specific mutation altering CDC28 function was
identified that constitutively causes most of the filamentous form
characteristics, including cell shape and cell polarity, in addition to cell
division cycle parameters. Second, the several elements of the protein kinase
cascade that phosphorylates CDC28 on tjrrosine 19 were foimd to regulate
filamentous growth, both as potential downstream regulators of the effects of
Elmlp, and in otherwise wild tjrpe cells exposed to the invasive growth
regime. The data support the h3^othesis that modulation of CDC28 function
in response to environmental signals is in large part responsible for the
transition between the yeast form and the filamentous form.
88
Results
Cloning and characterization of the mutation cdclS^ll?
S. cerevisiae stiains bearing elm7-l constitutively display several
characteristics of the filamentous growth form including elongated cell shape
(Fig. 1A,B), simultaneous emergence of buds on related mother and daughter
cells, and enhanced invasive growth (Blacketer et al. 1995). The following
results revealed that elm7-l is an allele of CDC28. The wild type allele of
elm7-l was cloned by complementation of another phenotype resulting from
the mutation, which is a cold-sensitive growth defect. One transformant
obtained after transformation of an elm7-l strain with a wild type genomic
DNA library that was able to grow at 15®C also displayed the ovoid cell shape
typical of yeast form cells. This strain contained plasmid pl27c, which was
responsible for complementation of elm7-l because derivative clones in
which the plasmid was lost during vegetative growth regained both the
abnormal cell shape and cold-sensitive growth phenotypes. Nucleotide
sequence analysis revealed that the genomic insert in pl27c contained CDC28.
Plasmid p28WT-URAc, in which CDC28 is the only intact genetic element in
the insert DNA, was introduced into elm7-l strains and found to restore
normal yeast form morphology, again in a plasmid dependent maimer (data
not shown). Thus, CDC28 complements elm7-l when present as part of a
centromeric plasmid.
89
!=•
CDC28 cdOB-m cdc28-127
bum±EU2
»30« 20-
ilJi
1.0- 151.5 U
2i
ZS' 3.0- 3i- 4.0- «3i) 3.5 4i) 45 5i)
LengthAwidth ratio
Volume ((un3 X10-2)
Figure 1. Effects of cdc28-127 on cell size and morphology. Haploids strains
were grown overnight on YPD plates and photographed suspended
in 1.2 M sorbitol. The black bar represents 10 fun in actual length.
(A) Strain AY2287 {cdc28-127). (B) Strain AY2285 (CDC2S). (C) Strain
AY2153 {cdc28-127 reconstructed in the D273-10B/A1 background).
(D) Strain AY2204 {cdc28-127 reconstructed in ttie Z backgroimd). (E)
Strain AY2194 {cdc28-127 reconstructed in the W303 background).
(F) Distribution of length/widtti ratios and approximated cell
volumes. The cdc28-127 and CDC28 strains analyzed are AY2287
and AY2285, shown in panels A and B, respectively; the cdc28-127
bud2A::LEU2 strain is AY2241 shown in Fig. 2C.
90
The CDC28 genomic locus was found to be tightly linked genetically to
the elm7-l mutation. The wild tjrpe URA3 gene was inserted as a marker into
the genome adjacent to the wild type CDC28 gene in strain AYIOOX. This
strain was crossed to strain 127D6 bearing elm7-l, and tetrads were analyzed.
Both parents in this cross were uraS mutants so that uracil prototrophy
identified progeny bearing CDC28, and progeny bearing elm7-l could be
recognized by elongated cell morphology. CDC28 and elm7-l segregated in
opposition in 30 complete tetrads, indicating tight linkage of the two genetic
elements. The fact that CDC28 both complements and is tightly linked to
elm7-l indicates the mutation resides within this locus. Accordingly, elm7-l
here is renamed as cdc28-127.
The specific mutation in cdc2S-127 that causes constitutive filamentous
growth was found to be substitution of cysteine residue 127 with a tyrosine
(termed C127Y). The CDC28 locus was isolated firom a wild t5rpe and cdc28-127
strains by PCR amplification of genomic DNA. Nucleotide sequence analysis
of several independent clones showed that cdc28-127 differs firom the wild
type allele CDC28 only by the C127Y mutation. To prove that C127Y is the
causative agent of the observed phenotjrpes, the mutant gene was used to
replace CDC28 in three different wild type haploid genetic backgrounds,
namely D273-10B/Al, Z, and W303. In all instances the resultant strains
displayed the morphologic and growth defects observed for the original
cdc28-127 mutant strain (Fig. 1C,D,E). The genetic background, however, had
91
modifying affects on the particular cell morphology and also atfected growth
rate. In the D273-10B/A1 background cdc2-117 strains grew on rich agar plates
at a slightly reduced rate compared to congenic CDC28 strains. In the W303
backgroimd die mutant strains grew significantly slower than did the wild
type control strains, and in the £ background the mutant strains grew
extremely slowly compared to wild type, requiring 4-5 days at room
temperature to form a colony that could be transferred to a fresh plate.
Filamentous growth characteristics caused by cdc28-127
The following data indicate that cdc28-127 causes all of the known
characteristics of pseudohyphal growth, with one exception. These
observations were made on haploid strains grown to early exponential phase
in liquid cultures of rich medium (YPD), implying that filamentous growth
characteristics occur in the absence of the normal requirements for diploidy,
nitrogen limitation, and contact with agar medium. Cells bearing cdc28-127
are sigiuficantly elongated and greatly increased in voliune compared to
congenic CDC28 cells (Fig. IF). Enhanced ability to grow invasively in rich
agar medium was observed previously for a cdc28-127 strain (Blacketer et al.
1995), and this was observed again in the cdc28-127 mutants constructed by
gene replacement (data not shown). Strains containing cdc28-127 do not form
large cell climips during growth in liquid mediiun. Thus, the delayed cell
92
separation, pheno^rpe characteristic of filamentous form growth is not caused
by cdc28-127.
The simultaneous bud emergence characteristic of pseudohyphal
growth also was found to result from cdc28'127. In contrast to wild type cells,
mother-daughters pairs of cdc28-127 mutants frequently contain buds very
similar in size (Fig. 2A,6)- Time-lapse photography of clonal growth
commonly showed buds of equal size on mother-daughter pairs in cdc28-127
mutant strains (Fig. 3A), whereas in congenic CDC28 strains a bud almost
always was visible on the mother cell prior to the daughter cell. The
cdc28-127 mutation, therefore, results in a pattern of sjmchronous
mother/daughter bud emergence and growth as opposed to the wild tjrpe
yeast form pattern of bud emergence and development on the mother cell
preceding that of the daughter.
Another filamentous growth characteristic arising from cdc28-127 is
reduced length of the Gl phase of the cell division cycle, and increased
duration of the G2 phase. Measiirement of DNA content per cell in a mixed
population revealed that the firequency of cells in G2 was sigruficantly
increased in a cdc28^127 mutant compared to a congenic wild type strain (Fig.
2D). One explanation for these results is that the Start checkpoint is
compromised in cdc28-127 mutants so that bud emergence carmot be delayed
in Gl. Start is operative in cdc28-127 strains to some extent, however, because
mutant cells are able to mate and thus must be capable of arresting in Gl
93
WSOA-IA
yzm
Figure 2. Cell cycle pareimeters. The black bar represents 5 nm in actual
length. (A) Strain AY2287 (cdc28-127). (B) Strain AY2285 (CDC28).
(C) Strain AY2241 (cdc28-127 bud2A::LEU2). Cells were grown
overnight in liquid YPD medium. (D) DNA content determined by
FACS analysis (Lew et al. 1992). The indicated strains were
converted to p° derivatives lacking mitochondricil DNA prior to
analysis.
94
aic28-127
e
a s 5 0
clb2::LEU2
t
100-
CDC28BUD2 ^
:
-I
Class:
°(§)®='lh^|(gsH|(g^
1
2
3
4
Figure 3. Bud site selection. (A) Strain AY2287 (cdc28-127). (B) Strain AY2249
(clb2::LEU2). (C) Strain aDUL (CDC28). Cells are photographed on
the surface of YPD agar medium. Black bars represents 25 jmi in
actual length. (D) Tabulation of data exemplified in panels A-C.
Bud site selection classes are defined at the bottom of the panel.
phase in response to mating pheromone. Mutant strains bearing cdc28-127
also arrest normally in stationary phase because unbudded cells accumulated
in the population after growth to saturation^, and because stationary phase
cells were able to withstand heat shock (data not shown). Furthermore,
.1
95
cdc28-127 mutant strains remained viable when depleted of nitrogen (data not
shown).
The mutation cdc28-127 also affects the mechanism of bud site
selection, causing cells to adopt the budding pattern typically observed in
diploid cells of the pseudohyphal form. Bud site selection was determined by
isolating cells with small buds on solid rich medium, and observing the
subsequent site of budding on both the mother and daughter cells in
time-lapse photography (Fig. 3). All strains analyzed were haploids, and thus
the normal budding pattern is axial (i.e., both mother and daughter cells form
buds adjacent to the previous cytokinesis site). As expected axial budding was
typical of wild type control strains (Fig. 3C). The mutant cells always formed
buds at one of the two poles of the elongated cells. Axial budding was
comparatively rare in the mutants, however, which instead budded with
high frequency in the bipolar budding pattern (i.e., both mother and daughter
cells bud at the pole opposite the cj^okinesis site) or in the unipolar budding
pattern (i.e., daughter's first bud at the pole opposite the previous C5^okinesis
site, mother's next bud adjacent to the previous c3rtokinesis site). The
unipolar budding pattern predominated over the bipolar pattern in the
mutant cells (Fig. 3D). This distribution of bud site selection patterns
cosegregated with the cell elongation phenotjrpe in progeny of
cdc28-127/CDC28 diploid strains, indicating this phenot5^e resulted
96
specifically 6rom cdc28-127 and not firom a second mutation present in this
genetic background.
CDC28 affects bud site selection through known polaiity^establishment
functions
CDC28 may affect bud site selection as an upstream regulator of the
"polarity-establishment functions" coded for by a large group of loci including
the BUD genes (for reviews, see Chant 1994; Drubin 1996). As an initial test of
this hypothesis one of the general bud site selection genes, BUD2, was deleted
in a haploid strain containing cdc28'127. Double mutant progeny as well as
cdc28-127 and bud2A::LEU2 single mutant siblings were analyzed for bud site
selection by time-lapse photography. As expected, bud site selection was
primarily random in bud2A::LEU2 strains (Chant and Herskowitz 1991) (data
not shown). In cdc28-127 bud2^:LEU2 double mutant strains buds usually
emerged from the longitudinal center of the elongated cells as opposed to the
poles (Fig. 2D), a condition that was never observed in BUD2 cdc28-127 cells.
Thus, the null mutation bud2A::LEU2 is epistatic to cdc28-127 regarding the
bud site selection phenotype, consistent with the hypothesis that filamentous
form differentiation involves a specific modification of the site-selection
functions mediated by CDC28.
The BUD2 deletion mutation also afiected cell shape. The cdc28-127
bud2A::LEU2 double mutants were significantly less elongated than congenic
97
cdc28'127 BUD2 cells (Fig. IF); this phenotype cosegiegated with the budZ
mutation in the progeny of a bud2A::LEU2/BUD2 CDC28fcdc28-127 diploid
(data not shown). The bud2A::LEU2 mutation did not affect the enlarged
voltmie of cdc28-127 cells (Fig. IF). Thus the general bud site-selection
functions may play an additional role in determination of cell shape in the
growing bud.
CLB2 plays a significant role in control of filamentous growth
The G2-expressed cyclin Clb2 is known to regulate cell shape (Lew and
Reed 1993), which suggests the hjrpothesis that the mutant protein
coded for by cdc28-127 causes filamentous growth characteristics because of
abnormal interactions with Clb2. To test this hypothesis, CLB2 first was
deleted in a D273-10B/A1 haploid strain. A slight degree of cell elongation
resulted from this mutation, although the phenotj^e is much less severe
than that resulting from cdc28-127 (Fig. 4A,B). This mild cell elongation
phenot5^e was similar to that observed in the haploid BF264-15DUa genetic
backgroimd (Richardson et al. 1992), however, a diploid strain in this
background lacking Clb2 displayed tmiformly elongated cells similar to
cdc28-127 strains (Lew and Reed 1993). Deletion of CLB2 in haploid cells
caused another phenotype that also results from cdc28-127, specifically,
alteration of the budding pattern firom axial to bipolar (Fig. 3B). These results
provide further evidence supporting the hypothesis that CX)C28 activity
98
Figure 4. Effects of altering CLB2 expression in cdc28-127 mutant strains. The
strains shown in panels A-D are sibling progeny from the cross of
strain AY2249 (MATa clb2::LEU2 leu2 uraS) to strain AYIOIX
{MATSL cdc28-127 leu2). The black bars represents 10 |im in actual
length, except for panel d where the bar represents 25 ^un.
Photomicrographs are of cells grown overnight on YPD plates and
susp^ded on a slide in 1.2 M sorbitol, ©ccept for panel D, which
shows a germinating spore clone after 2 days on YPD medium taken
with an inverted microscope. (A) CDC28 CLB2. (B) CDC28
clb2::LEU2. (C) cdc28-127 CLB2.
cdc28-127 clb2::LEU2. The clone
shown in panel D did not develop further and is representative of
15 deduced cdc28-127 clb2::LElI spores identified in tetratype and
nonparental dit3rpe tetrads from this cross. (E) Strain AY2252
icdc28-127 GAL:CLB2) grown in YPD liquid medium to early log
phase. (F) Strain AY2252 grown in YPGal liquid medium to early
log phase.
99
regulates function of the bud site selection apparatus, and are consistent with
the hypothesis that Qb2-CDC28 interaction is a critical factor in determination
of filamentous growth.
Further evidence suggesting CDC28^^"^ causes filamentous growth
characteristics because of an abnormal interaction witii CLB2 was provided by
observation of a S5mthetic lethal phenotype resulting from the clb2::LEU2
cdc28-127 double mutant combination. The requirement of cdc28-127 strains
for CLB2 was indicated by analysis of tetrads from a cdc28~127/CDC28
CLB21clb2::LEU2 diploid. From this diploid 11 of 14 tetrads were identified as
tetratj^es by the presence of one cdc28-127 CLB2 progeny clone (elongated
morphology, leucine auxotroph), one CDC28 CLB2 progeny clone (wild type
morphology, leucine auxotroph), and one CDC28 clb2::LEU2 progeny done
(slight cell elongation phenotype, leucine prototroph). In each instance the
fourth spore of the tetrad, predicted to carry both cdc28-127 and clb2::LEU2,
germinated but failed to develop a viable clone (Fig. 4A-D). Two of the tetrads
contained two CDC28 CLB2 progeny clones and two spores that did not form
colonies; again the two inviable spores are predicted to be cdc28-127 clb2::LEU2
double mutants. The terminal phenotj^e of germinating clb2::LEU2
cdc28-127 spores suggests a lethal effect specifically in G2, because large buds
invariably were present (Fig. 4D). Thus, the mutant CDC28 complex is
defective in a function sp>ecific to passage through the G2 phase of the cell
cycle.
100
A third indication that CDC28"^ is defective in its interaction with
Clb2 and thus causes constitutive filamentous growth is that overexpression
of CLB2 suppressed the cell elongation phenotype caused by cdc28-127. The
gene fusion GAL:CLB2 in which the CLB2 coding sequence can be
overexpressed
6:0m the GAL
promoter, was integrated into the LEU2 locus of
a cdc28-127 leu2 strain. Growth of this strain on galactose caused ceUs to
revert to the normal ovoid shape typical of the yeast form (Fig. 4E,F). Carbon
source had no effect on cell shape in other cdc28-127 strains lacking GAL:CLB2
(data not shown).
The protein kinase Sel2p is a regulator of filamentous growth characteristics
An independent line of genetic evidence implicating CDC28 as a
regulator of filamentous growth was provided by suppressor analysis of
mutations in the gene ELMl. Strain 104D576b exhibits constitutive
filamentous growth characteristics owing to presence of the recessive
mutation elml-1 (Blacketer et al. 1993). Ten spontaneously arising derivative
strains in which the cell morphology had reverted to the t3^ical yeast form
were collected. Ensuing genetic analysis revealed that all 10 strains contained
extragenic suppressor mutations responsible for restoration of the yeast form
cell shape. Three loci, termed SELl, SEL2, and SEL3 Oppressor of dml-1)
were represented in this collection. Four allelic recessive mutations were
101
obtained in SELl, five allelic dominant mutations were obtained in SEL2, and
one dominant mutation was obtained in SEL3.
SELl was identified by its genetic map position, bi the course of its
genetic characterization, the SELl locus was found to be linked to ELMl on
chromosome XI. Determination of genetic linkage with several cloned genes
on this chromosome (Table 1) indicated SELl was located in the vicinity of
open reading frame YKLlOlw designated in the S. cerevisiae genomic
sequence. This open reading firame was obtained £rom the ordered phage
done library available for the S. cerevisiae genome, and subcloned in plasmid
pN£30. Because sell mutations are dominant, it was necessary to rescue a
mutant allele, SELl-l, from the original suppressed strain for the purpose of
testing whether SELl was in fact the locus responsible for suppression of
elml-1. This was done, and the resultant gap-repaired plasmid pNE30GR was
introduced into the parental elml-1 strain 104D576b. All transformants were
completely restored to the yeast form morphology (Fig. 5), indicating that the
open reading frame YKLlOlw and SEU. are the same genetic element.
SELl has also been cloned as the gene HSLl and NIKl (Ma et al. 1996;
Tanaka and Nojima 1996). That study pointed out that the protein product
Sel2p, is most similar to the protein kinase Niml from S. pombe; over a span
of 304 aligned residues 48% are identical in Sel2p and Niml. The similar
regions of the two proteins are located within the protein kinase domain.
102
Table 1. Intergenic linkage data
Lod
Strains crossed.
TT:NPD:PD"
Map distance (cM)''
SEU'ELMl
NEY1494 x aDU
30;0-^
273
SEU-PUT3
NEY2018xNEY2065
11:1:3
56.7
SEL2-BUD2
NEY1494 x NEY2019
4:0:31
5.71
SEL2-GFA1
NEY2065 x NEY2020
2:0:50
1.92
' Tetratype (11), Nonparental ditype (NPD), Parental dit5rpe (PD).
Map distance was calculated from the formula:
[50aT+6NPD)l /TT+NPD+PD.
SEL2 comprises a reading frame of 1^19 codons, however, so that there are
extensive regions outside of the protein kinase domain available to provide a
number of additional functions besides this catal3^c activity. These
observations are of interest because Niml is known in S. pombe to promote
mitosis by inhibiting activity of the tyrosine kinase Weel, which in turn
phosphorylates and inhibits the cyclin-dependent kinase Cdc2 (Russell and
Nurse 1987; Russell and Nurse 1987). In S. cerevisiae the protein kinase
Swelp is known to function similarly to Weel downstream of Sel2p (Booher
et al. 1993; Ma et al. 1996).
The fact that mutations of SEL2 capable of suppressing elml-1 are
dominant suggests that activation of Sel2p, predicted to result in inhibition of
Swelp, is responsible for suppression of the constitutive Blamentous growth
103
-»-pRS316
Figure 5. Suppression of elongated cell morphology by SELZ-l. The elml-1
strain NEDN6d or the cdc28-127 strain AY2287 was transformed to
uracil prototrophy with the indicated plasmid. Transformant
colonies were grown overnight on selective agar medium,
suspended on a slide in 1^ M sorbitol, and visualized using DIC
optics.
phenot5^e. This h3^othesis predicts that inactivation of Sel2p, which would
result in hyperactivation of Swelp, will induce filamentous growth
characteristics. To test this hypothesis, SEL2 was deleted from the S.
cerevisiae genome. Strains bearing the mutation sel2::LEU2 were viable and
did not show any obvious growth defects. The mutant cells were significantly
i
104
elongated, however, and ttie budding pattern was switched in hapioid cells
£rom the normal axial pattern to the bipolar pattern typical of filamentous
growth (Fig. 6C). Both of these phenoiypes were observed during growth in
rich medium. Thus, activation of Sel2p by dominant mutation prevents
filamentous growth characteristics induced by elml-1, and inactivation of
Sel2p on its own induces filamentous growth characteristics. These data
implicate Swelp and its downstream target CDC28 as potential regulators of
filamentous growth.
The Swelp-CDC28 pathway is required for induction of filamentous growth
characteristics by elml or sell mutations
A sel2::LEU2 swel::URA3 double mutant strain was constructed to test
the hypothesis that the morphological effects of mutations in SEL2 are
mediated by Swelp. The cell shape and budding pattern phenotypes of this
strain were typical of the yeast form, and the filamentous growth
characteristics of the sel2::LEU2 SWEl strain were not observed (Fig. 6F).
Thus, Swelp is required for induction of filamentous growth characteristics
by the sel2 mutation. This observation predicted that Swelp function would
also be required for elml mutations to induce filamentous
growth
characteristics, according to the h3^othesis that the Sel2p-Swelp-CDC28
pathway acts downstieam of Elmlp. To test this hypothesis an elml::HIS3
swel::URA3 double mutant strain was constructed. As predicted, inactivation
105
Figure 6. Phosphorylation of CDC28 by Swelp is required for mutational
induction of filamentous growth characteristics. Straii\s were grown
overnight on YPD plates, suspended in 1.2 M sorbitol, and visualized
under DIC optics. (A) Strain NEY2340 (wild t5rpe). (B) Strain
NEY2338 (elml::HIS3). (C) Strain NEY2335 (sel2::LEU2). (D) Strain
NEY2344 {swel::URA3). (E) Strain NEY2341 (elml::HI$3
swel::URA3). (F) Strain NEY2339 (sel2::LEU2 swel::URA3). (G)
Strain AY2452 (CDC28 elml::HIS3 [p28Y19F-URAcl). (H) Strain
AY2451 icdc28::LEU2a elnil::HIS3 [p28Y19F-URAcl).
of SWEl also suppressed the induction of filamentous growth characteristics
by the elml mutation (Fig. 6E).
The activity of Swelp is to phosphorylate CDC28 on residue Y19 and
thus inhibit its cydin-dependent protein kinase activity (Booher et al. 1993).
106
The fact that elimination, of Swelp prevents filamentous
growth
characteristics induced by elml or sell mutations suggests accordingly that
Y19 phosphorylation is necessary for the morphologic pheno^^ to occur.
This h)^othesis was tested by replacing wild type CDC28 in an elml::HlS3
strain with a non-phosphorylated mutant protein,
in which the
tzirget tyrosine residue is replaced by a phenylalanine. Although CDCIS""" by
itself resulted in slightly elongated cells, this phenotype was clearly
distinguishable from that of elml CDC28 cells. The presence of the nonphosphorylated CDC28 mutant protein suppressed the cell elongation
phenot5rpe induced by elml (Fig. 6H), further implicating the Sel2p-SwelpCDC28 pathway as a controlling foctor for filamentous
growth, at least when
this morphologic phenotype is induced by elml mutations.
The Sel2p*Swelp pathway regulates filamentous growth in otherwise wild
t)^ cells
The invasive growth response was examined in strains containing
various mutations in the Sel2p-Swelp pathway, to test whether this kinase
cascade functions as a regulator of morphogenesis in response to specific
external sigimls. Congenic strains were grown on rich medium for 2 days, at
which time cells were washed off the surbce of the plates to revesil the extent
of invasive growth. The wild type control strain xmderwent invasive growth
as expected (Fig. 7A), although the response was much more extensive after
Figure 7. Invasive growth is affected by mutations of the Sel2p-Swelp
pathway. (A) bwasive growth assay. The indicated strains were
grown for four days on YPD plates, then cells were washed off of the
plate's surface. Strains are aDUL (Wd t)^), NEY2334 (sel2::LEU2),
and NEY2339 {sel2::LEU2 swel::lIRA3). Panels B and C show
colonies growing underneath the agar, from the plates shown in
panel A, photographed using an inverted microscope. (B) Strain
aDUL (wild type). (Q Strain NEY2334 (5e/2:;L£U2). Panels D to H
show coloiues growing imdemea^ the agaur, all taken &om a single
YPD plate washed after four days of growth. In each of these panels
the photograph was taken from the densest area of invasive growth.
(D) Strain NEY2340 (wild type). (E) Strain NEY2334 (sel2::LEU2). (F)
Strain NEY2353 {SEL2'1:LEU2). (G) Strain NEY2344 (swel::URA3).
(H) Strain NEY2339 {sel2::LEU2 swel::URA3).
108
Before
wash
After
wash
Wild type ••ii||^
sel2
u
set2swe1
C^f
'"•'ri V'?* V'^l^''''*" ^
V . ••/• ^ .. . •• -'
. •". ^*''"'"^
1^';-•->• -."-••>-••
' .i" .s.
:a
-7
-'-i
109
4 days of growth (data not shown). The sel2::LEU2 mutant strain exhibited a
much greater extent of invasive growth ttian did wild type cells (Fig. 7A),
with a response at 2 days similar to that of the maximal wild t3^e response at
4 days. Examination of cells growing beneath the agar medium showed the
mutant cells to be much more elongated than the wild type cells tmdergoing
invasive growth (Fig. 7B,C). Enhancement of invasive growth by the sel2
deletion mutation was dependent on Swelp function, because swel::URA3
sel2::LEU2 double mutants were markedly reduced in their degree invasive
growth compared to the sel2::LEUl single mutant (Fig. 7A,E,H). The extent of
invasive growth in the double mutant was the same as that observed in swel
single mutants, and was noticeably less than wild type cells (Fig. 7D,G). These
data are again consistent with ttie hypothesis that inhibition of CDC28 by
Swelp is required for filamentous growth, and that Sel2p action negatively
regulates Swelp. The hypothesis suggests that dominant mutatioiis of Sel2p,
originally identified as suppressors of filamentous growth characteristics
induced by elml mutations, also would inhibit invasive growth of otherwise
wild type cells. As predicted, the extent of invasive growth in strains bearing
the dominant allele SEL2-1 was significantly reduced compared to the wild
type strain (Fig. 7D,F).
110
cdc28'127 is epistatic to mutations in the Sel2p-Swelp pathway
The effects of mutations in SEL2 and SWEl regarding fUamentous
growth characteristics and the invasive growth response presiunably are
mediated by CDC28. This hypothesis predicts that the filamentous growth
characteristics resulting from cdc28-127 would occur independent of whether
the Sel2p-Swelp pathway was functiorud. To test this hypothesis the
dominant allele SEL2-1 was introduced into a cdc28-127 strain. Although this
mutation dearly suppressed the filamentous growth characteristics in an
elml-1 strain, there was no effect on the phenotype of the cdc28-127 strain
(Fig. 5). As another means of disrupting the Sel2p-Swelp pathway SWEl was
deleted in a cdc28-127 mutant strain. The cdc28-127 swel::URA3 strain
displayed constitutive filamentous growth characteristics identical to
cdc28-127 single mutants (data not shown), again indicating that the mutant
CDC28 protein is a downstream effector of filamentous growth characteristics.
Discussion
The data reported here reveal that a specific mutant form of CDC28,
namely CDC28*^^"^, simultaneously causes at least four cellular characteristics
that are specific to the filamentous form: apparent control of cell cycle
progression at a G2/M restriction point, elongated cell shape, specifically
altered cell polarity determination, and the ability to grow invasively in agar
medium. The fact that so many specific aspects of filamentous
form cells
Ill
result from this single amino add substitution is highly unlikely to be
coincidental, and instead argues that wild type cells imdergoing filamentous
growth in response to environmental signals have altered CDC28 function in
a way that is duplicated or mimicked by cdc28-127. Previous analysis of
cellular growth characteristics indicated that specific changes in cell cycle
regulation occur in filamentous form cells relative to yeast form cells (Kron et
al. 1994). That report proposed a signal transduction pathway comprising
components of the STE MAP kinase cascade is activated by nutritional signals
(Liu et al. 1993), and inhibits the G2 activity of the CDC28 complex to create a
restriction point at the G2/M boimdary. Passage of this restriction point is
proposed to depend on the bud having grown to approximately the same size
as the mother cell. Previous data also have shown that by altering G1 or G2
cyclin expression, CDC28 activity is a controlling factor for determination of
cell morphology (Lew and Reed 1993). The effects of CDC28"^ are consistent
with these studies, and indicate further that a modified CDC28 complex can
mediate not only these specific cell cycle characteristics, but also bud site
selection and invasive growth properties of the filamentous form. Thus, the
CDC28 complex is proposed to be a central downstream regulator of
filamentous form differentiation (Fig. 8). The fact that CDC28^-^^ does not
cause the delayed cell separation characteristic of filamentous form cells
might be explained by a separate signaling pathway not involving CDK being
responsible for this response. Alternatively, CDC28^^ may not fully mimic
112
RIamentous
growth signal
1
STE20. STE7,STE11
STEl2nEC1
I
FRE's
I
ELM1
cii o
Oiploici-specific
pathway
SWE1
\
COK alteration
CDK^,
t
i
l
COK^
l
YF cell shape
YF cell polarity
YF cell cycle
surface growth
I
I
I
I
FF cell shape
FF cell polarity
FF cell cycle
agar penetration
Figure 8. Model for the role of the CDC28 complex in control of filamentous
growth. CDKyp and CDK^p indicate two slightly different forms of
the CDC28 complex, whi^ mediate different downstream effects.
The SWEl pathway is proposed to be downstream of the STE MAP
kinase pathway because the diploid response occurs even in the
absence of Swelp. At least two means of changing the CDC28
complex from CDKyf to CDKpp are envisioned, one of which
depends on SWEl, and a second on a putative diploid-specific
pathway.
113
the changes in CDK that occur during filamentous form differentiation so
that some but not all of the effects occur.
The Sel2p-Swelp-CDC28 protein phosphorylation pathway also was
implicated in control of filamentous
growth, providing an independent line
of evidence that function of die CDC28 complex is a central controlling factor
in this process. Although direct evidence that Sel2p represses the activity of
Swelp by phosphorylation is not available, there is ample genetic evidence
that these two proteins function in a hierarchical cascade (Ma et al. 1996;
Tanaka and Nojima 1996). Likewise, the effects of Swelp are dependent on
phosphorylation of Y19 of CX)C28, and in this instance the enzyme-substrate
relationship has been defined (Booher et al. 1993). Thus, modification of
CDC28 is most likely the reason that mutations of SEL2 and SWEl can impart
filamentous growth characteristics or repress those phenotypes.
Phosphorylation of residue 19 of CDC28 by the Sel2p-Swelp pathway is
necessary for elml mutations to induce filamentous growth characteristics,
suggesting that Elmlp functions upstream of Sel2p and that this cascade
mediates the filam^tous growtfi phenotype, again by altering CDK function
in a defined manner. The Sel2p-Swelp-CDC28 cascade also must be intact for
filamentous growth characteristics to occur in response to the normal
environmental signal that induces morphologic differentiation when
haploid cells imdergo invasive growth, providing important confirmation
114
that this pathway is relevant to morphologic differentiation in wild tjrpe cells
and not only as a downstream effector of Elmlp.
The nature of the initiating signal that is upstream of this cascade
remains to be elucidated, however, perhaps some insight can be gained by the
examination of 5. pombe Niml. Several studies have implicated this kinase
in nutrient status signaling (Feilotter et al. 1991; Belenguer et al. 1997; Wu and
Russell 1997). In S. pombe, nitrogen starvation stimulates the mating
pathway, and results in the rapid degradation of Niml (Feilotter et al. 1991;
Hayles and Nvirse 1992; Belenguer et al. 1997; Wu and Russell 1997). The loss
of Niml results in the activation of Weel and ensuing Cdc2 t5rrosine 15
phosphorylation, therefore Weel plays a role in the nitrogen
starvation-dependent signaling of the mating response (Wu and Russell
1997). In S. cerevisiae, sell mutant strains have been found to be sensitive to
nitrogen concentration, such that increased amounts of nitrogen source
suppress the extreme elongated morphology observed in these mutants when
they reach stationary phase (Tanaka and Nojima 1996). Collectively, these
data suggest a potential scenario in which Sel2p degradation may occur in
response to nutrient deprivation, thereby eliciting a G2 delay in the cell cycle
and stimulation of filamentous
growth.
This possibili^ can not completely explain pseudohyphal growth,
however, because swel/swel diploids or a strain bearing only CDC28^"'' are
both able to respond normally to the induction signal (Kron et al. 1994). To
115
explain the fact that filamentous growth in haploids requires the Swelp
pathway, whereas the response in diploids does not, we suggest the existence
of a redimdant mechanism for alteration of CDK activi^ that is specific to
diploids. For example, as suggested previously ^on et al. 1994), activation of
the STE MAP kinase patiiway and subsequent transcriptional activation of
genes containing filamentous growth response elements (FREs) (Gavirias et
al. 1996; Madhani and Fink 1997) may activate an inhibitor of the Clb-CDC28
complex that functions independently of Y19 phosphorylation. If such an
inhibitor were active only in diploids, then pseudoh3^hal growth would not
be prevented by elimination of Swelp-dependent Y19 phosphorylation in
MATn/MATa cells, whereas the Swelp pathway would be necessary for
invasive growth of haploids. Even if such redundant pathways do exist,
however, both mechanisms are expected to be dependent on the STE MAP
kinase pathway, because disruption of this signal blocks the filamentous
growth response in both haploids and diploids (Liu et al. 1993; Roberts and
Fink 1994) (Fig. 8).
The exquisite regulation of CDC28 regulation throughout cell cycle
progression provides several possible alternative means by which CDK may
mediate filamentous growth. The fact tt\at the effects of CDC28^^ are
suppressed by overexpression of Clb2p suggests that decreasing activity of the
CDC28-Clb2p complex causes the filamentous characteristics. Thus, a
signaling mechanisms that altered Clb expression, is a possible means of
116
attaining the filcimentous fonn in that would be independent of Y19
phosphorylation. Clb expression is regulated transcriptionally throughout
the cell cycle and also by cell cycle-spedfic proteol5rtic degradation (Nasmyth
1993; King et al.1996), so down regulation of CDC28-Clb2 could occur by a
variety of mechanisms. Another possibility is that expression of the G1
cydins could be altered such that the Cln-CDC28 complex is hyperactive in
filamentous form cells compared to the yeast form. This condition is known
to cause hyperpolarization of cell growth (Lew and Reed 1993), and also to
delay expression of the Qb proteins (Nasmjrth 1993), so that the effects of Cln
overexpression may be similar to those of Clb imderexpression with regard to
causing filamentous growth characteristics.
As a last consideration regarding the mechanisms that might cause
alteration of CDC28 function so that filamentous growth characteristics result,
we expect that the changes will not be drastic, but instead should be subtle in
nature. Filamentous form cells grow at nearly the same rate as do yeast form
cells (Blacketer et al. 1993; Kron et al. 1994), and are tightly regulated regarding
cell size and shape. Drastic alterations in CDC28 function would cause
significant growth rate defects and frequent aberrant divisions. The uniform
nature of filamentous
form cells argues that although CDC28 function may be
altered, it still performs all essoitial functions albeit with slightly different
kinetics. Higher eukaryotes can regulate morphogenesis and cellular
differentiation by modifying the cell cycle program (OTarrell et al. 1989; Edgar
117
and Lehner 1996). Further analysis of the S. cerevisiae cell division cycle and
its control in filamentoiis growth is likely to provide significant insights into
how cell division in more complex organisms can be modulated to
accomplish specific functions during cellular differentiation.
Materials and methods
Media and genetic procedures
S. cerevisiae strains utilized in this study are listed in Table 2. Standard
genetic methods were used for mating S. cerevisiae strains, selection of
diploids, induction of meiosis, and tetrad dissection. (Rose et al. 1990). S.
cerevisiae transformation was by the lithium acetate procedure (Ausubel et al.
1989). One-step gene replacement and targeted integration of prototrophic
markers into the S. cerevisiae genome was performed as described (Rothstein
1991). Gene replacement was confirmed by DNA gel blot analysis, and/or by
obvious appearance of the specific phenot}^ known to result from deletion
of the gene undergoing modification (data not shown). Standard rich (YPD)
and synthetic (SD) agar (2%) media were used as described (Rose et al. 1990).
Unless otherwise noted media contained 2% glucose as the carbon source.
Isolation and genetic analysis of suppressor mutations defining the lod
SELl, SEL2, and SEL3 was as follows. In a related screen for dosage
suppressors of the elongated morphology phenotype induced by elml-1
118
Table 2. S. cerevisiae strains
Strain
Genotjrpe
Sotirce or derivation
W303
MATi/MATa ura3/ura3 leu2Aeu2
his3/his3 ade2/ade2 trpl/trpl
(Wallis et al. 1989)
MATi/MATa ura3/ura3 leu2Aeu2
L5487xL5684'
DDUL
MAT A/MAT a ura3/ura3 Ieu2/leu2
^53/-^
This study^
aDUL
MAT a uraS leu2
This study^
aDUL
MAT a. ura3 leu2
This stud)^^
aDL
MATAleu2
This study^
NEY2158
MATA ura3 leu2 his3
This study^
NEY2159
MATa ura3 leu2 his3
This study^
104D576b
MATA leu2 metS elml-1
(Blacketer et al. 1995)^
104DP10C
MATa. ura3 his3 elml-1
This stud)r^
NEDN6d
MATa ura3 leu2 metS elml-l
(Blacketer et al. 1995)~
NEY1489
MATa ura3 leu2 met6 elml-1
(Blacketer et al. 1995)^
127D6
MATa ura3 cdc28-127
(Blacketer et al. 1995)^
127D5
MATa leu2 cdc28-127
(Blacketer et al. 1995)^
AY2287
MATa ura3 leul cdc28-127
This study^
AYIOOX
MATa ura3 leu2 CDC28::URA3
Transformation of aDUL
with p28WT-URAi
AY102X
MATa ura3 leu2 bud2A::LEU2
Transformation of aDUL
AY2241
MATa cdc28-127 bud2A::LEU2 leu2
Segregant of AY102X x
AY2287
119
Table 2. (continued)
Strain
Genotype
Source or derivation
AY2242
MATa cdc28-127 BUD2 leu2
Segregant of AY102X x
AY2287
AY2249
MATa ura3 leu2 clb2::LEU2
Transformation of aDUL
AY2252
AlATa uraS leu2 cdc28~127
GAL-CLB2:LEU2
Transformation of AY2287
with YIpG2::CLB2'
AY2305
MAT^/MATa CDC28/cdc28::LEU2a
ura3/ura3 Ieu2/leu2 his3/+
Transformation of DDUL
AY2153
MATa cdc28::LEU2a ura3 leu2
[p28MUT-URAcl
Segregant of AY2305
transformant
AY2155
MATa cdc28::LEU2a ura3 leu2
[p28WT-URAcl
Segregant of AY2305
transformant
AY2399
MATa ura3 leu2 his3 cdc28::LEU2a
[p28Y19F-URAc]
Segregant of AY2305
transformant
WW28A/+
MATa/MATa CDC28/cdc28::LEU2a
ura3/ura3 Ieu2/leu2 his3/his3
trpl/trpl ade2/ade2
Transformation of W303
AY2195
MATa cdc28::LEU2a ura3 Ieu2his3
trpl ade2 [p28MUT-URAc]
Segregant of WW28A/+
transformant
AY2194
MATa cdc28::LEU2a ura3 leu2 his3
trpl ade2 [p28WT-URAc]
Segregant of WW28A/+
transformant
S228A/+
MATa/MATa CDC28/cdc28::LEU2a
ura3/ura3 Ieu2/leu2
Transformation of
AY2204
MATa cdc28::LEU2a ura3 leu2 his3
trpl ttde2 [p28MUT-URAc]
Segregant of 2Z28A/+
transformant
AY2203
MATa cdc28::LEU2a ura3 leu2 his3
trpl ade2 [p28WT-URAc]
Segregant of ZZ28A/+
transformant
120
Table 2. (continued)
Strain
Genotype
Source or derivation
NEY2170
MATa leu2 ura3 his3 elml::HIS3
Transfonnation of
NEY2158
AY2451
MATa leu2 ura3 his3 elml::HlS3
cdc28::LEU2a [p28Y19F-URAcl
Segiegant of AY2399 x
NEY2170
AY2452
MATa leu2 ura3 his3 elml::HIS3
CDC28 [p28Y19F-URAcl
Segregant of AY2399 x
NEY2170
NEY1494
MATa leul met6 elml-1 sel2-l
Mutation of 104D576b
NEY2018
MATa ura3 his3 elml-1 put3::URA3 Transfonnation of
104DF10C
NEY2019
MATa ura3 leu2 metS elml-1
bud2A::LEU2
Transformation of
NEDN6d
NEY2020
MATaura3leu2 met6 elml-1
GFA1:URA3
Transformation of
NEY1489
NEY2065
MATa ura3 elml-1 sel2-l
Segregant from NEDN6d x
NEY1494
NEY2170
MATa. uraS leu2 his3 elml::HIS3
Transformation of
NEY2158
NEYIOOX
MATa ura3 leu2 hi$3 sel2::LEU2
Transformation of
NEY2159
NEY2299
MATA ura3 leu2 his3 elml::HIS3
swel::URA3
Transformation of
NEY2170
NEY2317
MATa/MATa ura3/ura3 Ieu2/leu2 NEY2299 x NEYIOOX
his3/his3 elml::HIS3/+ sel2::LEU2/+
$wel::URA3/+
NEY2334
MATa ura3 leul his3 sel2::LEU2
Progeny of NEY2317
NEY2335
MATa. ura3 leu2 sel2::LEU2
Progeny of NEY2317
121
Table 2. (continued)
Strain
Genotype
Soiirce or derivation
NEY2338
MATdL ura3 leu2 his3 elmli'MISS
Progeny of NEY2317
NEY2339
MATa ura3 leul his3 sel2::LEU2
swelizURAS
Progeny of NEY2317
NEY2340
MATa. uraS leu2 his3
Progeny of NEY2317
NEY2341
MATa uraS leu2 his3 elml:-JilS3
swel::URA3
Progeny of NEY2317
NEY2344
MATa ura3 leu2 his3 swel::URA3
Progeny of NEY2317
NEY2335
MATa. ura3 leu2 sel2::LEU2
Progeny of NEYIOOX x
aOUL
NEY2353
MATa ura3 Ieu2-SEL2-1-LEU2
Integr. transform, of aDUL
with pNE33d
^ These parental strains in the Z background were provided by C. Styles,
Whitehead Institute.
^ The indicated strains are congenic in the D273-10B/A1 genetic background
(Tzagoloff et al. 1976). They were obtained by crossing tfie parental strain,
or a mutageruzed derivative ttiereof, to W303-1A (a meiotic segregant from
W303), then backcrossing the F1 progeny at least five successive times to
the parental D273-10B/Al parent^ strain. Diploid DDUL was obtained by
mating two of the F5 progeny.
^ Provided by D. Lew, Duke University.
122
(strain 104D576b), ten suppressed strains were obtained from 10,000 screened
colonies that remained in the yeast form even in absence of a plasmid. The
elongated morphology phenoQrpe was readily recovered in progeny of crosses
to a wild type tester strain, indicating in each instance an extragenic
suppressor mutation (generically termed sel), Dominance/recessiveness was
detennined by mating each original isolate to the elml-1 strain 104D6.
Tetrads from these diploids invariably produced two spore clones with tjrpiczil
yeast form cell morphology and two wi^ the filamentous form characteristics
typical of elml-1 strains, thus indicating the suppression phenotjrpe is a
single-gene trait Segregants from these crosses of the genotype MATA uraS
elml-1 sel were collected for each original isolate. These segregants were used
to determine allelism relationships of the 10 sel mutations by genetic linkage.
Each segregant was mated to each original isolate (MATA leuZ elml-1 sel).
Allelism was assigned if yeast form colonies were observed exclusively in the
progeny of such a cross, whereas non-allelic mutations were indicated by
recovery of colonies with filamentous form characteristics typical of elml-1
strains; at least 30 complete tetrads were examined for each cross.
Construction of plasmids containing CDC28, cdclS-ll?, or cdc28-Y19F
Standard molecular biology methods were employed for PCR
amplification and plasmid construction (Ausubel et al. 1989; Sambrook et al.
1989). CDC28 was obtained from D. Lew (Duke University) as a 2.03 kb
123
fragment extending from the Xhol site 342 bp upstream of the initiation
codon to the PvuTL site 790 bp downstream of the termination codon. This
fragment contains two base pairs inserted immediately upstream of the
CDC28 initiation codon that create an Ndel site not present in the native
gene. This CDC28 fragment was cloned in the Xhol and Smal sites of pRS306,
pRS316, or pRS305 (Sikorski and Hieter 1989) to construct p28WT-URAi,
p28WT-URAc, and p28WT-LEUi, respectively. Plasmid p28MUT-URAc,
containing the allele cdc28-127, was constructed by replacing the region
between the unique EcoNI and HindDI sites of p28WT-URAc with the
equivalent region feom a fragment PCR amplified from genomic DNA of a
cdc28-127 haploid sfrain.
Plasmid p28Y19F-URAc, carrying cdc28-Y19F, was constructed as
follows. Oligonucleotide primer 1022R from ttie noncoding strand of CDC28
contains a missense mutation that changes tyrosine codon 19 (TAC) to a
phenylalanine codon (TTC). This primer was used to amplify by PCR a 126 bp
region extending from 60 bp upstream of the CDC28 initiation codon to the
22nd codon of the open reading frame. The template for this reaction was
p28WT-URAc. This small fragment was then extended by PCR to beyond the
unique HindSn. site within CDC28. The Ndel-Hindm fragment excised from
the amplification product was used to replace the equivalent region of CDC28
in p28WT-LEUi, forming plasmid p28Y19F-LEUi. The nucleotide sequence of
the entire CDC28 gene in p28Y19F-LEUi was determined, confirming the
124
presence of the Y19F substitution mutation. The only other changes from the
wild t3rpe sequence are a silent mutation changing codon 22 hrom GTA to
GTT, and also the mutation forming the Ndel site at the initiation codon The
Xhol-EcoNl fragment of p28Y19F-LEUi containing the Y19F mutation was
used to replace the equivalent region of p28WT-URAc, forming plasmid
p28Y19F-URAc.
SEL2 plasmids
SEL2 was ^dsed from phage clone ATCC71138 (American Tj^e
Culture Collection, Rockville, MD) as a 7.26 kb BamHI/SstI fragment and
cloned in pRS316 to form plasmid pNE30. The dominant allele SEL2-1 was
obtained by in vivo gap repair as follows. pNE30 was digested at the unique
Sunl and Nrul sites within SEL2, which removes the entire open reading
frame but leaves significant lengths of the 5' and 3' flanking sequences. The
resulting linear fragment was used to transform the SEL2-1 strain NEY2065 to
uracil prototrophy The autonomously replicating plasmids plasmid
pNESOGR was recovered from one fransformant after amplification in E. coli.
This plasmid was shown to have been repaired from the dominant allele
SEL2-1 by its ability to suppress the filamentous growth characteristics of an
elml-1 strain. Plasmid pNE33d is a L£LZ2-marked integrative plasmid that
was constructed by excising the BamHI/Ssrt fragment bearing SEL2-1 from
pNE30GR and cloning it in pRS305.
125
Gene disruptions and tagging by integrative transformation
Recombinant DNA fragments used to replace wild type alleles with
deletion mutations were described previously for elml::HIS3 (Blacketer et al.
1993), budlt^iLEUl (Parket al. 1993), clb2::LEU2 (Richardson et al. 1992), and
put3::URA3 (Marczak and Brandriss 1989). The fragment used to introduce
swel::URA3 was constructed by digesting pSWEl-lOg (Booher et al. 1993) with
Xbal to remove the LEII2 insert from swel::LEU2, and replacing that region
with a 1.2 kb Nhel fragment containing URA3 (N.E., unpublished). The
resultant plasmid is pswel::URA3, from which a 2.5 kb disruption fragment
can be excised by digestion with ffindin and BamlU. The GFAl locus was
tagged with IIRA3 as a selective marker by first cloning genomic EcoRI
fragment containing GFAl (Watzele and Taimer 1989) in pRS306 (Sikorski
and Hieter 1989), linearizing ttie resultant plasmid at the imique Sphl site
within GFAl, then transforming strain NEY1489 to uracil prototrophy.
The deletion allele sel2::LEU2 was constructed as follows. A 5.34 kb
PvuU./Sstl fragment of the SEL2 locus was excised from pNE30 and cloned in
pUCllS (Vieira and Messing 1987) digested with Smal and Ssfl, The entire
ORF of SEL2 between the unique Nrul and Sunl sites was removed; after
digestion with these two enzymes the remaining plasmid was made blimt
ended by treatment with DNA polymerase I fQenow fragment and ligated to
an Nhel linker to form plasmid pNE37. The 2.2 kb Xbal fragment containing
126
LEU2 was excised from pSWEl-lOg (Booher et al. 1993) and inserted into this
unique Nhel site to form plasmid pNE38. Digestion of this plasmid with Sstl
and Hindni releases a 2.59 kb disruption fragment containing sel2::LBU2.
The deletion allele cdc28::LEUa was constructed as follows. The 2.03 kb
Xhol/PvuU genomic DNA fragment containing CDC28 was cloned in the
Xhol and Smal sites of pBluescript SK+ (Stratagene). The region of CDC28
between the unique Ndel and HmdHI sites (codons 1 to 247) was replaced with
the LEU2 gene obtained as a 2.8 kb BglU. fragment from plasmid YEplS
(Broach et al. 1979). To facilitate this cloning step the Nirfel/Hindlll-cleaved
CDC28 plasmid was made blimt-ended by treatment with DNA poljntnerase I
Klenow fragment (polIK) and ligated to a BglU linker. The resultant plasmid
pcdc28::LEU2 provides cdc28::LEU2a as a 4.1 kb fragment excised by digestion
with Apal and BamHl.
Size measurements and estimation of cell volume
Random fields of 30-50 cells were captured for analysis as digital files
using the program NIH Image; all unbudded and singly budded mother cells
were measured. Cell volumes were estimated by asstuning the cell shape
approximated a prolate spheroid emd applying the formula
V = (4/3)ic(a/2)(b/2)^ where a is the length of the cell and b is the maximimi
width. At least 200 cells were analyzed for each genotjrpe characterized.
127
Detennination of bud site selection
A segregating population was generated by crossing strain 127D6
(MATa ura3 cdc28-127) to strain aDL (MATa. Ieu2 CDC28) and collecting
progeny clones from complete tetrads. Wild type and mutant siblings, along
with the parental strains, were analyzed by time-lapse photography to
determine directly the location of bud emergence sites and the relative timing
of bud emergence on mother-daughter cell pairs. For this analysis cells were
first grown overnight on rich agar mediimi. Small budded cells then were
dragged to a new location on the plate using a micromanipulator. The
position of mother cells and their buds was noted. Images of the cells were
captured immediately after micromanipulation and at approximately 1 hour
intervals thereafter. For the parental strains at least 100 cell divisions were
examined for bud site selection. For progeny clones 32 divisions or more
were characterized. A mirumiim of 10 independent progeny clones of each
type were analyzed and at least 100 mother-daughter pairs were assessed for
each genotype.
The effect of clb2::LEU2 on bud site selection was determined similarly
using strain AY2249 (MATa ura3 leu2 clb2::LEU2) and progeny of this strain
crossed to strain 127D5 (MATa. Ieu2 cdc28-127); only progeny strains bearing
the wild type CDC28 allele were analyzed. The ejects of bud2A::LEU2 on bud
site selection in cdc28-127 strains was analyzed in progeny of a cross between
strain AY102X (MATa ura3 leu2 bud2A::LEU2) and strain 127D5 (MATa leu2
128
cdc28-127). Seven cdcZ8-lZ7 ^ui2A;:L£U2 progeny and five cdc2S-127 BUD2
siblings were analyzed for bud position and cell shape; distinctions in these
phenotypic parameters corresponded precisely with the genotype.
Acknowledgments
We thank D. Lew, M. Brandriss, and H.-0. Park, C. Styles, M. Gentzsch,
for supplying strains and/or plasmids. This work was supported by National
Science Foimdation Grant No. MCB-9319028 to A.MA1.
References
Ausubel, F.M., R. Brent, JLE. Kingston, D.D. Moore, J.A. Smith, J.G. Seidman,
and K. Struhl. 1989. Current Protocols in Molecular Biology, John Wiley
and Sons, New York.
Belenguer, P., L. Pelloquin, M.L. Oustrin, and B. Ducommun. 1997. Role of
the fission yeast niml protein kinase in the cell cycle response to nutrient
signals. Biochem. Biophys. Res. Commun. 232: 204-208.
Blacketer, M.J., C.M. Koehler, S.G. Coats, A.M. Myers, and P. Madaule. 1993.
Genetic control of dimorphism in S. cerevisiae: hivolvement of a the
novel protein kinase homologue Elmlp and protein phosphatase 2A.
Mol Cell Biol. 13; 5567-5581.
Blacketer, M.J., P. Madaule, and A.M. Myers. 1994. The S. cerevisiae mutation
elm4~l facilitates pseudohyphal differentiation and interacts with a
deficiency in phosphoribosylpyrophosphate synthase activity to cause
constitutive pseudohyphal growA. Mol. Cell. Biol. 14: 4671-4681.
Blacketer, M.J., P. Madaule, and A.M. Myers. 1995. Genetic analysis of
morphologic differentiation in S. cerevisiae. Genetics 140; 1259-1275.
Booher, R.N., R.J. Deshaies, and M.W. Kirschner. 1993. Properties of
Saccharomyces cerevisiae weel and its di^erential regulation of p34CDC28
in response to Gi and G2 cyclins. EMBO J. 12; 3417-3426.
129
Broach, JJL, J.N. Strathem, and J.B. Hicks. 1979. Transformation in yeast:
Development of a hybrid cloning vector and isolation of the CANl gene.
Gene 8; 121-133.
Chant, J. 1994- Cell polarity in yeast. Trends Genet, 10: 328-333.
Chant, J., and I. Herskowitz. 1991. Genetic control of bud site selection in yeast
by a set of gene products that constitute a morphogenetic pathway. Cell 65:
1203-1212.
Drubin, D.G. 1996. Origins of cell polarity. Cell 84: 335-44.
Edgar, B A., and CJ. Lehner. 1996. Developmental control of cell cycle
regulators: a fly's perspective. Science 274:1646-1652.
Feilotter, H., P. Nurse, and P.G. Yoimg. 1991. Genetic and molecular analysis
of cdrl/niml in Schizosaccharomyces pombe. Genetics 127: 309-318.
Friefelder, D. 1960. Bud formation in Saccharomyces cerevisiae. J. Bacterial
80:567-568.
Futcher, B. 1996. Cyclins and the wiring of the yeast cell cycle. Yeast 12:16351646.
Gavirias, V., A. Andrianapolous, C.J. Gimeno, and W.E. Timberlake. 1996.
SacchMomyces cerevisiae TECl is reqtiired for pseudohyphal growth.
Molec. Microbiol. 19: 1255-1263.
Gimeno, C.J., P.O. Ljungdahl, C.A. Styles, and G.R. Fink. 1992. Unipolar cell
divisions in the yeast S. cerevisiae lead to filamentous growth: regulation
by starvation and RAS. Cell 68:1077-1090.
Hayles, J., and P. Nurse. 1992. Genetics of the fission yeast
Schizosaccharomyces pombe. Annu. Rev. Genet. 26: 373-402.
King, R.W., R.J. Deshaies, J.-M. Peters, and M.W. Kirschner. 1996. How
proteolysis drives the cell cycle. Science 274: 1652-1659.
Koehler, C.M., and A-M. Myers. 1997. Serine-threonine protein kinase activity
of Elmlp, a regulator of morphologic differentiation in Saccharomyces
cerevisiae. FEBS Lett. 408: 109-114.
Kron, S.J., CA.. Styles, and GJl. Fink. 1994. S5munetric cell division in
pseudohyphae of the yeast Saccharomyces cerevisiae. h/lol. Biol. Cell 5:
1003-1022.
130
Lew, D.J., N.J. Marini, and SJ. Reed. 1992. Difierent G1 cyclins control the
timing of cell cycle commitment in mother and daughter cells of the
budd^g yeast S. cerevisiae. Cell 69: 317-327.
Lew, D.J., and SX Reed. 1993. Morphogenesis in the yeast cell cycle;
Regulation by Cdc28 and cycl^. /. Cell. Biol. 120:1305-1320.
Liu, H., C~A. Styles, and G JL Fink. 1993. Elements of the yeast pheromone
response pathway required for filamentous growth of diploids. Science
262:1741-1744.
Ma,
Q. Lu, and M. Gninstein. 1996. A search for proteins that interact
genetically with histone H3 and H4 amino termini uncovers novel
regulators of the Swel kinase in Saccharomyces cerevisiae. Genes & Dev.
10:1327-1340.
Madhani, H.D., and G.R. Fink. 1997. Combinatorial control required for the
specificity of yeast MAPK sigiuling. Science 275:1314-1316.
Marczak, J.E., and M.C. Brandriss. 1989. Isolation of constitutive mutants
affecting the proline utilization pathway in Saccharomyces cerevisiae and
molecular analysis of the PUTS transcriptional activator. MoL Cell. Biol.
9:4696-4705.
Nasm3rth, K. 1993. Control of the yeast cell cycle by the Cdc28 protein kinase.
Curr.Opin.Cell Biol. 5: 166-179.
Nurse, P. 1990. Universal control mechanism regulating onset of M-phase.
Nature 344: 503-508.
OTarrell, P.H., B.A. Edgar, D. Lakich, and CJ. Lehner. 1989. Directing cell
division during development. Science 246: 635-640.
Park, H.-O., J. Chant, and I. Herskowitz. 1993. BUD2 encodes a GTPaseactivating protein for Budl/Rsrl necessary for proper bud-site selection in
yeast. Nature 365: 269-274.
Pringle, J.R., and LJI. Hartwell. 1981. The Saccharomyces cerevisiae cell cycle.
In Molecular Biology of the Yeast Saccharomyces (ed. J.N. Strathem, E.W.
Jones, and J.R- Broach), pp. 97-142. Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY.
Richardson, H.E., D.J. Lew, M. Henze, K. Sugimoto, and SJ. Reed. 1992.
Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and
in G2. Genes & Dev. 6: 2021-2034.
131
Roberts, R.L., and G.R. Fink. 1994. Elements of a single MAP kinase cascade in
Saccharomyces cerevisiae mediate the two developmental programs in
the same cell type: Mating and invasive growth. Genes & Dev. 8: 29742985.
Rose, M.D., F. Winston, and P. Heiter. 1990. Methods in Yeast Genetics. A
Laboratory Course Manual, Cold Spring Harbor Laboratory Press,
Plainview, NY.
Rothstein, R. 1991. Targeting, disruption, replacement, and allele rescue:
integrative DNA transformation in yeast. Methods Enzymol. 194: 281-301.
Russell, P., and P. Nurse. 1987. The mitotic inducer nintl'*' functions in a
regulatory network of protein kinase homologs controlling iniation of
mitosis. Cell 49: 569-576.
Russell, P., and P. Nurse. 1987. Negative regulation of mitosis by weel'*', a
gene encoding a protein kinase homolog. Cell 49: 559-567.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY.
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces
cerevisiae. Genetics 122: 19-27.
Tanaka, S., and H. Nojima. 1996. Nikl: a Niml-like protein kinase of S.
cerevisiae interacts with the Cdc28 complex and regulates cell cycle
progression. Genes to Cells 1: 905-921.
Tzagoloff, A., A. Akai, and F. Foury. 1976. Assembly of the mitochondrial
membrcme system. XVI. Modified form of the ATPase proteolipid in
oligomydn-resistant mutants of S. cerevisiae. FEBS Lett. 65: 391-396.
Vieira, J., and J. Messing. 1987. Production of single stranded plasmid DNA.
Methods Enzymol. 153: 3-11.
Wallis, J.W., G. Chrebet, G. Brodsky, M. Rolfe, and R. Rothstein. 1989. A
hjrper-recombination mutation in S. cerevisiae identifies a novel
eukaryotic topoisomerase. Cell 58: 409-419.
Watzele, G., and W. Taimer. 1989. Cloning of the glutamine:fructose-6phosphate amidotransferase gene from yeast. /, Biol Chem. 264: 87538758.
132
Wittenberg, C., and SJ. Reed> 1996. Plugging it in: signaling circuits and the
yeast cell cycle. Curr, Opin. Cell Biol. 8: 223-230.
Wu, L., and P. Russell. 1997. Roles of Weel and Niml protein kinases in
regulating the switch from mitotic division to sexual development in
SMzosaccharomyces pombe. Mol. Cell. Biol. 17: 10-17.
i
133
CHAPTER 4. ANALYSIS OF SEL2/HSL1 REVEALS A LINK
BETWEEN CYTOKINESIS AND CELL CYCLE CONTROL IN
SACCHAROMYCES CEREVISIAE
A paper submitted to Molecular and Cellular Biology
Nicholas P. Edgington and Alan M. Myers
Abstract
The serine/threonine kinase Sel2p has been previously shown to be
part of a regulatory pathway that impinges on the activity of the cell cycle
CDK Cdc28p. The cellular localization pattern of Sel2p was investigated, and
revealed that Sel2p is localized as a ring structure at the bud neck in a cell
cycle-dependent manner. In addition, Sel2p overexpression was found to be
detrimental to cell growth resulting in defects in cytokinesis, and in a block of
cell cycle progression late in mitosis. The growth inhibition was determined
to be partially dependent on the presence of the tjnrosine kinase Swelp, and
was suppressed by mutations in the G2 cyclin CIb2p, and Cdc28p. Sel2p may
function as a repressor of a novel Swelp-dependent cjrtokinesis surveillance
mecharusm in the G2/M phase of the cell cycle to ensure coordination
between nuclear division and cytokinesis.
134
Introduction
The process of cell growth and division requires the concerted
temporal and spatial organization of components that govern DNA
replication and segregation, rearrangement of the cytoskeleton, and
deposition of cell growth materials. One mechanism to ensure the correct
ordering of cell cycle events, is the limited availability and substrate spedfidty
that the cyclin regulatory subimits impart on the master regulatory CDK,
Cdc28p. Cyclin abtmdance, as their name implies, varies during the cell cycle
due to tight transcriptional and posttranslational regulation (35, 66, 67). Both
G1 and G2 cyclins contain motifs that target their destruction by proteolysis.
The S. cereuisiae G1 cyclins Clnlp, Cln2p, and Cln3p, contain PEST motifs
that target G1 cyclins to the ubiqtiitin 26S proteosome (51,73,87,91, 94). The
G2 cyclins, or Clbs, contain a N-terminal destruction box motif that directs
ubiquitination and subsequent degradation via the anaphase-promoting
complex or APC (24,25,37,47,48).
A second mechanism that ensiu^s correct ordering of the events that
occur during the cell cycle is the existence of surveillance mechanisms that
serve to monitor for defects that may occur during events such as spindle
formation, DNA replication or damage, and bud emergence. Their activation
results in a delay or block of further progression of the cell cycle to allow
defects in previous steps to be corrected (23, 29, 55, 65,81). In S. pombe, the
kinase Weel is involved in signaling DNA damage by phosphorylating the
135
cell cycle CDK Cdc2 on tyrosine 15, thereby inducing a delay in the G2/M
transition to allow DNA repair to occur (74). A tyrosine phosphatase, Cdc25
removes the phosphate to allow cell cycle progression (77). Anotiier kinase,
Niml was determined to be an inducer of mitosis, since Niml directly
phosphorylates and inhibits Weel (21, 31,68, 92). Homologues of Weel and
Cdc25 exist in S. cerezrisiae as Swelp (14) and Mihlp (77), respectively,
however lliey appear to have different functions since they not a part of the
DNA damage checkpoint in S. cerevisiae (4, 77,83, 84). Therefore, it was not
known what physiological role they played imtil a purpose for this pathway
was implicated when it was demonstrated that Swelp was necessary to
produce a G2 delay as part of a bud emergence checkpoint pathway, by
phosphorylating Cdc28p on tyrosine 19, which is equivalent to the Cdc2
tyrosine 15 residue (55,81), and specifically ii\hibiting Cdc28p/Qb complexes
when bud emergence is blocked.
A S. cerevisiae homologue of the S. pombe niml* kinase,
SEL2/NIK1/HSL1 was recently cloned, and exhibited genetic interactions with
Swelp and Cdc28p, that suggests tiiat Sel2p functions to inhibit
Swelp-dependent inactivation of Cdc28p/Clb complexes (28, 61, 85). Sel2p
may play a role in the suppression of invasive growth, as mutations in Sel2p
result in elongated cells that exhibit a G2 delay in cell division and enhanced
invasive growth phenotypes that are suppressed by deletion of SWEl (28).
136
Significantly, Sel2p does not appear to be a part of the Swelp-dependent bud
emergence surveillance mechanism (52).
Late in the cell cycle, separation of sister chromatids must be
coordinated with C3rtokinesis and cell abscission. Di the abscence of a
surveillance mechanism at this stage of cell division, multinucleate and
anucleate progeny would be formed by the as3mchronous execution of
cytokinesis and nuclear division. Cytokinesis, and redistribution of actin and
other morphogenesis proteins to the bud neck region, appear to require the
proteolytic degradation of the G2 cyclins by the APC (54). Thus,
overexpression of a stable version of Clblp or Clb2p lacking the destruction
box motif, results in constitutive depolarization of the actin cytoskeleton, and
cells arrest in telophase (36,56).
The septins, Cdc3p, CdclOp, Cdcllp, Cdcl2p , appear at the incipient bud
site as a ring structure that presumably act to direct or restrict the deposition
of cell wall growth material to the bud neck area (18, 60). Septin function is
also required late in mitosis, as mutation of the septins results in defective
cytokinesis, in which mother and daughter pairs remain attached and exhibit
an elongated morphology reminiscent of filamentous growth (13, 33, 38, 46).
Detailed mechanisms for the coordination between cytokinesis, G2
cyclin inactivation and nuclear division remain poorly characterized. Since
Sel2p is not a part of the Swelp-dependent bud emergence pathway, but
appears to negatively regulate Swelp function, we sought to further define
I
137
SeI2p function by determining Sel2p cellular localization. Significantly, Sel2p
was found to be localized as a ring structure at the bud neck region between
mother and daughter cells in a cell qrcle-dependent manner. In addition, the
Sel2p C-terminal is required for localization, and Sel2p kinase activity was
found to further refine localization. Overexpression of Sel2p resulted in
growth cirrest in late mitosis, with cells exhibiting a peanut-shaped cellular
morphology indicative of a defect in cjrtokinesis. The growth arrest
phenotype was partially suppressed by Swelp, and fully suppressed by
mutations in Qb2p and Cdc28p, suggesting that i) inhibition of Sel2p maybe
necessary for qrtokinesis to occur, ii) Swelp and Cdc28p may be the target of a
novel cytokinesis surveillance mechanism that is repressed by Sel2p function
during the G2/M phase of the cell cycle, and iii) Sel2p may be one of a
growing ntmiber of noncyclin proteins whose degradation in the G2/M phase
is dependent on destruction box sequences and APC function.
Materials and Methods
Strain background, media, and genetic methods
Strains in this study are derived from the D273-10B/A1 genetic
backgroimd obtained from A. Tzagoloff (Columbia University), and listed in
Table 1. Auxotrophic markers were obtained by outcrossing the parent to
138
Table 1. 5. cerevisiae strains
Strain
Genome
Derivation
AMY2249
MAT a leu2 ura3 clb2::LEU2
This laboratory
MBY5130
MAT aleu2 uraS cdc28-127
This laboratory
NEY2157
MAT SiuraS leu2
(13)
NEY2158
MAT Aura3 leu2 his3
(13)
NEY2159
mat
a ura3 leu2 his3
(13)
NEY2173
mat
^faura3/ura3 Ieu2fleu2 his3/+
Mating of NEY2157 x
NEY2159
NEY2410
MAT iura3 leu2 his3 sel2::lIRA3
Transformation of
NEY2158
NEY2411
mat
a ura3 leu2 his3 sel2::URA3
Transformation of
NEY2159
NEY2556
mAT
a ura3 leu2 his3 sweli:LEU2
Transformation of
NEY2159
NEY2646
MAT A/a ura3/ura3 Ieu2/leu2 his3/his3 Mating of NEY4210 x
sel2::URA3/sel2::URA3
NEY2411
139
strain W303-1A (89), and subsequently backcrossing meiotic progeny at least
six times to D273-10B/A1.
The following media were used: YPD (1% yeast extract, 2% peptone, 2%
glucose); YPGAL and YPSUC are identical to YPD except 2% galactose or
2%sucrose were substituted; SD (2% glucose, 0.17% yeast nitrogen base
without cunino acids and ammonia, 0.5 % ammonium sulfate, supplemented
as required widi histidine, leucine, methionine, and uracil at20 mg/ml each).
Solid media for yeast contained 2% agar. Yeast strains were cultured at 30°C
unless specified otherwise. Galactose inductions were carried out as follows.
Strains were incubate for overnight growth in liquid YPD or YPSUC media at
30°C. Cultures were then diluted to an ODg<„ of 0.05-0.1 into YPD or YPSUC
(noninducing) or YPGAL liquid (inducing). Cells were then observed directly
at different time points for cell morphologies (GFP fusions), or fixed for FACS
analysis. Standard methods were used for genetic manipulations (76).
DNA manipulations and strain construction
DNA manipulations, nucleotide sequence analysis, polymerase chain
reaction, and S. cerevisiae transformation were performed according to
standard methods (5). The construction of the SEL2 deletion plasmids, pNE38
(psel2::LEU2) and pNE39 (psel2::URA3) have been described elsewhere (28).
SEL2 was deleted in yeast strains by the transformation of 3.59-kb and 2.59-kb
SstI/Hindin digested fragments from pNE38 and pNE39, respectively.
140
Disruption of SWEl was performed by transformation of yeast strains with a
2.8-kb HindJE/BamHl fragment from
pSWEl-lOg (14),
The plasmid pNE32, a plasmid containing the SEL2 promoter, coding,
and 3'-regions on a 5.34-kb Pt7uII/SstI fragment cloned into a derivative of
pRS315 (82), was utilized to make in-frame SEL2 truncation mutants by
digesting at unique restriction sites within the C-terminal of SEL2, filling-in
with Klenow fragment, and religating the resulting plasmid. Thus, pNE45
was constructed by removing a 1,37-kb Apal/Mlul fragment pNE32, and then
blunting, and religating the plasmid. A 2.1-kb Apal/Nsil fragment within
pNE32 was removed and modified in the same manner to create the plasmid
pNE46, which results in a franscript that continues into the polylinker due to
a frameshift.
The in-frame SEL2 truncation plasmid pNE47 was created by
cutting, filling and religating the Stul and Nsil sites within pNE32, removing
2.95-kb fragment.
Digestion and religation of pNE32 with Nsil removed a
708-bp fragment, to form pNE48.
To facilitate the introduction of a green fluorescent protein (GFP) (69)
fusion and a triple HA epitope tag (32,86), to SEL2, a Notl site was introduced
into pNE43, a plasmid which contains the the C-terminal of SEL2, and the
predicted stop condon. PCR mutagenesis using the primer 5'-GCC GGA CGT
AGCGGCCGC TAA ATT C-3' (Notl site underlined), was used to create the
plasmid pNE44. This plasmid was digested with Notl and ligated to a 750-bp
Notl fragment containing the in-frame GFP®^gene from the plasmid
141
pSF-GPl (J. Hiisch, Columbia University) to create the plasmid pNE49, and to
a 111-bpNofI 3XHA fragment from pMPY3XHA (80) to form pNE50. A 1.07-kb
Mlul/Sstl fragment from the plasmid pNE34, which contains a 5.4'kb
PvulL/Sstl fragment of SEL2 cloned into the 2 micron plasmid pRS425 (82),
was removed and replaced separately witii ttie 1.75-kb Mlul/Ssfl fragment
from pNE49 containing the GFP®*^ fusion to create the plasmid pNE52, and
with the 1.1-kb M2tfl/Sstl fragment from pNE50, to create pNE53. An
integrative L£U2-marked SEL2-GFP construct was made by removing a
1.07-kb Mlul/Sstl fragment from
the plasmid pNE33 (pRS305-SEL2), and
replacing it with the 1.75-kb Mlul/Sstl GFP®®^ fusion fragment from
pNE44,
to create pNE63. This construct was linearized with BstXl for integrative
transformation.
The catalytic activity of SEL2 was abrogated by PGR mutagenesis of the
conserved lysine at amino acid position 110, to an arginine residue using the
primer 5'-A GCT GCC ATT AQA ATT GTC-3' (the adenine to guanine amino
add substitution is imderlined) on the template pNE32. The PGR reaction
resulted in a second introduced conservative mutation of the lysine at
position 99 to an arginine, thus the final construct, pNE45-M4 contains a K to
R substitution at positions 99 and 110. To construct a Idnase dead' GFP-tagged
SEL2 construct, SEL2'®-GFP, a 2.0-kb HindJH/Stul fragment from
pNE45-M4
was cloned into a similarly digested pNE63 plasmid to form pNE69. The
plasmid pNE63NS was formed by the digestion of pNE63 with Stul and Nsil,
142
filled with Klenow fragment, and religaied to form an in-frame truncated
SEL2-GFP construct. These plasmids were digested with BstXl for integration
into yeast strains.
SEL2 was put under control of ttie galactose-inducible GALl-10
promoter in two steps. First, a partial Ncol and AvrVi digested pNE32 was
cloned into the integrative vector pYX013 (Novagen, Madison, WI) to form
the plasmid pNE57, which removes 27 amino adds from the N-terminal of
the SEL2 coding region and most of the C-terminal sequence, but leaving the
kinase domain intact. A 3.21-kb AvrIL/Ssfl fragment containing the epitope
tagged C-terminal region from pNE53 was doned into pNE57 to form pNE55,
which lacks only the 27 amino adds from the N-terminal of SEL2. The
plasmid pNE60 was formed by ligating a 4.82-kb partial Ncol/Mlul cut
fragment from pNE32 into pYX013.
A galactose-inducible SEL2-GFP construct, pNE62, was made by
subdorung a 4.0-kb AvrU/Sstl fragment from
pNE52 into a similarly digested
pNE57. A galactose-indudble SELZ'^ plasmid, pNE67, was construded by
removal of a 321-bp Ncol fragment from
321-bp Ncol fragment from
pNE55 and replacement with a
pNE45-M4. All pYX013-derived vedors were
linearized with PstI prior to integrative fransformation
into yeast strains.
143
FACS analysis and morphological observations
FACS analysis was performed with propidium iodide staining as
described previously (53, 75). Strains for FACS analysis were made rho° by
growth in media containing ethidiiun bromide (39). Cell morphologies were
observed using light or Normarski microscopy on an Olympus BX40
microscope with the PM-30 exposure control unit to facilitate photography.
GFP constructs were visualized by fluorescence microscopy by resuspending
live cells in mounting media (72). Nocodazole (Sigma, St.Loius, MO) arrest
was performed as described previously (64). For the galactose-induced
overexpression of SEL2, cells that lacked wild-type constricted bud necks were
defined as having abnormal c3^okinesis, and were thus divided by the total
number of ceUs coimted (> 100 cells) to determine the percent cytokinesis
defective for each strain.
Results
Structure/function analysis of Sel2p
Sel2p consists of 1318 amino adds, and is nearly three times larger
than its S. pombe homologue Niml. This suggests that in comparison with
Niml, Sel2p may have additional roles or modes of regulation. Thus, to gain
further insight into Sel2p function, database searches were performed in
regions other than the previously described N-terminal serine/threonine
144
kinase domain (28, 61, 85) (Chapter 3). The Oterminal region of Sel2p was
found to contain several putative domains including at least five PEST
regions, two nuclear localization signals, a five amino add repeat, a B-cydin
type destruction box, and three proline rich SH3-binding domains (Fig. lA).
To determine what role if any the C-terminal region might play in Sel2p
function, tnmcations were made and introduced into NEY2410 (sel2::URA3)
to test for complementation of the elongated cell morphology (Fig. IB). None
of the tnmcation mutants were capable of complementation (Fig.lB, and data
not shown), suggesting that the Oterminal is required for some aspect of
Sel2p function.
The C-teiminal domain is required for Sel2p localization to the bud neck
Insight into the function of Sel2p may be gained by determining its
cellular location. This was performed by the introduction of a fully
functional (Fig. IB and Fig. 2) Sel2p-GFP fusion, pNE63, into the diploid strain
NEY2646 (sel2::URA3 /sel2::lIRA3). Observations by fluorescence microscopy
of an asynchronous population of cells showed that Sel2p-GFP was visible
when expressed under its own promoter and in low copy, and localized to the
bud neck region. Two lines of evidence suggest that this localization is
identical to the localization of the wild-tjrpe Sel2p. First, when this plasmid is
introduced into SEL2 wild-type strains, the signal is weaker presimiably due
to competition from the wild-type copy of Sel2p. Second, tnmcations in the
i
Figure 1. Sequence analysis of Sel2p. (A) Comparison of the amino acid sequence
of Sel2p reveals a serin^tii^nine kinase domain @ , PEST region^
S , a 'SKRSL' repeat 1, putative nuclear localization sequences _ ,
SH3-binding domains S, and a 6-type cyclin destruction box S .
PEST scores were determine by the program PEST-HND. (B) Constructs
used in this study. The 3XHA epitoTO tag! , and GFP fusions are also
shown • . An asterix denotes the SEL2^'1IOR mutation. Constructs
contain either their native (NAT) promoters or were fused to GALl
(GAL) promoter sequences. 'Expression' indicates the copy nximber
of the constructs; low copy(CEN)/ high copy (2fiM), and inte^tive (INT).
Complementation of a sei2 null stram was scored as a "V',
or not
apphcable (N).
i
146
NcoINcoI
I I
PEST scofcs:
AvrnSlal
I
SillS
Apal
6^
MUMS
IL
734/8.91
5J7 i95
NmO SttI
Promotu/
Ecpcession/
Constract Complem.
I pNE32
NATCEN +
bapNESS
GAL INT N
iBpNE67
GAL INT N
pNE48
NAT GEN -
^NE60
GAL INT N
pNE45
NAT GEN -
pNE46
NAT GEN -
pNE47
NATCEN -
pNE57
GAL INT N
pNE52
NAT 2nM N
pNE63
NAT INT +
pNE69
NAT INT -
pNE63NSNAT INT pNE62
GAL INT N
147
r.
\
I)
Figure 2. Sel2p localization during the cell cycle. The strain NEY2646
(sel2::URA3/sel2::URA3) was transformed witii the plasmid pNE63
(SBL2-GFP), and grown to early log phase in YPD (A-D) or arrested
with 10 ^g/ml nocodazole for 3 hours at 30°C in \PD (E and F). The
cells were then observed by Normarski (A and C), bright field (E) or
fluorescence (B, D, and F) microscopy. The arrow (B) shows the lack
of staining in imbudded cells.
148
Sel2p-GFP construct fail to localize to the bud neck, even though these
plasmids contain the identical GFP fusion. Sel2p-GFP fluorescence was
observed only dunng ttie budded period of cell division, which corresponds
to the G2/M phase of the cell cycle. The appearance of Sel2p-GFP fluorescence
appeared immediately after, but not preceding bud emergence, is in contrast
to appearance of die neck ring septins at tfie incipient bud site just prior to
bud emergence (60).
Haploid and diploid filamentous differentiation rely, in part, on
different signaling components (50), and deletion of Sel2p results in enhanced
haploid invasive growth (28) (Chapter 3). To examine if Sel2p is expressed
and localized in haploid strains, Sel2p-GFP was examined in haploid cells,
and was fotmd to be identical to the localization in diploid cells. In addition,
consistent with the diploid result suggesting that Sel2p is not present during
the G1 phase of the cell cycle, fluorescence was not observed in nutritionally
deprived diploid or haploids or o-factor arrested MAT a haploid strains (data
not shown). Near the end of cjrtokinesis, the neck ring septins, Bud3p, Bud4p,
and BudlOp localize as a double ring structure, with one ring in the mother
cell, and one ring in the daughter cell (15, 60,71). Similarly, asynchronous
and nocodazole arrested cells exhibited a Sel2p-GFP signal as a double ring
late in the cell cycle (data not shown, and Fig. 2F). Sel2p was cloned as a
dominant suppressor of the serine/threonine kinase Elmlp, thus it is possible
that Elmlp regulates Sel2p localization as part of a conserved signaling
149
cascade (28) (Chapter 3). To test this hypothesis, Sel2p-GFP was introduced
into an elml mutant strain, and was found to be localized identically as in
wild-t3rpe cells, suggesting ttiat Sel2p localization is not dependent on the
presence of Elmlp (data not shown).
The large C-terminal domain of Sel2p consists of 76% of the entire
protein, is required for Sel2p function, and thus may contain a bud neck
localization domain. To examine this, an in-frame tnmcated version of
Sel2p-GFP, pNE63NS was introduced into NEY2646. This construct, which
lacks most of the Oterminal domain, was imable to complement a sel2
deletion strain (Fig. 1B,3C,D), and did not localize to the bud neck (Fig. 3C,D).
SEL2 is predicted to encode a serine/threonine kinase, and therefore catalytic
activity may be required for Sel2p localization to the bud neck. A Sel2p
'kinase dead' mutant construct, Sel2p'®-GFP, which contains a lysine to
arginine mutation at positions 99 and 110 within the kinase domain, was
introduced into the diploid strain NEY2646. As Figure 3F and 3H
demonstrate, Sel2p'®-GFP does not complement the cell elongation
phenotype of sell mutant strains, but does localize to the bud neck. In
addition, some cells exhibited Sel2p'®-GFP staining at the tips of daughter
cells, and also as a diffuse second ring just below the bud tip, suggesting that
kinase activity may serve to refine bud neck localization.
150
m
\
}1
f"
.
Figure 3. The Sel2p C-tenninal region, but not Sel2p kinase activity, is
required for bud neck localization. The strain NEY2646
(sel2::LIRA3/ sel2::URA3) was transformed with the plasmid
pRS315 (A and B), pNE63NS (SEL2 trunc.-GFP) (C and D), pNE69
(SELZ'^'GFP) (E-^, and grown to early log pha% in YPD. The cells
were then observed by Normarski (A and C), bright field (E and G)
or fluorescence (B-H) microscopy. The arrows (F and H) show
diffuse Sel2p staining in the daughter cell.
151
Effects of Sel2p oveiexpiession
The effects of Sel2p oveiexpression were observed by integrating full
length and truncated versions of Sel2p under control of the
galactose-indudble GALl-10 promoter (Fig. 4). Wild-^^ strains that
contained the plasmids pNE55, pNE57, and pNE60, were induced for six
hours in liquid galactose medium. Es^ression of Sel2p protein was
confirmed by Western blot analysis using antibodies against the
hemagglutinin epitope tag (data not shown). The terminal morphologies of
these strains were quite dissimilar. The full length Sel2p construct, pN£55,
resiJted in cells attsiiiung a peanut-shaped morphology (Fig. 4B,D). Induction
of the construct pNE57, in which Sel2p lacks most of the C-terminal, results
in nearly wild-type shaped cell (Fig. ffi), and overexpression of a construct
that deletes 236 amino adds from the C-terminal region of Sel2p (pNE60),
results in a cellular morphology that resembles filamentous growth (Fig. 4F).
To determine the cellular localization of Sel2p when overexpressed, a
Sel2p-GFP was placed in a high-copy vector (pNE52), and imder control of the
GALl-10-10 promoter (pNE62), and observed by fluorescence microscopy.
High-copy expression of Sel2p-GFP results in mild pertubations of cellular
morphology in wild-type cells, including the presence of wide bud necks (data
not shown, and Fig. 5A,B). In addition to the bud neck localization pattern
that resembles low-copy SeI2p-GFP, high-copy Sel2p-GFP also results in cells
that contain spots of fluorescence that appear to be on the surface of the cells
152
Figure 4. Galactose-induced overej^ression of Sel2p results in defects in
bud neck formation. Strain NEY2532 (wild-type) was
transformed with the plasmids pRS316 (A anci C), pNE55 (B and
D), pNE57 (E), and pNE60 (F). The resisting strains were induced
in galactose for 6 hours at 30°C and photographed.
153
,
'
N
^
1)
Figiire 5. Overexpression of Sel2p results in mislocalization and aberrant
morphology. The strain NEY2173 (wild-type) was transformed with
the plasmid pNE52 (2 \iM SEU-GFP) (A and B), and pNE62 (GALlSEL2-GFF) (C-F), and grown to early log phase in SD-leudne (A and
B) or grown to early log phase in YPSUQ and then transferred to
YPG^ for 6 hours at 30°C (C-F). The cells were then observed by
bright field (A, C and E)) or fluorescence (B, D, and F) microscopy.
154
as determined by moving the focal plane up and down (Fig. 5B). Galactose
induction of the Sel2p-GFP construct pNE62 results in a nearly uniform
phenotype (Fig. 5C-F) that is identical to the HA-tagged version (Fig. 4B), and
exhibits spots of fluorescence that appears to be on the surface of the cells.
Cells that exhibit a constriction between the mother and daughter cells exhibit
a ring pattern similar to Sel2p-GFP expression under the SEL2 promoter (Fig.
5F). Thus, it appears that overexpression of Sel2p results in its own
mislocalization, and results in cell with severe defects in forming a
constriction between mother and daughter cells.
It was observed that galactose-induction of Sel2p from the plasmids
pNE55, pNE57, and pNE60 resulted in lower cell densities in the wild-type
streun NEY2159 when compared to empty vectors or other Sel2p truncations
(data not shown and Fig. 6). Thus the cell growth kinetics were monitored by
observing the ODgoo of strains carrying these plasmids (Fig. 6). Sel2p
genetically interacts with, and is predicted to inhibit the tyrosine kinase
Swelp. Therefore to determine if ftie effects of Sel2p overexpression were
mediated via its action on Swelp, the same plasmids were integrated into the
strain NEY2556 {swelvUEUl). The C-terminal truncated Sel2p (pNE57) had
the greatest growth inhibition phenot3rpe, beginning at about 5 hours (Fig.
6C). The full length construct also exhibited a growth inhibition phenotype
with similar kinetics, but to a lesser degree. However, in addition to the
growth inhibition, pNE55 also resulted in cells that arrested in a
Figure 6. Overexpression of Sel2p results in growth inhibition and a
cytokinesis defect The strains NEY2159 (wild-type) (A, C, and E),
and NEY2556 {swel:-XEU2) (B and D), were transform^ with the
plasmids pRS316 (diamond), pNE55 (square), pNE57 (triangle), or
pNE60 (cross), and grown overnight in YPD. Strains were washed
three times in YPGAL, and diluted to an ODgo<, of 0.05 in YPD (A and
B) or YPGAL (C, D, and E) and grown for the indicated times.
156
B
0.01
2
4
6
8
10
Growth m glucose (houis)
t1
2"
4"
6"
8" iT 11"
Growtii in glucose (hours)
0.01
0
2
4
6
8
10
Growth m gcilactose (hours)
4"
6"
8" 1*3
Growtfi in galactose (houis)
4
6
Time (hours)
n
157
peanut-shaped moiphology, indicative of a cytokinesis defect (Fig. 6E).
Although the strain carrying the construct pNE60 resulted in cells with an
elongated phenot}^, the cell division rate seemed to be only mildly affected
during the first six hours of induction. No difiierences were observed in the
swel::LEU2 strains (NEY2556) except for the strain canying pNE57, which
exhibited near normal growth rate in stark contrast to pNE57 in the wild-type
strain (Fig. 6D). Thus, the growth inhibition exhibited by the overexpression
of the Sel2p kinase domain is dependent on the presence of Swelp.
To determine at what stage of the cell cycle Sel2p overexpression
caused an arrest, the strains described in the growth curves containing pNE55
and pNE57 were induced in 2% galactose for 5 and 10.5 hours, fixed, and
analyzed for flow cjrtometry (Fig.7). These strains exhibited a greater
proportion of cells with a 2N DNA content indicative of cells in G2/M phase
of the cell cycle, which increased with time compared to the uninduced
straii\s (T=0) and NEY2159 transformed with pRS316 (Fig. 7A,C,E). In
addition, swel deletion was able to suppress the G2 delay exhibited by the
coi^truct pNE57, containing oidy the Sel2p kinase domain (compare Fig.TE
and 7F), consistent with the observed suppression of the growth inhibition
(Fig. 6D). Partial suppression of the G2 delay by overexpression of the full
length Sel2p, was also observed in the swel mutant strain NEY2556 (compare
Fig.7C and 7D). Thus, the growth inhibitory effects and a G2 delay due to the
158
swel
W.T.
B
T^lO^hr
pRS316
TsShr
TslOJhr
pNE55
T=5hr
T=103hr
pNE57
T=5hr
IN 2N
Figure 7.
Overexpression of Sel2p results in a G2 delay. Histograms of
flow qrtometric analysis of propidium iodide-stained cells. The strains
NEY2159 (wUd-type) (A, C, and E), and NEY2556 {swel::LEU2) (B, D and F),
were made rho°, transformed with die plasmids pRS316 (A and B), pNE55 (C
and D), or pNE57 (E and F), and grown overnight in YPD. Strains were
washed three times in YPGAL, and diluted to an OD^ of 0.1 in YPD (T=0) or
YFGAL (T=5 and 10.5) and grown for the indicated times. Cells were then
fixed, stained and analyzed for FACS analysis.
159
overexpression of Sel2p, can be partially overcome by deleting a putative
target of Sel2p^ namely Swelp.
Qb2p deletion suppresses Sel2p overexpression
Budded cells that have nearly completed mitosis must tmdergo a
rearrangement of the actin cytoskeleton from being circumferential to being
redirected to the bud neck to execute cytokinesis (54,56). This actin
rearrangement is dep^dent on the inactivation of Cdc28p/G2 cyclin
complexes, thiis if the actin rearrangement is not allowed by the
overexpression of a stabilized Clb2 lacking the destruction box motif, then
cells arrest in late anaphase or telophase with delocalized actin spots (36).
Examination of the nuclear and actin staining of the peanut-shaped cells
overexpressing full length Sel2p revealed nuclear staining consistent with
cells in late anaphase and telophase, and delocalized actin staining (data not
shown). These data suggest that Sel2p overexpression results in a terminal
phenotype similar to chronic Clb2p activation. Accordingly, the
overexpression of Sel2p may result in the chronic activation of Clb2 or
prevent Clb2p degradation. An allele of the cell cycle CDK CDC28, cdc28-127,
recovered in a screen to identify elongated cell morphology mutants (12,13),
exhibits a G2 delay and appears to have a diminished capacity to interact with
G2 cyclins (28) (Chapter 3). Thus, if overexpression of Sel2p results in chronic
activation of Clb2p, then mutations that reduced the efficacy of Clb2p activity
160
may relieve the Sel2p-dependent cell cycle arrest phoio^pe. To test this
hypothesis, pNE55, pNE57, and a galactose-inducible Sel2p*® construct,
pNE67, were introduced into the strains NEY-2159 (wild-type), AMY2249
{clb2::LEU2,) and MBY5130(cdc2^-127). These strains were grown in rich
liquid media containing 2% sucrose, and then serially diluted and plated on
rich medium agar plates containing either 2% glucose or 2% galactose, and
grown for 2 days (Fig. 8). As shown previously (Fig. 6C), pNE55 and pNE57
strongly inhibited colony growth in wild-type strains (Fig. 8B, first row), but
the Sel2p'^ construct did not suggesting that Sel2p kinase activity is required
for growth inhibition. Deletion of CLB2 and the cdc28-127 allele were both
competoit to suppress the growth inhibitory effect of full length Sel2p
overexpression, but not in the C-terminally truncated Sel2p construct.
Intriguingly, overexpression of the Sel2p'® construct did inhibit colony
growth of the strain MBY5130.
Discussion
Sel2p localizes to the mothei/bud neck
Sel2p now joins a growing number of proteins that locedize to the
mother/bud neck region, and function to regulate aspects of cellular
morphogenesis. Significantly, Sel2p is a member of the GIN4
serine/threonine kinase family that includes Gin4p and YCL024w (1, 42).
161
pRS316
pNE55
pNE57
pNE67
GLC NEY2159
AMY2249
MBY5130
B
GAL NEY2159
AMY2249
MBY5130
Figure 8. Sel2p growth inhibition from overexpression requires kinase
activity and is suppressed by clb2 deletion. The strains NEY2159
(wild-type), AMY2249 iclb2:±EU2), and MBY5130 (cdc28-127), were
transformed with pRS316, pNE55 (GAL1'SEL2), pNE57 (GALlS£L2trunc.)/ and pNE67 (GAL1'SEL2'^), and grown to esirly log
phase in YPSUC. Approximately 5x10*, 5x10^, and 500 cells were
plated on YPD (A) or YPGAL (B) plates, incubated for 2 days at 30°C,
and photographed. The triangles represent 10-fold serial dilutions.
162
Gin4p and YCL024w share the greatest degree of homology with each other
(47.4% identity over 1,001 amino adds), as compared to Sel2p (44% identity
over 550 amino adds, and 40.6% identic over 539 amino adds, respectively).
Due to their sequence similarity, Gin4p and YCX024w may be involved in a
redimdant function that is distinct from Sel2p fimction. Gin4p localizes to
the mother/bud neck region as a ring structure independent of actin function
(6), and exhibits genetic interactions with the septins (70). Gin4p was doned
by a genetic screen for fusion proteins that when overexpressed, resiilted in a
growth arrest (1). In contrast to the growth inhibition exhibited by full length
and the N-terminal kinase domain of Sel2p (Fig. 6 and Fig. 7), growth
inhibition by Gin4p was due to a C-terminal fusion lacking the entire kinase
domain (1). Thus, members of the GIN4 family collocalize to the mother/bud
neck, but may play distinct roles in cytokinesis.
The proteins Bud3p, Bud4p, Bud6/Aip3p, and Budl0p/Axl2p, localize
to the mother/bud neck as a ring structure, and regulate bud-site selection in
S. cerevisiae (15, 27, 71). Bud6p localizes independently of the septins (2, 96),
but BudSp and Bud4p require prior assembly of the septins (16, 78,79).
Although Sel2p may not play a direct role in bud-site selection, sell mutant
haploid strains do exhibit a defective budding pattern in which cells bud in a
bipolar rather than axial pattern (NJP. Edgington, A.M. Myers, unpublished
results). Regarding the timing of localization, Sel2p-GFP became visible after
cells became budded (Fig. 2). The septins localize to presumptive bud sites.
163
and thus Sel2p may require the prior assembly of the septins for proper
localization. Sel2p localization follows the appearance of Bud6p and BudlOp,
but precedes BudSp and Bud4p. In addition, Sel2p localization remained
intact in the absence of kinase activity, but exhibited diffuse staining in the
bud (Fig. 3 ), suggesting that kinase activity may act to refine Sel2p
localization, perhaps due to interactions with substrate molecules in the neck
region. Alternatively, autophosporylation of Sel2p may stabilize the binding
of Sel2p to other proteins located at the mother/bud neck region, presumably
via the Sel2p C-terminal domain (Fig. 3). Recently, immtmofluorescence
microscopy of the Sel2p S. pombe homologue Niml revealed that moderately
overproduced Niml was localized to tiie cjrtoplasm (93). The fact that the
mechcmisms of Niml and Sel2p appear to be conserved with regard to the
inhibition of the Weel/Swelp homologues, is supported by the sequence
conservation in the kinase domains, genetic interactions (28, 61), and partial
complementation of a cdc25-22^ S. pombe mutant strain by Sel2p
overexpression (85). However, Niml lacks the large C-terminal portion that
Sel2p contains, suggesting that the Niml/Weel/Cdc2 kinase cascade is
conserved between S. cerevisiae and S. pombe , but that it responds to
different input signals which may reflect the intrinsic differences in cell
morphogenesis, division, and 'lifestyle' that these two divergent yeasf s have
evolved.
164
Sel2p oveiexpiession results growth arrest in late mitosis
Sel2p is not localized during the unbudded G1 portion of ttie cell cycle.
This suggests that Sel2p may be regulated transcriptionally or
posttranslatioiudly, or both, as cells enter Gl. Consistent with this hypothesis,
Sel2p overexpression results in cells arresting in anaphase and telophase,
suggesting that the down regulation of Sel2p may be necessary for cell cycle
progression from telophase to Gl. Sel2p overexpression perturbed cellular
morphology restating in peanut-shaped cells with wide bud necks (Fig. 4 and
Fig. 5). A variety of other mutations in genes that are involved in
morphogenesis also restdt in a similar phenotype. These include certain
mutant actin alleles (26), bnil/EhoA strains (49), strains overexpressing of
Bnilp (30), bud6/bud6 diploids (2), peal and spal mutants in the presence of
mating pheromone (88), and cla4 steZO mutant strains (22). The
peanut-shaped double cla4 stelO mutant strains exhibit actin concentrated at
the bud tips, which may be a result of inappropriate growth on the mother
side of the bud (22). In contrast, Sel2p overexpression results in almost an
identical morphologic phenot5rpe, but exhibits delocalized actin staining,
suggesting that these mutations both result in abnormal bud neck formation,
but the cells are existing or arresting in different physiological states. This is
consistent with the hypothesis that the Ste20-like kinases are required at the
165
early step of establishment or maintenance of the bud neck, whereas Sel2p
may play a late role in monitoring qrtokinesis.
Bnilp, Qa4p, and Ste20 display genetic interactions with the septins,
and 6ud6p localizes as a double ring structure at cytokinesis (2,22,70). Sel2p
may be localized to the bud neck via protein-protein interactions mediated
via three putative SH3-binding domains located in its C-terminal region (Fig.
1 and Fig. 9A). SH3-binding domains interact with proteins containing Src
homology 3 (SH3) domains (63). Morphogenesis proteins that contain SH3
domains, and thus have the potential to interact with Sel2p, include Rvsl67p
(8), Abplp (58), Beelp (57), Bemlp (17), Boilp, and Boi2p (10,62). The feet that
i) Sel2p collocalizes to the region of the septins, ii) contains putative domains
that may mediate bud neck localization, and iii) displays cellular phenotypes
in common with other morphogenesis proteins involved in polarized
growth and cjrtokinesis, strongly supports a role for Sel2p in these processes.
Overexpression of Sel2p-GFP resulted in an pimctate pattern of
fluorescence on the cell jjeriphery (Fig. 5), suggesting that Sel2p may be
associated with the membrane, presumably indirectly since Sel2p does not
contain any predicted transmembrane domains. The mislocalization of Sel2p
may result in the titration of other neck ring components or Sel2p substrates
that are required for the proper restriction of growth at the bud neck.
Alternatively, the c3rtokinesis defects of Sel2p overexpression may be due to
the inappropriate presence of Sel2p during Gl, when Sel2p is not normally
166
CONSENSUS
Sel2p
Bein3p
Srv2p
BudSp
Abplp
Slalp
BoUp
Boi2p
B
(566)
(619)
(623)
(364)
(568)
(337)
(199)
(516)
(192)
(402)
(439)
CONSENSUS
S. cermsiiie Sel2p
S.cereoisiae Clblp
5. cereoisiae Clb2p
S. cereoisiae CIb3p
S. cereoisiae CIb4p
S. ponAe cdcl3
S. cereoisiae Pdslp
S.cereoisiaeAselp
S.ponOiecuQ.
z p a V pz p
X
Zi
B
P
R
R
P
K
X= any residue
as hydrophobic residue
p= piefeiied ptoiine
P= conservedproline
r
B r
RZZXiODZZll
(828)B&
(36) QT
(25) Q Zi
(43) B V
(51) H V
(59) B R
(85) Q tt
(760)Dq Xi r P X P XiB
(33) B A P H Q S T X Q
(52) BT VQQo K ST
S.cereoisiaeCdt2Sp (148)Q8 sgn 8 L oQ
Figure 9. Sel2p contains putative SH3-binding domains (A), and a destruction
box (B). Consensus sequences are given on the top line, and
sequence aligiunents were made by visual inspection. Conserved
residues are highlighted in black.
167
present at the bud neck. It has been previously suggested that the G1 cyclins
may play a role in cjrtokinesis, by performing some function in Gl, such as
organizing the bud neck, that becomes vital for the subsequent execution of
cytokinesis (22). If Sel2p interfered wi& the initial establishment of the bud
neck during Gl, then defects in cjrtokinesis might be ecpected late in the cell
cycle.
Growth arrest induced by the overexpression of the Sel2p kinase
domain was fotmd to be dependent on the presence of Swelp (Fig. 7). This
overexpression may be predicted to strongly inhibit the activity of Swelp,
which would be predicted to block any Swelp-d^>endent inhibition of
Cdc28p/ab complexes, and thus promote mitosis, rather that delay mitosis.
Presumably, the construct containing only the Sel2p kinase domain does not
localize to the bud, as evidenced by the lack of fluorescence of the pNE63NS
construct at the bud neck (Fig. 3), and the lack of an abnormally wide bud neck
during overexpression of the kinase domain (Fig. 4E). A possibility exists that
the truncated Sel2p may not have cellular access to Swelp, and the
overexpression of Sel2p kinase domain may indirectly result in the activation
of another Swelp-d^)endent surveillance mechanism ttiat is active after
nuclear segregation.
Further insight into the function of Sel2p may be gained by the analysis
of the genes ZDSl, and ZDS2. These genes are homologous to each other but
otherwise do not exhibit similarity to any other proteins in other organisms.
168
They have been isolated in a large variety of diverse genetic screens (11, 95),
and sigiiificantly, collocalize and potentially regulate Cdc42p, a Rho-related
small GTPase that is essential for polarized growth (9,11). High-copy
expression of ZDSl suppresses the G2 delay and the elongated morphology
that sell strains &chibit (61). Deletion of ZDSl results in a mild defect in
cellular morphology that is suppressed by deletion of Swelp (61). Double zdsl
zds2 deleted mutants are delayed in G2 of the cell cycle (95), and exhibit
grossly abnormal morphology which is suppressed by mutating the target
residue of Swelp, namely the tyrosine 19 amino acid of Cdc28p to a
nonphosphorylatable phenylalanine {CDC28^^) (19). Strains containing sell
or elml (kinases), or cdc55, or pphll pphll mihl (phosphatase suburuts)
mutations all exhibit elongated cell morphologies which are suppressed by
the CDC28^^^ allele, and exhibit genetic interactions with each other (12, 28,
59,90). These data suggest that the Zds proteins may act downstream or
parallel to Sel2p, and potentially in conjunction with other kinases or
phosphatases to repress the activation of a Swelp-dependent morphologic
surveillance mechanism that exists during bud growth.
Potential mechanisms of Sel2p function
Sel2p overexpression results in a terminal phenot3rpe similar to
chronic Qb2p activation (i.e. late anaphase/telophase arrest with delocalized
actin). Clb2p contains a destruction box motif that targets G2 cyclins for
169
degradation (3,37,48). This region is required for recognition by the
anaphase-promoting complex (AFC) and subsequent addition of ubiquitin to
neighboring lysine residues, which restilts in degradation by the 26S
proteosome (40). Noncyclin proteins that contain destruction boxes have
been identified (45), and several play roles in cell cycle progression, such as
sister chromatid separation during anaphase, and spindle disassembly during
telophase (20,34,44). Sel2p contains a putative destruction box that is most
closely related to the B-type cyclins, and matches the QbSp destruction box in
seven out of nine residues (Fig. 9B). The presence of this putative domain in
Sel2p suggest that Sel2p may be regulated temporally in a manner analogous
to the B-type cyclins, in which degradation occurs near anaphase and
continues throughout the G1 phase (24). Sel2p localization during the cell
cycle, and the growth arrest of Sel2p overexpressing strains in late anaphase
support this h3rpothesis.
If overexpression of Sel2p results in chroiuc activation of Clb2p, then
mutations that reduce Clb2p activity may alleviate the Sel2p-dependent cell
cycle arrest phenotype. Deletion of CLB2, or the presence of an allele of
CDC28, cdc28-127, that appears to have a diminished capacity to interact with
G2 cyclins (28) (Chapter 3), releases cells from the growth inhibitory effects of
Sel2p overexpression (Fig. 8). These data suggest two possible scenarios of G2
cyclin regulation by Sel2p in which the removal of Sel2p must precede Clb2
degradation, or that Sel2p may interfere with, or protect Clb2 from
170
degradation by the APC or 26S proteosome, either directly or indirectly. This
effect is not likely to be due to nonspecific titration of AFC components, since
i) Sel2p kinase activity is required for growth inhibition (Fig. 8B),
ii) overexpression of the noncatalytic Sel2p C-terminal does not exhibit a
similar growth defect (data not shown), and iii) whereas titration of the APC
components by the overexpression of a peptide containing a destruction box
prevents anaphase 6:0m occurring in cycling Xenopus extracts (41), strains
overexpressing Sel2p progress ttirough anaphase. A hjrpodietical 'protector'
of CIb2p has been postulated to save a small fraction of the Qb2p protein from
AFC-mediated degradation until telophase is complete (43). Intriguingly,
Sel2p contains a C-terminal region of limited homology ( 25.5% identity over
165 amino adds) to the ubiquitin-spedfic protease Ubp3p, an enz3mne which
removes ubiquitin 6:0m ubiquinated substrates (7, 40). In addition, the
C-terminal of Sel2p contains four potential Cdc28p phosphorylation
concensus sites, and has been shown to bind Cdc28p in vitro (85). A highly
speculative model of Sel2p action is that Sel2p can sequester a small amount
of ubiquinated Clb2 at the bud neck to locally regulate cell morphogenesis, by
preventing the relocation of actin and other morphogenesis proteins to the
bud neck region. When cjrtokinesis is complete, Clb2p and Sel2p may then
both degraded to allow cell abscission and transition to Gl. Further
investigations into the relevance of the domain homologies that exist within
171
Sel2p, should facilitate a fuller understanding of the regulation of Sel2p, and
its relationship to the progression of &e cell cycle.
Acknowledgments
We thank the instnunoitation center manager, Kristi Harkins for
assistance in performing the flow cytometric aiudysis, and members of the
Myers lab for useful discussions. This work was supported by award no.
MCB-9319028 from the National Science Foundation.
References
1.
Akada, R., J. Yamamoto, and I. Yamashita. 1997. Screening and
identification of yeast sequences that cause growth inhibition when
overexpressed. Mol. Gen. Genet. 254:267-274.
2.
Amberg, D. C.,,]. E. Zahner^ J. W. MttlhoUand, J. R. Pringle, and D.
Botstein. 1997. Aip3p/Bud6p, a yeast actin-interacting protein that is
involved in morphogenesis and the selection of bipolar budding sites.
Mol. Biol. Cell 8:729-753.
3.
Amon, A., S. Imiger, and K. Nasm3rth. 1994. Qosing the cell cycle in
yeast: G2 cyclin proteolysis initiated at mitosis persists tmtil the
activation of G1 cyclins in the next cycle. Cell 77:1037-1050,
4.
Amon, A., U. Surana, I. Muioff, and K. Nasmyth. 1992. Regulation of
p3^CDC28 t3rrosine phosphorylation is not required for entry into
mitosis in S. cerevisiae. Nature 355:368-9371.
5.
Ausubel, F. M., R. Bren^ R. E. Kingston, D. D. Moore, 1. Seidman, A.
Smith, and K. Stnihl. 1989. Current Protocols in Moleciilar Biology.
John Wiley and Sons, New York.
6.
Ayscough, K. R., J. Stryker, N. Pokala, M. Sanders, P. Crews, and D. G.
Drubin. 1997. High rates of actin filament turnover in budding yeast
and roles for actin in establishment and maintenance of cell polarity
172
revealed using the actin inhibitor latrunculin-A. J. Cell Biol. 137:399416.
7.
Baker, R. T., J. W. Tobias, and A. Varshavsky. 1992. Ubiquitin-specific
proteases of Saccharomyces cerevisiae. J. Biol. Chem. 267:23364-23375.
8.
Bauer, F., M. Urdad, M. Aigle, and M. Crouzet 1993. Alteration of a
yeast SH3 protein leads to conditional viability with defects in
c3rtoskeletal and budding patterns. M.CB. 13:5070-5084.
9.
Bender, A., and J. R. Pringle. 1989. Multicopy suppression of the cdcli
budding defect by CDC^ cind three nevvrly identified genes including
the Ras-related gene RSRl. Proc. Natl. Acad. Sd. USA 86:9976-9980.
10.
Bender, L., S. Lo, H. Lee, V. Kokojan, J. Peterson, and A. Bender. 1996.
Associations among PH and SH3 domain-containing proteins and
Rho-type GTPases in yeast. J. Cell Biol. 133:879-894.
11.
Bi, E., and J. R. Pringle. 1996. ZDSl and ZDS2, genes whose products
may regulate Cdc42p in Saccharomyces cerevisiae. Mol. Cell. Biol.
105264-5275.
12.
Blacketer, M. J., C. M. Koehler, S. G. Coats, A. M. Myers, and P.
Madaule. 1993. Regulation of dimorphism in Saccharomyces
cerevisiae: Involvement of the novel protein kinase homolog Elmlp
and Protein Phosphatase 2A. M.C.B. 13:5567-5581.
13.
Blacketer, M. J., P. Madaule, and A. M. Myers. 1995. Mutational analysis
of morphologic ditferentiation in Saccharomyces cerevisiae. Genetics
140:1259-1275.
14.
Booher, R. N., R. J. Deshaies, and M. W. Kirschner. 1993. Properties of
Saccharomyces cerevisiae weel and its differential regulation of
p34CDC28 in response to G1 and G2 cyclins. EMBO J. 12:3417-3426.
15.
Chant, J. 1996. Generation of cell polarity in yeast. Curr. Opin. Cell Biol.
8557-565.
16.
Chant, J., M. Mischke, E. Mitchell, L Herskowitz, and J. R. Pringle. 1995.
Role of Bud3p in producing the axial budding pattern of yeast. J. Cell
Biol. 129:767-778.
17.
Chenevert, J., K. Corrado, A. Bender, J. Pringle, and L Herskowitz. 1992.
A yeast gene (BEMl) necessary for cell polarization whose product
contains two SH3 domains. Nature 356:77-79.
173
18.
Cid, V. J., A. Durw, F. Del Ray, M. P. Snyder, C Nombela, and M.
Sinchez. 1995. Molecular basis of cell integrity and morphogenesis in
Saccharomyces cerevisiae. Microb. Rev. 59:345-386.
19.
Clark, D. J. 1996. Thesis. University of Virginia, Charlottesville.
20.
Cohen-Fix, O., J.-M. Peters, M. W. Kirchner, and D. Koshland. 1996.
Anaphase initiation in Saccharomyces cerevisiae is controlled by the
APC-dependent degradation of the anaphase inhibitor, Pdslp. Genes
Dev. 103081-3093.
21.
Coleman, T. R., Z. Tang, and W. G. Dunphy. 1993. Negative regulation
of the Weel protein kinase by direct action of the niml/cdrl mitotic
inducer. Cell 72rf>19-929.
22.
Cvrckovi, F., C. De Viigilio, E. Manser, J. R. Ptingle, and K. Nasmyth.
1995. Ste20-like protein kinases are required for normal localization of
cell growth and for cytokinesis in buddmg yeast. Genes Dev. 9:18171830.
23.
DUrso, G., and P. Nurse. 1995. Checkpoints in the cell cycle of fission
yeast. Curr. OPin. in Gen. Dev. 5:12-16.
24.
Deshaies, R. J. 1997. Phosphorylation and proteolysis: partners in the
regulation of cell division in budding yeast. Curr. Opin. Genet. Devel.
7:7-16.
25.
Deshaies, R. J. 1995. The self-destructive personality of a ceU cycle in
transition. Curr. Opin. Cell Biol. 7:781-789.
26.
Drubin, D. G., H. D. Jones, and K. F. Wertman. 1993. Actin structure
and function; roles in mitochondria organization and morphogenesis
in budding yeast and identification of Ihe phalloidin-binding site. Mol.
Biol. Cell 4:1277-1294.
27.
Drubin, D. G., and W. J. Nelson. 1996. Origins of cell polarity. Cell
84:335-344.
28.
Edgington, N. P., Blacketer, M. J., Bierwagen, T., and A. M. Myers. 1997.
Control of Saccharomyces cerevisiae filamentous growth by a protein
kinase pathway including Sel2p and Cdc28p. Submitted.
29.
Elledge, S. J. 1996. Cell cyle checkpoints: Preventing an identity crisis.
Science 274:1664-1672.
174
30.
Evangelista, M., K. Blundell, M. S. Longtine, C J. Chow, N. Adames, J.
R. Pringle, M. Peter, and C. Boone. 1997. Bnilp, a yeast fonnin linking
Cdc42p and the actin cjrtoskeleton duiing polarized morphogenesis.
Science 276:118-122.
31.
Feillotter, H., P. Nurse, and P. Young. 1991. Genetic and molecular
analysis of cdrl'*'/niml'*'in Schizosaccharomyces pombe. Genetics
127:309-18.
32.
Field, J. F., J. Nikawa, D. Broek, B. MacDonald, L. Rodgers, I. A. Wilson,
R. A. Lemer, and M. Wigler. 1988. Purification of a RAS-responsive
adenylyl cyclase complex from Saccharomyces cerevisiae by use of an
epitope addition method. Mol. Cell. BioL 8:2159-2165.
33.
Ford, S. K., and J. R. Pringle. 1991. Cellular morphogenesis in the
Saccharomyces cerevisiae cell cycle: Localization of ttie CDCll gene
product and the timing of events at the budding site. Dev. Gen. 12.
34.
Funabiki, H., H. Yamano, K. Kumada, K. Nagao, T. Hunt, and M.
Yanagida. 1996. Cut2 proteolysis required for sister-chromatid
separation in fission yeast. Nature 381:438-441.
35.
Futcher, B. 1996. Cyclins and wiring of the yeast cell cycle. Yeast 12:16351646.
36.
Ghiara, J. B., H. E. Richardson, K. Sugimoto, M. Henze, D. J. Lew, C.
Wittenberg, and S. L Reed. 1991. A cyclin B homolog in S. cerevisiae:
Chronic activation of ttie Cdc28 protein kinase by cyclin prevents exit
from mitosis. Cell 65:163-174.
37.
Glotzer, M., A. Murray, and M. Kixschner. 1991. Cyclin is degraded by
the ubiquitin pathway. Nature 349:132-138.
38.
Haarer, B. K., and J. R. Pringle. 1987. Immunofluorescence localization
of the Saccharomyces cerevisiae CDCll gene product to the vicinity of
the 10-nm filaments in the mother-bud neck. Mol. Cell Biol. 7:36783687.
39.
Hauswirth, W. W., L. O. Lim, B. Du|on, and G. Turner. 1987. Methods
for studying the genetics of mitochondria. In V. M. Darley-Usmar, D.
Rickwood, and M. T. Wilson (ed.). Mitochondria: a practical approach.
IRL Press Limited, Washington, D.C.
175
40.
Hochstrasser. 1995. Ubiquitin, proteosomes, and the regulation of
intracellular protein degradation. Curr. Opin. Cell Biol. 7:215-223.
41.
Holloway; S. L., M. Glotzer, R. W. King^ uid A. W. Murray. 1993.
Anaphase is initiated by proteolysis rather than by the inactivation of
maturation-promoting factor.
73:1393-1402.
42.
Hunter, T., and G. D. Plowman. 1997. The protein kinases of budding
yeast six score and more. Trends Biochem. Sd. 22:18-22.
43.
Imiger, S., S. Piatti, C. Michaelis, and K. Nasmyth. 1995. Genes
involved in sister chromatid separation are needed for B-type cyclin
proteolysis in budding yeast.
81:269-277.
44.
Juang, Y.-L., J. Huang, J.-M. Peters, M. E. McLaughlin, C-Y. Tai, and D.
Pellman. 1^7. APC-mediated proteolysis of Asel and the
morphogenesis of the mitotic spindle. Science 275:1311-1314.
45.
Kaplon, T., and M. Jacquet. 1995. The cellidar content of Cdc25p, the ras
exchange factor in Saccharomyces cerevisiae, is regulated by
destabilization through a cyclin destruction box. J. Biol. Chem.
270:20742-20747.
46.
Kim, H. B., B. K. Haarer, and J. R. Pringle. 1991. Cellular morphogenesis
in the Saccharomyces cerevisiae cell cycle: Localization of the CDC3
gene product and the timing of events at the budding site. J. Cell Biol.
112:535-544.
47.
King, R. W., R. J. Deshaies, J. Peters, and M. W. Kirschner. 1996. How
proteolysis drives the cell qrcle. Science 274:1652-1659.
48.
King, R. W., M. Glotzer, and M. W. Kirschner. 1996. Mutagenic analysis
of the destruction signal of mitotic cylins and structural
characterization of ubiquinated intermediates. Mol. Biol Cell 7:13431357.
49.
Kohno, H., K. Tanaka, A. Mine, M. Umikawa, H. Imamura, T.
Fujiwara, Y. Fujita, K. Hotta, H. Qadota, T. Watanabe, and Y. Takai.
1996. Bnilp implicated in cytoskeletal control is a putative target of
Rholp small GTP binding protein in Saccharomyces cerevisiae. EMBO
J. 15:6060-6068.
50.
Kron, S. J., and N. A. R. Gow. 1995. Budding yeast morphogenesis;
signalling, cytoskeleton, and cell cycle. Curr. Opi. Cell Biol. 7:845-855.
176
51.
Lanker, S., M. H. Valdivieso, and C. Wittenberg. 1996. Rapid
degradation of the
cyclin Cln2 induced by CDK-dependent
phosphorylation. Science 271:1597-1601.
52.
Lew, D. 1997. Personal communication.
53.
Lew, D. J., N. J. Maiini, and S. L Reed. 1992. Different G1 cyclins control
the timing of cell cycle commitment in mother and daughter cells of
tfie buddMg yeast S. cerevisiae. Cell 69:317-327.
54.
Lew, D. J., and S. L Reed. 1995. Cell cycle control of morphogenesis in
budding yeast. Curr. Opin. Gen. Dev. 5:17-23.
55.
Lew, D. J., and S. L Reed. 1995. A cell cyle checkpoint monitors cell
morphogenesis in budding yeast. J.C.B. 129:739-749.
56.
Lew, D. J., and S. L Reed. 1993. Morphogenesis in the yeast cell cycle:
Regulation by Cdc28 and cyclins. J.C.B. 120:1305-1320.
57.
Li, R. 1997. Beelp, a yeast protein with homology to Wiscott-Aldrich
syndrome protein, is critical for the assosibly of cortical actin
cytoskeleton. J. Cell Biol. 136:649-658.
58.
Lila, T., and D. G. Drubin. 1997. Evidence for physical and functional
interactions among two Saccharomyces cerevisiae SH3 domsiin
proteins, an adenylyl cydase-assodated protein and the actin
c5^oskeleton. Mol. Biol. Cell 8:367-385.
59.
Lin, F. C., and K. T. Amdt. 1995. The role of Saccharomyces cerevisiae
type 2A phosphatase in the actin cj^oskeleton and in entry into mitosis.
EMBO J. 14:2745-2759.
60.
Longtine,
S., J. L^eA^anm,
L* ^^alencik, ^5. S. ^^1—^^^var, H. Fares,
C. De Viigillo, and J. R. Pringle. 1996. The septins: roles in cytokinesis
and other processes. Curr. Opin. Cell Biol. 8:106-119.
61.
Ma, X-J., Q. Lu, and M. Grunstein. 1996. A search for proteins that
interact genetically with histone H3 and H4 amino termini uncovers
novel regulators of the Swel kinase in Saccharomyces cerevisiae.
Genes Dev. 10:1327-1340.
62.
Matsui, Y., R. Matsui, R. Akada, and A. Toh-e. 1996. Yeast src homology
region 3 domain-containing proteins involved in bud formation. J.
CeU Biol. 133:865-878.
177
63.
Mayer, B. J., and M. J. Eck. 1995. Minding your p's and q's. Curr. Biol.
5:364-367.
64.
Minshiill, J., A. Straight, A. D. Rudner, A. F. Demburg, A. Belmont,
and A. W. Murray. 1996. Protein phosphatase 2A regulates MPF activity
and sister chromatid cohesion in budd^g yeast. Curr. Biol. 6:1609-1620.
65.
Murray, A. W. 1995. The genetics of cell cycle checkpoints. Curr. Opin.
CeU Biol. 5:5-11.
66.
Nasm3rth, K. 1996. At the heart of the budding yeast cell cycle. Trends
Genet. 12:405-412.
67.
Nasm3rth, K. 1996. Viewpoint: Putting the cell cycle in order. Science
274:1643-1645.
68.
Parker, L. L., S. A. Walter, P. G. Young, and H. P. Piwnica-Worms. 1993.
Phosphorylation and inactivation of die mitotic inhibitor Weel by the
niml/cdrl kinase. Nature 363:736-738.
69.
Prasher, D. C., V. K. Eckenrode, W. W. Ward, F. G. Prendergast, and M.
J. Cormier. 1992. Primary structure of the Aequoria victoria greenfluorescent protein. Gene 111:229-233.
70.
Pringle, J. R. Personal communication.
71.
Pringle, J. R., E. Bi, H. A. Harkins, J. E. Zahner, C. De Virgillo, J. Chant,
K. Corrado, and H. Fares. 1996. Establishment of yeast cell polarity.
C.SJi. Symp. Quant. Biol. 60:729-744.
72.
Pringle, J. R., R. A. Preston, A. E. M. Adams, T. Steams, D. G. Drubin, B.
K. Haarer, and E. W. Jones. 1989. Fluorescence microscopy methods for
yeast. Methods Cell Biol. 31:357-435.
73.
Rechsteiner, M., and S. W. Rogers. 1996. PEST sequences and regulation
by proteolysis. Trends Biochem. Sd. 21:267-271.
74.
Rhind, N., B. Fumari, and P. Russell. 1997. Cdc2 tyrosine
phosphorylation is required for the DNA damage checkpoint in fission
yeast. Gen. Dev. 11:504-511.
75.
Richardson, H. E., D. J. Lew, M. Henze, K. Sugimoto, and S. I. Reed.
1992. Cyclin B homologs in Saccharomyces cerevisiae: function in S
phase and in G2. Genes Dev. 6:2021-2034.
178
76.
Rose, M. D., F. Winston, and P. Better. 1990. Methods in yeast genetics.
A genetic laboratory course manual. Cold Spring Harbor Laboratory
Press, Plainview, NY.
77.
Russell, P., S. Moreno, and S. I. Reed. 1989. Conservation of mitotic
controls in fission and budding yeasts. Cell 57:295-303.
78.
Sanders, S. L., and C. M. Field. 1995. Bud-site selection is only skin deep.
Curr. Biol. 5;1213-1215.
79.
Sanders, S. L., and I. Herskowitz. 1996. The Bud4 protein of yeast,
required for axial budding, is localized to the mo&er/bud neck in a cell
cyde-dependent manner. J. Cell BioL 1324:413-427.
80.
Schneider, B. L., W. Seufert, B. Steiner, Q. H. Yang, and A. B. Futcher.
1995. Use of polymerase chain reaction epitope tagging for protein
tagging in Saccharomyces cerevisiae. Yeast 11:1265-1274.
81.
Sia, R. A. L., H. A. Herald, and D. J. Lew. 1996. Cdc28 tyrosine
phosphorylation and the morphogenesis checkpoint in budding yeast.
Mol. Biol. CeU 7:1657-1666.
82.
Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast
host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122:19-27.
83.
Sorger, P. K., and A. W. Murray. 1992. S-phase feedback control in
budding yeast independent of tjrrosine phosphorylation of p34"''^2S
Nature 355:365-368.
84.
Stueland, C S., D. J. Lew, M. J. Cismowski, and S. L Reed. 1993. Full
activation of p34^^^^^® histone HI kinase activity is unable to promote
entry into mitosis in checkpoint-arrested cells of the yeast Saccaromyces
cerevisiae. Mol. Cell. Biol. 13:3744-3755.
85.
Tanaka, S., and H. Nojima. 1996. Nikl: a Niml-like protein kinase of S.
cerevisiae interacts with the Cdc28 complex and regulates cell cycle
progression. Genes Cells 1:905-921.
86.
Tyers, M., G. Tokiwa, and B. Futcher. 1993. Comparison of the
Saccharomyces cerevisiae G1 cyclins: Cln3 may an upstream
activator of Clnl, Cln2, and other qrclins. EMBO J. 12:1955-1968.
179
87.
Tyers, M., G. Tokiwa, R. Nash, and B. Futcher. 1992. The Cln3-Cdc28
kinase complex of S. cerevisiae is regulated by proteolysis and
phophorylation. EMBO J. 11:1773-1784.
88.
Valtz, N., and 1. Herakowitz. 1996. Pea2 protein of yeast is localized to
sites of polarized growth and is required for efficient mating and
bipolar mating. J. Cell BioL 135:725-739.
89.
Wallis, J. W., G. Chrebe^ G. Brodsky, M. Rolfe, and R. Rottistein. 1989.
A hyper-recombination mutation in S. cerevisiae identifies a novel
euk^ryotic topoisomerase. Cell 58:409-419.
90.
Wang, Y., and D. J. Burke. 1997. Cdc55p, the B-t5rpe regulatory subunit
of protein phosphatase 2Ar has multiple functions in mitosis and is
required for the kinetochore/spindle checkpoint in Saccharomyces
cerevisiae. Mol. Cell. Biol. 17:620-626.
91.
Willems, A. R., S. Lanker, E. E. Patton, K. L. Craig, T. F. Nason, N.
Mathias, R. Kobayashi, C. Wittenberg, and M. Tyers. 1996. Cdc53 targets
phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic
pathway. Cell 86:453-463.
92.
Wu, L., and W. Russell. 1993. Niml kinase promotes mitosis by
inactivating Weel tyrosine kinase. Nature 363:738-741.
93.
Wu, L., K. Shiozaki, R. Aligue, and P. Russell. 1996. Spatial
organization of the Niml-Weel-Cdc2 mitotic control network in
Schizosaccharomyces pombe. Mol. Biol. Cell 7:1749-1758.
94.
Yaglom, J., M. H. K. Linskens, S. Sadis, D. Rubin, B. Futcher, and D.
Finley. 1995. p34^<^c28-mediated control of Cln3 cyclin degradation.
M.C.B. 15:731-741.
95.
Yu, Y., Y. W. Jiang, R. J. Wellinger, K. Carlson, J. M. Roberts, and D. J.
Stillman. 1996. Mutations in the homologous ZDSl and ZDS2 genes
affect cell cyde progression. Mol. Cell. Biol. 10:5254-5263.
96.
Zahner, J. E., H. A. Harkins, and J. R. Pringle. 1996. Genetic analysis of
the bipolar pattern of bud site selection in the yeast Saccharomyces
cerevisiae. Mol. Cell. Biol. 16:1857-1870.
180
CHAPTER 5. GENERAL CONCLUSIONS
The resiilts from diis dissertation, provide insights into die
mechanisms of budding yeast cell morphogenesis and polarity. The
serine/threonine kinase Elmlp is required for maintenance of the yeast form
growth (12), however sequence analysis has suggested that Elmlp is not
closely related to conserved kinase families with defined biological roles.
Thus, to identify potential substrates or downstream signaling components of
Elmlp, genetic screens were employed to identify other potential components
of the Elmlp>-dependent signaling pathway in S. cerevisiae. Utilizing
transposon mutagenesis, BCYl, the negative regulatory subimit of PKA, was
isolated (15). The imrestricted activity of PKA resulted in media- and MAT
locus-dependent suppression of elml strains. The suppression of haploid
elml strains was lost in the presence of the transcriptional repressor al/cx2,
which is present only in diploids, and in rich media. Parallel results were
obtained in wild-tjrpe strains imdergoing two forms of filamentous growth,
namely haploid invasive growth and diploid pseudohyphal growth. High
PKA activity significantly enhanced diploid pseudohyphal growth, but
blocked cell elongation during haploid invasive growth. Snflp acts in
opposition to PKA activity, thus deletion of Snflp results in many of the
same phenotypes as overexpression of PKA activity (56,132). Mutations in
the serine/threonine kinase Snflp resulted in suppression of haploid cell
181
elongation during invasive growth, suggesting tiiat Snflp may also play a role
in cell morphogenesis, in addition to roles in carbon source utilization.
These data suggest that multiple pathways regulate a cell fate with regard to
the execution of the filamentous growth response.
A second genetic screen for spontaneous suppressors of elml strains,
resulted in the isolation of a dominant allele of SEL2, a serine/threonine
kinase which shares significant homology to the nimV protein kinase in the
evolutionarily distant fission yeast S. pombe. (39,121) The protein kinase
nimV allows entry into mitosis by inhibiting the tyrosine kinase weeV,
which in turn inhibits mitotic progression by phosphorylating the cell cycle
CDK cdc2*. Genetic evidence suggests that Sel2p performs the analogous
inhibition of the S. cerevisiae weeV homologue Swelp, however the
biological roles of nimV and Sel2p kinases appear to be distinct. Sel2p is
almost three times Icirger than niml* due to a large C-terminal extension,
which contains homology to conserved domains including a SH3-binding
domain, potential PEST regions, nuclear localization domains, and positively
charged regions that may be involved in mediating protein-protein
interactions, and a destruction box. Evidence that supports the role of the
C-terminal of Sel2p is suggested by the fact that even small deletions of this
region result in noncomplementation of the sell null allele. Cellular
localization has demonstrated ttiat Sel2p is located at the bud neck during bud
development, that catalytic activity is not required for localization, and that
182
deletions in the C-tenninal portion, block bud neck localization. Supporting a
role in bud neck formation or c3rtokinesis, overexpression of Sel2p results in
growth arrest late in mitosis, and the formation of an abnormally wide bud
neck that is suppressed by mutations in the G2 cyclin CLB2, and an allele of
CDC28,cdc28'127, that presumably is defective in G2 cyclin interactions.
Collectively, the investigation into the mechanism of Elmlp function
has revealed the differential effects of PKA on filamentous
growth, and has
resulted in the discovery of a conserved kinase cascade that impinges on the
regulation of the cell cycle CDK, Cdc28p, and plays a role in cytokinesis or bud
neck formation late in the cell cycle.
183
APPENDIX. IDENTBFICATION OF ELMS
Introduction
Mutational analysis is a powerful tool for the elucidation of the
underlying mechanisms of signal transduction in budding yeast.
Filamentous growth is a differentiation pathway that is regulated by nutrient
status, and involves complex signaling integration that results in changes in
cell shape, cell cycle control, budding pattern, cell separation, and agar
penetration, hi order to identify gene products that regulate cell morphology
and filamentous growth, our lab performed a genetic screen to isolate
recessive mutations that result in constitutive cell elongation (6). The screen
identified fourteen gene lod, of which, three have been previously identified.
New alleles of GRRl, CDC28, and CDC12 were isolated. These gene encode
proteins that perform functions in cell division. Grrlp has been implicated in
targeting G1 cyclin degradation (3); The cell cycle cyclin dependent kinase
(CDK), Cdc28p performs essential functions necessary for cell division (19, 20),
and Cdcl2p, a bud neck-assodated septin, is required for cytokinesis (15). A
novel serine/ threonine kinase, ELMl has also been identified, and is
essential for maintenance of yeast form cell morphology (5). The substrates of
Elmlp remain to be identified however. The further cloning and
characterization of other ELM gene loci will provide additional information
regarding the determinants of morphogenesis.
184
During the genetic analysis of the ELM mutations, several gene loci
were found to be chromosomally linked to one another. The identification of
ELM7 as being allelic to CDC28 (9), suggested that the isolation of ELMS
would be possible, since the two gene loci were separated by a genetic map
distance of 18 centimorgans on chromosome II (6). Genetic mapping and
complementation analysis has resulted in the cloning of ELMS. The ELMS
gene is allelelic to HSL7 (16), and is predicted to encode a novel 95.2kD
membrane protein that has homologues in higher eukaryotes.
Materials and Methods
Strains and media
Strains used are congenic, derived from D273-10B-A1, and are listed in
Table 1. Standard yeast methods and media were used (2). The invasive
growth assay was performed as previously described by Roberts and Fink (21).
Cloning of ££A15
The plasmid pJH4-67-13-2C (YCp50::CKSl) obtained from S. Reed (12),
was transformed into the elmS-1 strain 123D65d to determine if this plasmid
\
185
Table 1. 5. cerevisiae strains and plasmids
Strain
Genotype
NEY2157
MAT Si ura3 leu2 metS
NEY2157NK5
MATa ura3 leu2 met6 EIM5-URA3
123D65d
MATA ura3elm5-l
Plasmid
Derivation
PJH4-67-13-2C
gift from S. Reed (12)
pNKl
720bp Xhol/EcoRI subclone of pJH into pRS316
pNK3
pJH4 cut with HindUI and religated, thus removing CKSl ORF
pNK4
943bp HindDl/Nrul from pNK3 cloned into pRS316 (22)
pNK5
3.08kb HindHl/EcoRl ftom pNK3 subcloned into pRS306 (22)
contained the ELMS locus by observing complementation of the cell
elongation phenot)^ of elmS-l strains. The plasmid pNKl, containing only
the CKSl ORF, consisted of a 720 bp Xhol/EcoRI fragment from
pJH4-67-13-2C
cloned into pRS316 (22). The plasmid pNK3 was constructed by cutting the
plasmid pNKl with HindUl to remove a 453bp sized fragment, and then
religating the plasmid, resulting in the exdsion of all but 11 amino adds of
the CKSl ORF, but containing the 5'-flanking region and ORF of YBR133c.
The 943bp HindlH/Nrul restriction fragment from
pNK3 containing the
186
YBR133C ORF was subcloned into pRS316 to make the plasmid pNK4. An
integrative ELMS plasmid/ pNK5, was constructed by subdoning a 3.08kb
Hindin/EcoKl fragment from pNK3 into pRS306 to make the plasmid pNK5.
A BLAST search was performed utilizing the algoritiim provided by the NJCBI
(1). The GAP program is a part of the Wisconsin GCG package (Madison, WI).
Southern Blot Hybridization
Southern blot hybridization was performed as previotisly described (2).
The ATrwI-linearized plasmid pNK5 was transformed into the streiin (D273),
and integration was selected by growth on minimal media lacking uradl.
Genomic DNA was isolated from URA3+ strains by glass bead disruption (2),
digested with EcoBl, and separated by agarose gel electrophoresis. Southern
hybridization was performed using a 1.9kb Nrul/Xbal fragment of the
plasmid pNK5 that was labeled with a-P^ dCTP by the random primer
method. Correctly tagged integrants resulted in hybridizing band of sizes
7.5kb and a 3.54kb, as compared to wild-t5rpe control strains which resulted in
a single 3.54kb band.
187
Results
aoiiing£LM5
The identification of ELM7 as being allelic to CDC28, resulted in the
opportunity to clone ELMS, since the two gene loci were linked, and
represented by a genetic map distance of 18 cM (6,9). The genetic map
distance was roughly correlated to actual physical distance on chromosome 11,
and these observations suggested several open reading frames, based on the
recently completed genome sequencing project that may be allelic to the ELMS
locus. Several previously identified ELM genes are involved in aspects of cell
division, thus the search for the ELMS gene locus was narrowed down to
previously identified ORFs on chromosome II that may play roles in cell
division or morphogenesis. One gene, CKSl is highly evolutionarily
conserved, and was located roughly in the correct physical location in relation
to the CDC28 gene locus. CKSl is a subimit of CDC28 that performs an
unknown function, but appears to necessary for both the G1 and G2 phases of
the cell cycle (11,23). CKSl was a possible candidate for being allelic to ELMS,
and if not then CKSl would serve as a useful genetic marker for mapping the
position of ELMS. The plasmid pJH-4-67-13-2C was transformed into the
strain 123D56d to determine if CKSl could complement elmS-1. This plasmid
did result in complementation of ttie recessive allele elmS-1 (Fig.l),
suggesting that either ELMS was contained within the plasmid or that the
188
plasmid contained a low-copy extragenic suppressor. The plasmid contained
at least two ORFs, and a possible third small overlapping ORF. Therefore to
localize the complementing region within the plasmid, subclones were
constructed that contained only one ORF (Fig. 1). A plasmid that contained
only CKSl was imable to complement elm5-l, thus one of the two other ORFs
was responsible for the suppression. The complementing activity was
narrowed to a region containing only the ORF YBR133c, and was identical to
the recently described HSL7 gene (16).
Segregation Test
To confirm that the complementing activity depended on the presence
of the plasmid pNK3, a segregation test was performed, in which the plasmid
was allowed to be lost by overnight growth in nonselective rich liquid media.
The yeast cells were then plated on rich media plates for overnight growth for
single colonies. Individual colonies were then replica plated to minimal
media plates lacking uracil. Those colonies which were unable to grow on
the selective minimal plates always had an elongated morphology,
confirming that the suppression of cell elongation in elm5-l strains was
plasmid-dependent.
189
noio
EcoMTao
'ZZZZZt4
•0
HlnOMM3
pNKI
725 bp
pNK4
943 bp
pNKa^NKS
3009 bp
Figiire 1. Plasmid construction and complementation tests in the elm5-l
mutant strain 123D56d. (A) Plasmid constructs showing relevant
inserts, restriction enzyme sites and ORFs. (B) Plasmid names of
the juxtaposed constructs. (C) The plzismids were transformed into
the strain 123D56d, and selected for on minimal media lacking
uracil. Yeast colonies were picked from transformant plates,
patched to a minimal plate, grown for 2 days at 30X, and
photographed. A rotmd yeast form shape indicates
complementation.
190
Allelism
To detennine if the suppression of elmS-l by the ORF YBR133c was due
to complonentation or extragenic suppression, YBR133c was cloned into an
integrative vector pRS306 to form pNK5, linearized with Mlul, and
transformed into the strain NEY2157. Potential integrants were selected on
minimal media lacking uradl. DNA was isolated from these transformants
by glass bead disruption in phenol, purified, digested with the restriction
ertz3ane EcoRI, and separated by electrophoresis in a 1% agarose gel. The
DNA was transferred to nylon, and probed witii a 1.9kb Nrul/Xbal
radiolabeled DNA fragment from the plasmid pNK5. Six out of a total of six
strains contained two bands, 3.54kb and 7.48kb, which exemplified correctly
integrated YBR133c with the URA3 gene, as compared to the wild-type locus
of a single band of 3.54kb (Fig. 2). The resulting strain, NEY2157NK5 was
mated to the strain 123D65D. The resulting diploid was sporulated, and sixty
tetrads were dissected. If ELMS and YBR133c are identical, then the predicted
Mendelian segregation would suggest that the phenotype of round/L/RA3+,
will always segregate in opposition to an elongated/uraS- phenot)^. If the
two gene lod are not allelic, then uracil prototrophy and cell shape will
segregate independently. Out of a total of sixty complete tetrads, uracil
prototrophy and an elongated cell morphology always segregated in
opposition, thus YBR133c is ELMS.
191
W.T.
1
2
3
4
5
6
ELMS'URA3
tagged (7.5kl>)
ELMS
wild-type (334kb)
Figure 2. Southern blot to confirm. ELM5-URA3 integration into the strain
NEY2157. Genomic DNA was isolated £com the NEY2157 strain
trai\sformed with Nrul-linearized pNK5, and digested with the
restriction enz3rme EcoRI. The digested DNA was separated on an
agarose gel and probed witii a random primed 1.9 kb Nrul/Xbal
DNA fragment from the plasmid pNK5. The autoradiogram was
exposed for two days, dien developed.
BLAST search with ElmSp predicted amino add sequence
To determine if any unique amino add sequences existed that would
potentially allow the cellular location of ElmSp to be determined, the
program PSORT was utilized. PSORT will examine if consensus sequences
exist that are indicative of fransmembrane
domains, targeting sequences for
the nudeus, mitochondria, peroxisome, and the vacuole, and if there are
sequences for lipid modification. The results of the program suggested that
there were no transmembrane domains, or any other signals, and that there
was a 65% chance that the protein was localized into the cjrtoplasm. In
contrast, the NCBI Entrez database suggests that there is a single
transmembrane domain located from amino add 167, and ending at 183, with
the full length protein being 827 amino adds. A BLAST search was
performed to determine if any significant homologies with other sequences
192
existed that might give insight into the function of EhnSp (1). The result of
this search demonstrated that significant homology existed over the entire
length of a putative protein in C elegans (Fig.3). The 146.8kd predicted
protein, U10402 scored a probability of 2.8e-40, is uncharacterized, and is
located on chromosome m. Comparison of the two ORFs using the GCG
program GAP shows that the proteins are 47.67 percent similar, and 24.52
percent identical over a 1158 amino add region.
Invasive growth phenotj^e
Filamentous growth in haploid strains is manifested by invasive
growth in rich media (YPD) (21). ELMS was recently cloned as as part of a
screen for genes diat are required in the absence of N-tenninal segments of
the histone H3 and H4 genes (16). The authors demonstrated that elm5
mutants exhibited a G2 HSL7 delay. Kron and Fink demonstrated that yeast
cells imdergoing pseudohyphal growth also exhibited a G2 delay (13). These
data suggested that Elm5p may play a inhibitory role in invasive growth
differentiation. To test if elmS-1 cell exhibited enhanced invasive growth,
congenic ELMS and elmS-1 haploid strains were tested imder conditions of
invasive growth. CeU were patched on YPD plates and grown for 3 days at
30°C. After this time, cells located in the surface were removed by washing
under a gentle stream of water, such that only the cells that had penetrated
\
!
t
•
Figure 3. A comparison of ElmSp protein sequence with the C elegans
homologue U10402. The aligiunent was performed by the GCG
software program GAP, thus gaps were introduced to achieve
optimal ^gnment. Solid lines represent identities, and colons
represent conserved residues. The two proteins had 24.52 percent
identical residues, and were 47.67 perc^t similar to each otiier over
their entire lengths. The underlined residues represent the
putative transmembrane domain of ElmSp.
194
ELM5
1 MHSNVEVGVKPGENHKQHSKKSRFLQlVSSHSPELPSmDOTLLPrrmi 50
D10402
1
:rr
.:r: . . .|
H.
-H- | ..
: . |.
MLLLSVRniieSSSm,SSTItI£CSF»BSG8UlE!n)UJFSAa)HFP 46
ELMS
51 YXEtVGQVFKDFQRQSIQKHKFLQIPEFQljQDICIPFFIlVRiaCMDDIPS 100
010402
47 VIDLFDVQUNDUffiSYVVtatVNSV'
PFLILZTPSKEIV 84
ELMS 151 MT^.vrywTvm-j.nWBTVP>ap«f.TTcrrsr.p UEDSOFUI
191
=|..:|
. . :|r.r 1|
:.|
010402 130
ILKranWTRNSKFTONVQLFSAIEKCnniHFTZaiVDL 168
ELKS
192
VEUmmoOCSXBPSUrZSUajiaSXPSm/IFailMPVSCLLVSSSI 241
l-t|--.|||- 1 1 1 1 - I I - 1 - -- = --11 l l l = - . : r : . | r . -
010402 169 WmaiTRXQV^
EUG
ALTISSELPISLTELKLVDRMiaEFLMFVlESGL 214
242 F»SNCnanFVLHKENQIlLILKEQKVIIGDSQII...(aiELCVILHaiEiaMt 289
I I-.
I h - II -=-! :--|:: -- •
NGEKSILaRTIMLRIVUWtTinTK
244
010402 21S PIS(Sl
EUC
290 NVKQGSSKZLEraKLLnCTRVUISNSiraQFLLQBJSRIMPFLKPHSm 339
-I-I = =-- = =
--1--= -
- = =11-1 1=1
010402 245 YIfTSlKSEX5aALSBKV!Srai..:{RSRFDVa)VIE»CDVLQAFLQPLSBI 292
ELMS
340 LUiSTYLTFEKDLVKmuSSAILEkLCIDUFIUSAKRFLVILVAGAGKG 389
010402 293
ELMS
I .r.l lll-l ••H|-.|-..|=:r||.|
LOSGVYinFEQI
]QIKXI]VYGEA.WGUJCD
390 ?LVI>RTFKI...ISHLFMDS
..
| . r \-.\\\
lYLLQQCStG 330
1IV5ZIAIEKNFQAYL^!LC3KI)NFDCMD 432
1|.:. :|||i.|.r
010402 331 nStKILKSEBEXiartFRQQQESLICSKLYXVEKIiaiAIV
ELH5
j.::
K 370
433 NBVKLIKEDeiTKMQIHEFSEKRIQIDLCXSELLSSFQaiELSEGCIHSlE 482
•ll-:|..||
---
I h . = 11111111 I l l l l l l l
010402 371 BHVTIIESCMRSI.PGIAKDHQ?liOPDIIVSRLUWPat)MELSPEa.
416
ELH5
513
483 KKHSHNDTIFXPRSVSSyiAPISSPLnfOKL
-I II--1-I1-- II 1- -.-I.:
010402 417
ELMS
DISIPQKXTSYVKPDISTHIHQTimQSIPIfLSBAXPSHraGE 459
514
SQ1I0lSLEAmiVHRVFVCILSSRVNEWIRFEBP..MXCimTV<QD 556
:= ! -
-I- I
h llll
I-
I
010402 460 PELDEQHVHNHMDQrfWYLSKXXFLKEXTKFVFCFBatlFtOISSHEHSD 509
ELMS
557 EDDFTVEFSQSSLNE...FKIKBItlSEIHSFIGFFSA...HL'OOIIFLSTLP 601
I -
010402 510
ELMS
=-1
I= = : : | - l
I
-
SIEPFJIGmiLQLXKrVMLSIEPSTBTPGtlVSWFPAVIFLSDQLRVCaSD 559
602 MDSTVBLKPSEEnieiTRREBlLUJCIJJHTtMU'S.•.WSPIIFPLKQPI 648
.
I
.1| .1
: -.| . : .[
010402 560 RITGVWYEMRVE...KKRIMIZSVSTPIQNniGBSYUIRIMLINSSRLSl 606
ELMS
649
SFICDSELSVL
I
-j:
MSRIBSDTEQICVIREMSLESFiyLMLSNV.
.TSAV 692
| : | =-- =
I - : ---I
010402 607 SSTaCEVPGVQRRqfKagQBKQOKPWLPPSiaCgLTOWHMWOKPEDg 656
ELMS
693
TMSmnPRSXVnXTIXILABMRHySKnHQKLENQZDLOQDIE
..|
: -r . . . . | . . ..j
|
j.
N 737
1
010402 657 EmVISLSEVFRAEUQNASBQQGIEA....MKEAEALELI)ELIM3NEKRN 703
ELMS
738 EEEQCTLSNLSTGHQSVQ
010402 704
ELMS
OIBQL
SEtAKPQBLDS 771
III:: . .
::::|
|. .j.:::.
EEEDAKEIKSAKSRQiagCLniQFLVQVIISU
.VQIUCLKFSKHSICSQQFTI 753
772 DOCPMFDLKSTKALEPSNE
790
<HMVHn(ELSKKLIK»imFIIQBDFVA 803
010402 754 SVLNQUmCK£EiQL0KmX3m
ELMS
791
LPRREDLEEDVPEVHVBV
• l.j:. II:.1
II
KTSVSTLBMVCGRAFSL 825
:|.:.
010402 304 EIGEIVHSQRHnEElflEDITERLSKVSKiaiiGORTWIRWPPYLKSIVLGN 853
195
the agar remained. As shown in Figure 4, elm5-l mutants were significantly
elongated compared to an isogenic wild-type strain, and compared to the cell
length of elmS-l cells not undergoing invasive growth (data not shown).
Therefore, ElmSp may be required for repressing invasive growth imder
conditions of adequate nutrients. The supressive effect may also include
diploid pseudohyphal growth, because ELM5/elm5 heterozygotes in the Z
background strong induction of tiiis differentiation pathway (6).
Discussion
The recent identification of ELM7 as being allelic to CDC28 has
facilitated the isolation of ELMS, due to the fact that CDC28, and ELA15 are
genetically linked by a genetic map distance of 18 centimorgans.
Complementation and allelism demonstrated that ELMS was not allelic to the
CDK subunit CKSl, but instead to a novel uncharacterized predicted protein
YBR133c. Homology searches revealed that ElmSp may contain a single
transmembrane spanning domain, and has homologues in C. elegans, S.
pombe, and in H. sapiens (Fig. 3) (10,16).
Ma and collegues cloned ELMS as HSL7 based on a synthetic lethal
screen with N-terminal deletions in the histone genes H3 and H4. Deletion
of HSL7 resulted in a G2 delay in the cell cycle, and an elongated cell
morphology. Swelp, a tryosine kinase, has been previously shown to cause a
G2 delay in the cell cycle due to the inhibitory phosphorylation of the T5rr-19
196
Figiire 4. Invasive growth properties of wild-tj^ (A,C) and elm5-l (B,D)
haploid strains on rich media. Isogenic strains, NEY2157 and
123£)65d were patched on rich agar medium, and allowed to grow
for 3 days at 30°C. The surface colonies were removed by a stream of
water, such that only cells which had penetrated the agar would
remain. Plates containing the invasive colonies were then
photographed on an inverted microscope. (A,B) represent colonies
just beginning invasive growth, and (C,D) represent more mature
invasive colonies on the same plate.
197
residue of Cdc28p, the master regulatory kinase required for cell cycle
progression (7). Deletion of Swelp blocked the elongated cell morphology of
hsl7 mutated strains, suggesting that the delay in the cell cycle was due to
Swelp activity (16). Thus, ElmSp may play a role in the inhibition of Swelp,
or ElmSp may be a part of a cell cycle checkpoint, in which the absoxce of
ElmSp results in the activation of Swelp to delay mitosis. A high copy
suppressor of HSL7/ELM5 was cloned, and demonstrated to be allelic to ZDSl,
a protein involved in regulating cell morphology (4,16, 24). These genetic
studies, in addition to the enhanced filamentous growth phenotypes of elm51 strains observed in ihis study (Fig. 4), suggest that ElmSp is involved in
maintaining or regulating the yeast form cell morphology in S. cerevisiae.
Recently, the ELMS homologue in 5. pombe has been cloned as the gene skb
(10). This gene product was cloned on the basis of a 2-hybrid protein-protein
interactions with the S. pombe gene shkl, a S. cerevisiae Ste20p homologue.
Ste20p is a member of the PAK kinase family, and plays a role in the mating
response and in mediating filamentous growth (14,18, 21). These data
strongly suggest that Elm5p may physically interact with Ste20p, Cla4p, or
Shklp, to regulate filamentous
growth (8,17).
The genetic screen to identify elongated morphology mutants resulted
in the isolation of four alleles of the ELMS locus (6), however the nature of
these mutations have not been investigated. Future work will be to
determine the subcellular localization of ElmSp, to identify domains of ElmSp
198
that are required for function,, and to determine the relationship of ElmSp
with other proteins that regulate cell morphology. The fact that ElmSp is
conserved throughout evolution suggests that the function of this family of
proteins is important in executing some as yet tmknown aspect of cellular
morphogenesis.
Acknowledgments
I thank K. Olsen for assistance in plasmid construction;^ and S.Reed for
providing plasmids.
References
1.
Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.
Basic local aligrunent search tool. J. Mol. Biol. 215:403-410.
2.
Ausubel, F. M., R. Bient, R. E. Kingston, D. D. Moore, 1. Seidman, A.
Smith, and K. Struhl. 1989. Current Protocols in Molecular Biology.
John Wiley and Sons, New York.
3.
Barral, Y., S. Jentsch, and C. Mann. 1995. G1 cyclin turnover and
nutrient uptake are controlled by a common pathway in yeast. Genes
Dev. 9-399-409.
4.
Bi^ E., and J. R. Fiingle. 1996. ZDSl and ZDS2, genes whose products
may regulate Cdc42p in Saccharomyces cerevisiae. Mol. Cell. Biol.
10:5264-5275.
5.
Blacketer, M. J., C. M. Koehler, S. G. Coats, A. M. Myers, and P.
Madaule. 1993. Regulation of dimorphism in Saccharomyces
cerevisiae: Involvement of the novel protein kinase homolog Elmlp
and Protein Phosphatase 2A. M.C.B. 13:5567-5581.
199
6.
Blacketer, M. J., P. Madaule, and A. M. Myeis. 1995. Mutational analysis
of morphologic differentiation in Saccharomyces cerevisiae. Genetics
140:1259-1275.
7.
Booher, R. N., R. J. Deshaies, and M. W. Kiischner. 1993. Properties of
Saccharomyces cerevisiae weel and its differential regtilation of
p34CDC2S in response to G1 and G2 cyclins. EMBO J. 12:3417-3426.
8.
Cvrckova, F., C De Viigilio, E. Manser, J. R. Pringle, and K. Nasmyth.
1995. Ste20-like protein kinases are required for normal localization of
cell growth and for cytokinesis in budding yeast. Genes Dev. 9:18171830.
9.
Edgington, N. P., M. J. Blacketer, T. Bierwagen, and A. M. Myers. 1997.
Control of Saccharomyces cerevisiae filamentous growth by a protein
kinase pathway including Sel2p and Cdc28p. Submitted .
10.
Gilbreth, M., P. Yang, D. Wang, J. Frost, A. Polverino, M. H. Cobb, and
S. Marcus. 1996. The highly conserved skbl gene encodes a protein that
interacts with Shkl, a fission yeast Ste20/PAK homolog. Proc. Natl.
Acad. Sd. USA 93:13802-13807.
11.
Hadwiger, J. A., C, Wittenberg, M. D. Mendenhall, and S. I. Reed. 1989.
The Saccharomyces cerevisiae CKSl gene, a homolog of the
Schizosaccharomyces pombe sucl'*' gene, encodes a subunit of the
Cdc28 protein kinase complex. M.C.B. 9:2034-2041.
12.
Hadwiger, J. A., C. Wittenberg, H. E. Richardson, M. De Bairos Lopez,
and S. I. Reed. 1989. A family of cyclin homologs that control G1 phase
in yeast. P.N.A.S. USA 86:6255-6259.
13.
Kron, S. J., C. A. Styles, and G. R. Fink. 1994. Symmetric cell division in
pseudohyphae of the yeast Saccharomyces cerevisiae. M.B.C. 5:10031022.
14.
Liu, H., C. A. Styles, and G. R. Fink. 1993. Elements of the yeast
pheromone response pathway required for filamentous growth of
diploids. Science 262:1741-17^.
15.
Longtine, M. S., D. J. DeMarini, M. L. Valendk, O. S. Al-Awar, H. Fares,
C. De Viigilio, and J. R. Pringle. 1996. The septins: roles in cjrtokinesis
and other processes. Curr. Opin. Cell Biol. 8:106-119.
200
16.
Ma, X-Jv Q' Lu, and M. Grunstein. 1996. A search for proteins that
interact genetically with histone H3 and H4 amino termini uncovers
novel regulators of the Swel kinase in Saccharomyces cerevisiae.
Genes Dev. 10:1327-1340.
17.
Maitiii, H., A. Mendoza, J. M. Rodriguez-Pachon, M. Molina, and C.
Nombela. 1997. Characterization of SKMl, a Saccharomyces cerevisiae
gene encoding a novel Ste20/PAK-like protein kinase. Mol. Microbiol.
23:431-444.
18.
Mosch, H., R. Roberts, and G. R. Fink. 1996. Ras2 signals via the
Cdc42/Ste20/mitogen-activated protein kinase module to induce
filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sd.
USA 935352-5356.
19.
Nasm3rth, K. 1996. At the heart of the budding yeast cell cycle. Trends
Genet. 12:405-412.
20.
Nasmyth, K. 1996. Viewpoint Putting the cell cycle in order. Science
274:1643-1645.
21.
Roberts, R. L., and G. R. Fink. 1994. Elements of a single MAP kinase
cascade in Saccharomyces cerevisiae mediate two developmental
programs in the same cell type: mating and invasive growth. Gen. Dev.
8-.2974-2985.
22.
Sikorski, R. S., and P. Hieter. 1989. A system of shuttle vectors and yeast
host strains designed for efficient manipulation of DNA in
Saccharomyces cerevisiae. Genetics 122:19-27.
23.
Tang, Y., and S. I. Reed. 1993. The Cdk-assodated protein Cksl
functions both in G1 and G2 in Saccharomyces cerevisiae. Genes Dev.
7:822-832.
24.
Yu, Y., Y. W. Jiang, R. J. Wellinger, K. Cadson, J. M. Roberts, and D. J.
Stillman. 1996. Mutations in the homologous ZDSl and ZDS2 genes
affect cell cycle progression. Mol. Cell. Biol. 10:5254-5263.
201
REFERENCES
1.
Adams, A. E., D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R.
Pringle. 1990. CDC42 and CDC43, two additional genes involved in
budding and the establishment of cell polari^ in die yeast
Saccharomyces cerevisiae. J. Cell Biol. 111:131-142.
2.
Amberg, D. C, J. E. Zahner, J. W. MulhoUand, J. R. Pringle, and D.
Botstein. 1997. Aip3p/Bud6p, a yeast actin-interacting protein that is
involved in morphogenesis and the selection of bipolar budding sites.
Mol. Biol. Cell 8:729-753.
3.
Atherton-Fessler, S., G. Hannig, and H. Piwnica-Wotms. 1993.
Reversible tjrrosine phosphorylation and cell cycle control. Sem. Cell
Biol. 4:433-442.
4.
Ayscough, K. R., and D. G. Drubin. 1996. Actin; general principles from
studies in yeast. Annu. Rev. Cell Dev. Biol. 12:129-160.
5.
Ayscough, K. R., J. Stryker, N. Pokala, M. Sanders, P. Crews, and D. G.
Drubin. 1997. High rates of actin filament turnover in budding yeast
and roles for actin in establishment and maintenance of cell polarity
revealed using the actin inhibitor latnmculin-A. J. Cell Biol.
137:399-416.
6.
Bardwell, L., J. G. Cook, C. J. Inouye, and J. Thomer. 1994. Signal
propagation and regulation in the mating pheromone response
pathway of the yeast Saccharomyces cerevisiae. Dev. Biol. 166:363-379.
7.
Barral, Y., and C Maim. 1995. Degradation des cyclines G1 et
differendation cellulaire chez Saccharomyces cerevisiae. C.R. Acad. Sd.
Paris 318:43-50.
8.
Bauer, F., M. Urdad, M. Aigle, and M. Crouzet. 1993. Alteration of a
yeast SH3 protein leads to conditional viability with defects in
cytoskeletal and budding patterns. M.C.B. 13:5070-5084.
9.
Bender, A., and J. R. Pringle. 1989. Multicopy suppression of the cdc24
budding defect by CDC42 and three newly id^tified genes induding
the Ras-related gene RSRl. Proc. Natl. Acad. Sd. USA 86:9976-9980.
202
10.
H., S. Raths, F. Crausaz, and H. Riezman. 1994. The ENDS
gene encodes a protein that is required for the internalization step of
endocytosis and for actin cytoskeleton organization in yeast. Mol. Biol.
CeU 5:1023-1037.
11.
Benton, B. K., A. H. Tinkelenbetg, D. Jean, S. Driscoll-Plump, and F. R.
Cross. 1993. Genetic analysis of C]n/Cdc28 regulation of cell
morphogenesis in budding yeast EMBO J. 12:5267-5275.
12.
Blacketer, M. J., C M. Koehler, S. G. Coats, A. M. Myers, and P.
Madaule. 1993. Regulation of dimorphism in Saccharomyces
cerevisiae: bivolvement of the novel protein kinase homolog Elmlp
and Protein Phosphatase 2A. M.C.B. 13:5567-5581.
13.
Blacketer, M. J., P. Madaule, and A. M. Myers. 1995. Mutational analysis
of morphologic ditferentiation in Saccharomyces cerevisiae. Genetics
140:1259-1275.
14.
Booher, R. N., R. J. Deshaies, and M. W. Kirschner. 1993. Properties of
Saccharomyces cerevisiae weel and its diHerential regulation of
p34CDC28 in response to G1 and G2 cyclins. EMBO J. 12:3417-3426.
15.
Broach, J. R. 1991. RAS genes in Saccharomyces cerevisiae: signal
transduction in search of a pathway. T.I.G. 7£28-32.
16.
Brown, C M., and J. S. Hough. 1965. Elongation of yeast cells in
continous culture. Nature 206:676-678.
17.
Chant, J. 1994. Cell polarity in yeast. Trends Genet.10:328-333.
18.
Chant, J., K. Corrado, J. Pringle, and I. Herskowitz. 1991. Yeast BUD5,
encoding a putative GDP-GTP exchange factor, is necessary for bud site
selection and interacts with bud formation gene BEMl. Cell
65:1213-1224.
19.
Chant, J., and I. Herskowitz. 1991. Genetic control of bud site selection
in yeast by a set of gene products that constitute a morphogenetic
pathway. Cell 65:1203-1212.
20.
Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J. R. Pringle. 1995.
Role of 6ud3p in producing the axial budding pattern of yeast. J. Cell
Biol. 129:767-778.
203
21.
Chant, J., and J. R. Prinze. 1991. Budding and cell polarity in
Saccharomyces cerevisiae. Curr. Op. Gen. E>ev. 1:342-350.
22.
Chant> and J. R. Pringje. 1995. Patterns of bud-site selection in the
yeast Saccharomyces cerevisiae. J. Cell Biol. 129:751-765.
23.
Chenever^ K. Coxrado, A. Bender, J. Pringle, and I. Heiskowitz. 1992.
A yeast gene (BEMl) necessary for cell polarization whose product
contains two SH3 domains. Nature 356:77-79.
24.
Choder, M., and R. A. Young. 1993. A portion of RNA polymerase n
molecules has a component essential for stress responses and stress
survival. Mol. Cell. Biol. 13:6984-6991.
25.
Cid, V. J., A. Dur^, F. Del Ray, M. P. Snyder, C Nombela, and M.
Swchez. 1995. Molecular basis of cell integrity and morphogenesis in
Saccharomyces cerevisiae. Microb. Rev. 59:345-386.
26.
Cook, J. G., L. Bardwell, S. J. Kron, and J. Thomer. 1996. Two novel
targets of the MAP kinase Kssl are negative regulators of invasive
growth in the yeast Saccharomyces cerevisiae. Genes Dev. 10:2831-2848.
27.
Cooper, J. A., and D. P. Kiehart 1996. Septins may form a ubiquitous
family of C3rtoskeletal filaments. J. Cell Biol. 134:1345-1348.
28.
Cross, F. R. 1995. Starting the cell cycle: what's the point? Curr. Opin.
CeU Biol. 7:790-797.
29.
Cvrckova, F., C De Viigilio, E. Manser, J. R. Pringle, and K. Nasmyth.
1995. Ste20-like protein kinases are required for normal localization of
cell growth and for c3rtokinesis in budding yeast. Genes Dev.
9:1817-1830.
30.
Cvrckova, F., and K. Nasmyth. 1993. Yeast G1 cyclins CLNl and CLN2
and a GAP-like protein have a role in bud formation. EMBO J.
12:5277-5286.
31.
Dorin, D., L. Cohen, K. Del Villar, P. Pouliet, R. Mohr, M. Whiteway, O.
Witte, and F. Tamanoi. 1995. Kir, a novel R^family G-protein,
induces invasive pseudohyphal growth in Saccharomyces cerevisiae.
Oncogene 11:2267-2271.
204
32.
Diubin, D. G. 1991. Development of cell polarity in budding yeast. Cell
65:1093-1096.
33.
Diubin, D. G., and W. J. Nelson, 1996. Origins of cell polarity. Cell
84:335-344.
34.
Dunens, P., E. Revaidel, M. Bonneu, and M. Aigle. 1995. Evidence for a
branched pathway in the polarized cell division of Saccharomyces
cerevisiae. Cuir. Genet. 27:231-216.
35.
Eide, D., and L. Guazente. 1992. bicieased dosage of a transcriptional
activator goie enhances iron-limited growth of Saccharomyces
cerevisiae. J. Gen. Microbiol. 138:347-354.
36.
Estruch, F., and M. Carlson. 1990. Increased dosage of die MSNl gene
restores invertase expression in yeast mutants d^ective in the SNFl
protein. Nucleic Adds Res. 18:6959-6964.
37.
Evangelista, M., K. Blundell, M. S. Longtine, C. J. Chow, N. Adames, J.
R. Fringle, M. Peter, and C. Boone. 1997. Bnilp, a yeast formin linking
Cdc42p and the actin cytoskeleton during polariz^ morphogenesis.
Science 276:118-122.
38.
Evans, D. R. H., and M. J. R. Stark. 1997. Mutations in the
Saccharomyces cerevisiae type 2A protein phosphatase catalytic subunit
reveal roles in cell wall integrity, actin cytoskeleton organization and
mitosis. Genetics 145:227-241.
39.
Feillotter, H., P. Nurse, and P. Young. 1991. Genetic and molecular
analysis oi cdrV^/niml'^ m Schizosaccharomyces pombe. Genetics
127:309-18.
40.
Field, J. F., J. Nikawa, D. Broek, B. MacDonald, L. Rodgeis, I. A. Wilson,
R. A. Lemer, and M. Wigler. 1988. Purification of a RAS-responsive
adenylyl cyclase complex from Saccharomyces cerevisiae by use of an
epitope addition method. Mol. Cell. Biol. 8:2159-2165.
41.
Finger, F. P., and P. Novick. 1997. Sec3p is involved in secretion and
morphogenesis in Saccharomyces cerevisiae, Mol. Biol. Cell 8:647-662.
42.
Flescher, E. G., K. Madden, and M. Snyder. 1993. Components required
for c3rtokinesis are important for bud site selection in yeast. J. Cell Biol.
122:373-386.
205
43.
Ford, S. K., and J. R. Pringle. 1991. Cellular morphogenesis in the
Saccharomyces cerevisiae cell cycle: Localization of ^e CDCll gene
product and the timing of events at the budding site. IDev. Gen. 12.
44.
Fujita, A., C. Oka, Y. Arikawa, T. Katagari, A. Tonrichi, S. Kuhara, and
Y. MisiunL 1994. A yeast gene necessary for bud site selection encodes a
protein similar to insulin degrading enzymes. Nature 372:567-570.
45.
Gavrias, V., A. Andrianopoulos, C. J. Gimeno, and W. E. Timberlake.
1996. Saccharomyces cerevisiae TECl is required for pseudohyphal
growtti. Mol. Microbol. 19:1255-1263.
46.
Ghiaia, J. B., H. E. Richardson, K. Sugimoto, M. Henze, D.J. Lew, C.
Wittenberg, and S. L Reed. 1991. A cyclin B homolog in S. cerevisiae;
Chronic activation of die Cdc28 protein kinase by cyclin prevents exit
&om mitosis. Cell 65:163-174.
47.
Gimeno, C. J., and G. R. Fink. 1994. biduction of pseudoh3rphal growth
by overexpression of PHDl, a Saccharomyces cerevisiae gene related to
transcriptional regulators of fungal development. M.C.B. 14:2100-2112.
48.
Gimeno, C. J., P. O. Ljungdahl, C. A. Styles, and G. R. Fink. 1992.
Unipolar cell divisions in the yeast 5. cerevisiae lead to filamentous
growth: Regulation by starvation and RAS. Cell 68:1077-1090.
49.
Hadwiger, J. A., C. Wittenberg, H. E. Richardson, M. De Barros Lopez,
and S. I. Reed. 1989. A family of cyclin homologs that control G1 phase
in yeast. P.N.A.S. USA 86:6255-6259.
50.
Halme, A., M. Michelitch, E. L. Mitchell, and J. Chant. 1996. BudlOp
directs axial cell polarization in budding yeast and resembles a
transmembrane receptor. Current Biology 6:570-579.
51.
Hartwell, L. H., J. Culotti, J. R. Pringle, and B. J. Reid. 1974. Genetic
control of cell division cyde in yeast. Science 183:46-51.
52.
Hartwell, L. H., and M. W. Unger. 1977. Unequal division in
Saccharomyces cerevisiae and its implications for the control of cell
division. J. Cell Biol. 75:422-435.
206
53.
Healy, A. M., S. Zolnieiowicz, A. E. Stapleton, M. GoeM, and A. A.
Depaoli-Roach. 1991. CDC55, a Saccharomyces cerevisiae gene involved
in cellular morphogenesis: identification, characterization and
homology to the B subunit of mammalian type 2A protein
phosphatase. Mol. Cell. Biol. 115767-5780.
54.
Heiskowitz, 1.1988. Life cycle of the budding yeast Saccharomyces
cerevisiae, Microbiol. Rev. 52:536-553.
55.
Herskowitz, 1.1995. MAP kinase pathways in yeast for mating and
more. Cell 80:187-197.
56.
Hubbard, E. J. A., X. Yang, and M. Carlson. 1992. Relationship of the
cAMP-dependent protein kinase pathway to the SNF2 protein kinase
and invertase expression in Saccharomyces cerevisiae. Genetics
130:71-80.
57.
Hunter, T., and G. D. Plowman. 1997. The protein kinases of budding
yeast six score and more. Trends Biochem. Sd. 22:18-22.
58.
Jansen, R.-P., C. Dowzer, C. Michaelis, M. Galova, and K. Nasmyth.
1996. Mother ccU-spedfic HO expression in budding yeast depends on
the unconventional myosin Myo4p and other cytoplasmic proteins.
CeU 84:687-697.
59.
Johnson, D. I., and J. R. Pringle. 1990. Molecular characterization of
CDC42, a Saccharomyces cerevisiae gene involved in the development
of cell polarity. J. CeU Biol. 111:143-152.
60.
Johnston, G. C., C. W. EhrhardI; A. Lorincz, and B. L. A. Carter. 1979.
Regulation of cell size in the yeast Saccharomyces cerevisiae. J.
Bacteriol. 137:1-5.
61.
Kellog, D. R., A. Kikuchi, T. Fujii-Nakata, C. W. Turck, and A. W.
Murray. 1995. Members of the NAP/SET fomily of proteins interact
specifically with B-t5rpe cyclins. J.C.B. 130:661-673.
62.
Kellog, D. R., and A. W. Murray. 1995. NAPl acts with clb2 to perform
mitotic functions and to suppress polar bud growth in budding yeast.
J.C.B. 130:675-685.
207
63.
Khazak, V., P. P. Sadhale, N. A. Woychik, R. Bren^ and E. A. Golemis.
1995. Human. RNA polymerase H s^unit hsRPB7 functions in yeast
and influences stress survival and cell morphology. M.B.C. 6:759-775.
64.
Kilmattin, J. V., and A. E. M. Adams. 1984. Structural rearrangements
of tubulin and actin during the cell cycle of the yeast Saccharomyces. J.
Cell Biol. 98:922-933.
65.
Kim, H. B., B. fC Haaier, and J. R. Pringle. 1991. Cellular morphogenesis
in the Saccharomyces cereoisiae cell cycle: Localization of the CDC3
gene product and the timing of events at ttie budding site. J. Cell Biol.
112535-544.
66.
King, R. W., R. J. Deshaies, J. Peters, and M. W. Kirschner. 1996. How
proteolysis drives the cell cycle. Science 274:1652-1659.
67.
King, R. W., P. K. Jackson, and M. W. Kirschner. 1994. Mitosis in
transition. Cell 79:563-571.
68.
Kobayashi, O., H. Suda, T. Ohtani, and H. Sone. 1996. Molecular cloning
and analysis of the dominant flocculation gene FLOS from
Saccharomyces cereoisiae. MoL Gen. Genet. 251:707-715.
69.
Koehler, C. M. 1995. Iowa State University.
70.
Kohno, H., K. Tanaka, A. Mino, M. Umikawa, H. Imamura, T.
Fujiwara, Y. Fujita, K. Hotta, H. Qadota, T. Watanabe, and Y. Takai.
1996. Bnilp implicated in cytoskeletal control is a putative target of
Biholp small GTP binding protein in Saccharomyces cerevisiae. EMBO
J. 15:6060-6068.
71.
Konopka, J. B., C. DeMattei, and C. Davis. 1995. AFRl promotes apical
morphogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 15:723-730.
72.
Kron, S. J., and N. A. R. Cow. 1995. Budding yeast morphogenesis:
signalling, cjrtoskeleton, and cell cycle. Curr. Opi. Cell Biol. 7:845-855.
73.
Kron, S. J., C. A. Styles, and G. R. Fink. 1994. Symmetric cell division in
pseudohyphae of ttie yeast Saccharomyces cerevisiae. M.B.C.
5:1003-1022.
208
74.
Kuriyama, H., and J. C. Slaughter. 1995. Control of cell morphology of
the yeast Saccharomyces cerevisiae by nutrient limitation in
continuous culture. Lett. Appl. Microb. 20:37-40.
75.
Lambrechts, M. G., F. F. Bauer, J. Marmur, and I. S. Pretorius. 1996.
Mud, a mudn-like protein that is regulated by MsslO, is critical for
pseudohyphal differentiation in yeast. Pro. Natl. Acad. Sd. USA
93:8419-8424.
76.
Law, S. F.,J. Estojak, P Wang, T. MysUwiec, G. Kruh, and E. Golemis.
1996. Human enhancer of filamentation 1, a novel plSC^-like docking
protein, associates with focal adhesion kinase and induces
pseudohyphal growth in Saccharomyces cerevisiae. Mol. Cell. Biol.
16:3327-3337.
77.
Leberer, E., D. Y. Thomas, and M. Whiteway. 1997. Pheromone
signalling and polarized morphogenesis in yeast. Curr. Opin. Genet.
Dev. 7:59-66.
78.
Lee, K. S., L. K. Hines, and D. E. Levin. 1993. A pair of functionally
redtmdant yeast genes (PPZI abd PPZ2) encoding type 1-related protein
phosphatases function within the PKCI-mediated pathway. Mol.
CeU.Biol. 13:5843-5853.
79.
Lee, R. H., and S. M. Honigberg. 1996. Nutritional regulation of late
meiotic events in Saccharomyces cerevisiae through a pathway distinct
from initiation. Mol. Cell Biol. 16:3222-3232.
80.
Levin, D. E., and B. Errede. 1995. The proliferation of MAP kinase
signaling pathways in yeast. Curr. Opin. Cell Biol. 7:197-202.
81.
Lew, D. J., and S. Kombluth. 1996. Regulatory roles of cyclin dependent
kinase phosporylation in cell cycle controL Curr. Opin. Cell Biol.
8:795-804.
82.
Lew, D. J., and S. L Reed. 1995. Cell cycle control of morphogenesis in
budding yeast. Curr. Opin. Gen. Dev. 5:17-23.
83.
Lew, D. J., and S. L Reed. 1995. A cell cyle checkpoint monitors cell
morphogenesis in budding yeast. J.C.B. 129:739-749.
84.
Lew, D. J., and S. L Reed. 1993. Morphogenesis in the yeast cell cycle:
Regulation by Cdc28 and cydins. J.C.B. 120:1305-1320.
209
85.
Lillie, S. H., and J. R. Piingle. 1980. Reserve carbohydrate metabolism in
Saccharomyces cerevisiae: responses to nutrient limitation. J. Bacteriol
143:1384-1394.
86.
Liu, H., C. A. S^les, and G. R. Fink. 1993. El^ents of the yeast
pheromone response pathway required for filamentous growth of
diploids. Science 262:1741-1744.
87.
Liu, H., C. A. SQrles, and G. R. Fink. 1996. Saccharomyces cerevisiae
S288C has a mutation in FLOS, a gene required for filamentous growth.
Genetics 144:967-978.
88.
Ljungdahl, P. O., C. J. Gimeno, C A.Styles, and G. R. Fink. 1992. SHR3;
A novelcomponent of the secretory pa^way specifically required for
the proper localization of amino add permeases in yeast. Cell
71:463-478.
89.
Longtine, M. S., D. J. DeMaiini, M. L. Valendk, O. S. Al-Awar, H. Fares,
C. De Virgilio, and J. R. Pringle. 1996. The septins: roles in cytokinesis
and other processes. Curr. Opin. Cell Biol. 8:106-119.
90.
Lorincz, A., and B. L. A. Carter. 1979. Control of cell size at bud
initiation in Saccharomyces cerevisiae. J. Gen. Microbiol 113:287-295.
91.
Lorincz, A. T., and S. L Reed. 1984. Primary structure homology
between the product of yeast division control gene CDC28 and
vertebrate oncogenes. Nature 307:183.
92.
Madaule, P., R. Axel, and A. M. Myers. 1987. Characterization of two
members of the rho gene family from the yeast Saccharomyces
cerevisiae. Proc. Natl. Acad. Sd. USA 84:779-783.
93.
Madden, K., C. Costigan, and M. Snyder. 1992. Cell polarity and
morphogenesis in Saccharomyces cerevisiae. T.I.C.B. 2:22-29.
94.
Madden, K., and M, Snyder. 1992. Specification of sites for polarized
growth in Saccharomyces cerevisiae and the infuence of external
fectors on site selection. Mol. Biol. Cell 3:1025-1035.
95.
Madhaid, H. D., and G. R. Fink. 1997. Combinatorial control required
for the spedfidty of yeast MAPK signaling. Sdence 275:1314-1317.
210
96.
Manser, E., T. Leungs H. Salihuddiiv Z. S. Zhao, and L. Lim. 1994. A
brain serine/threonine protein kinase activated by Cdc42 and Racl.
Nature 367:40-46.
97.
Marsh, Lv A. M. Neiman, and I. Heiskowitz. 1991. Signal transduction
during pheromone response in yeast. Aimu. Rev. Cell Biol. 7:699.
98.
Martin, H., A. Mendoza, J. M. Rodriguez-Pachon, M. Molina, and C.
Nombela. 1997. Characterization of SKMl, a Saccharomyces cerevisiae
gene encoding a novel Ste20/PAK-like protein kinase. Mol. Microbiol.
23:431-444.
99.
McCa£^y, M., J. S. Johnston, B. Gottd, A. M. Myers, J. Rossier, M. R.
Popoff, P. Macule, and P. Boquet. 1991. The small GTP-binding protein
Rholp is localized on the golgi apparatus and post-golgi vesicles in
Saccharomyces cerevisiae. J. Cell Biol. 115:309-319.
100.
Michelitch, M., and J. Chant 1996. A mechanism of Budlp GTPase
action suggested by mutational analysis and immunologicalization.
Current Biology 6:446-454.
101.
Mitchell, A. P. 1994. Control of meiotic gene expression in
Sacharomyces cerevisiae. Microbiol. Rev. 58:56-70.
102.
Mosch, H., R. Roberts, and G. R. Fink. 1996. Ras2 signals via ttie
Cdc42/Ste20/mitogen-activated protein kinase module to induce
filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sd.
USA 93:5352-5356.
103.
Mosch, H.>U., and G. R. Fink. 1997. Dissection of filamentous growth by
transposon mutagenesis in Saccharomyces cerevisiae. Genetics
145:671-684.
104.
Nasmyth, K. 1996. At the hecirt of the budding yeast cell cycle. Trends
Genet. 12:405-412.
105.
Nasmyth, K. 1993. Control of the yeast cell cycle. Curr. Opin. Cell Biol.
5:166-179.
106.
Nasmyth, K. 1996. Viewpoint; Putting the cell cycle in order. Science
274:1643-1645.
211
107.
Nurse, P. 1994. Ordeiing S phase and M phase in the cell cycle. Cell
79547-550.
108.
Pack, H., E Bi, J. R. Pringle, and L Heiskowitz. 1997. Two active states of
the Ras-ielated Budl/R^l protein bind to different effectors to
determine yeast cell polarity. Proc. Natl. Acad. ScL USA 94:4463-4468.
109.
Park, H., J. Chant, and I. Hexskowitz. 1993. BUD2 encodes a
GTPase-activating protein for Budl/Rsrl necessary for proper bud-site
selection in yeast. Nature 365:269-274.
110.
Peterson, J., Y. Zheng, L. Bender, A. Myers, R, Cerione, and A. Bender.
1994. biteractions between the bud emergence proteins Bemlp and
Bem2p and Rho-type GTPases in yeast. J. Cell Biol. 127:1395-1406.
111.
Powers, S., E. Gonzales, T. Christensen, J. Cubert, and D. Broek. 1991.
Fimctional cloning of BUD5, a CDC25-related gene from a S. cerevisiae
that can supress a dominant-negative RAS2 mutant. Cell 65:1225-1231.
112.
Pringle, J. R., E. Bi, H. A. Haddns, J. E. Zahner, C. De Virgillo, J. Chant,
K. Cortado, and H. Fares. 1996. Establishment of yeast cell polarity.
C.SJI. Symp. Quant, Biol. 60:729-744.
113.
Reed, S. 1.1980. Selection of S. cerevisiae mutants defective in the start
event of cell division. Genetics 95:561-77.
114.
Richardson, H. E., C. Wittenberg, F. Cross, and S. I. Reed. 1989. An
essential G1 function for cyclin-like proteins in yeast. Cell 59:1127-1133.
115.
Roberts, R. L., B. Bowers, M. L. Slater, and E. Cabib. 1983. Chitin
synthesis and localization in cell division cycle mutants of
Saccharomyces cerevisiae. Mol. Cell. Biol. 3:922-930.
116.
Roberts, R. L., and G. R. Fink. 1994. Elements of a single MAP kinase
cascade in Saccharomyces cerevisiae mediate two developmental
programs in the same cell type: mating and invasive growth. Gen. Dev.
8:2974-2985.
117.
Robinson, L., M. Menold, S. Garret^ and M. Culbertson. 1993. Casein
kinase I-like protein kiruises encoded by YCKl and YCK2 are required
for yeast morphogenesis. M.C.B. 13:2870-2881.
212
118.
Roemer, T., K. Madden, J. Chang, and M. Snyder. 1996. Selection of
axial growth sites in yeast requires Axl2p, a novel plasma membrane
glycoprotein. Genes Dev. 10:7^-793.
119.
Ruggieii, R., A. Bender, Y. Matsui, S. Powers, Y. Takai, J. Prii^e, and K.
Matsumoto. 1992. RSRl, a ras-like gene homologous to
K.rev-l{smg21A/raplA): Role in ttie development of cell polarity and
interactions with the Ras pathway in Saccaromyces cerevisiae. Mol.
CeU. Biol. 12:758-766.
120.
Russell, P., S. Moreno, and S. I. Reed. 1989. Conservation of mitotic
controls in fission and budding yeasts. Cell 57:295-303.
121.
Russell, P., and P. Nurse. 1987. The mitotic inducer
funtions in
a regulatory network of protein kinase homologies controlling
initiation of mitosis. Cell 49:569-96.
122.
Sanders, S. L., and C. M. Field. 1994. Septins in common? Curr. Biol.
4.^07-910.
123.
Sanders, S. L., and I. Herskowitz. 1996. The Bud4 protein of yeast,
required for axial budding, is localized to the moAer/bud neck in a cell
cycle-dependent manner. J. Cell Biol. 1324:413-427.
124.
Schultz, J., B. Ferguson, and G. F. Sprague. 1995. Signal transduction
and gro\^ control in yeast. Cvur. Opin. Genet. Dev. 5:31-37.
125.
Sia, R. A. L., H. A. Herald, and D. J. Lew. 1996. Cdc28 tyrosine
phosphorylation and the morphogenesis checkpoint in budding yeast.
Mol. Biol. CeU 7:1657-1666.
126.
Simon, M.-N., C. De Virgillo, B. Souza, J. R. Pringle, A. Abo, and S. I.
Reed. 1995. Role for the Rho-family GTPase Cdc^ in yeast
mating-pheromone signal pathway. Nature 376:702-705.
127.
Sivadon, P., F. Bauer, M. Aigle, and M. Cxouzet 1995. Actin cytoskeletal
and budding pattern are altered in the yeast rvslBl mutant: the Rvsl61
protein shares common domains with the brain amphiphysin. Mol.
Gen. Genet. 246:485-495.
128.
Surana, U., H. Robitsch, C Price, T. Schuster, I. Fitch, A. B. Futcher, and
K. Nasm3rth. 1991. The role of CDC28 and cyclins during mitosis in the
budding yeast S. cerevisiae. Cell 65:145-161.
213
129.
S3nnoii8, M. 1996. Rho family GTPases: the qrtoskeleton and beyond.
Trends Biochem. Sd. 21:178-181.
130.
Tedfoid, K., S. Kim, D. Sa, K. Stevens, and M. Tyeis. 1997. Regulation of
the mating pheromone and invasive growth responses in yeast by two
MAP kinase substrates. Curr. Biol. 7:228-238.
131.
Thevelein, J. M. 1994. Signal transduction in yeast. Yeast 10:1753-1790.
132.
Thompson>Jaeger, S., J. Francois, J. P. Gaughian, and K. Tatchell. 1991.
Deletion of SNFl affects the nutrient response of yeast and resembles
mutations which activate the adenylate cyclase pathway. Genetics
129:697-706.
133.
Toda, T., S. Cameron, P. Sass, M. Zoller, and M. Wigler. 1987. Three
different genes in S.cerevisiae encode the catal3rtic subimits of the
cAMF-dependent protein kinase. Cell 50:277-287.
134.
Valtz, N., and I. Herskowitz. 1996. Pea2 protein of yeast is localized to
sites of polarized growtti and is required for efficient mating and
bipolar mating. J. Cell Biol. 135:725-739.
135.
Van Zyl, W., W. Huang, A. A. Sneddon, M. Stark, S. Camier, M.
Werner, C. Marck, A. Sentenac, and J. R. Broach. 1992. Inactivation of
the protein phosphatase 2A regulatory subunit A results in
morphological and transcriptional defects in 5. cerevisiae. Mol. Cell.
Biol. 12:4946-4959.
136.
Ward, M. P., C. J. Gimeno, G. R. Fink, and S. Garrett 1995. SOK2 may
regulate cyclic AMP-dependent protein kinase-stimulated growth and
pseudohyphal development by repressing transcription. M.C.B.
15:6854-6863.
137.
Welch, M. D., D. A. Holtzman, and D. G. Drubin. 1994. The yeast actin
cytoskeleton. Curr. Opin. Cell Biol. 6:110-119.
138.
Wemer-Washbume, M., E. Braun, G. C. Johnston, and R. A. Singer.
1993. Stationary phase in the yeast Saccliaromyces cerevisiae. Microb.
Rev. 57:383-401.
214
139.
Wigler, M., J. Field, S. Powers, D. Bioel^ T. Toda, S. Cameron, J.
Nikawa, T. Micha^, J. Colicelli, and K, Ferguson. 1988. StucUes of RAS
function in yeast Saccaromyces cerevisiae. C. S. H. S. Quant. Biol.
58:649-655.
140.
Wittenbeigr C, K. Sagimoto, and S. I. Reed. 1990. Gl-spedfic cyclins of
S. cerevisiae: Cell cycle peroididty, regulation by mating pheromone,
and association witfi the p34CDC28 protein kinase. Cell 62:225-237.
141.
Wright, R. M., T. Repine, and J. E. Repine. 1993. Reversible
pseudohyphal growth in haploid Saccharontyces cerevisiae is an
aerobic process. Curr. Genet. 23:388-391.
142.
Yamochi, W., K. Tanaka, H. Nonaka, A. Maeda, T. Musha, and Y.
Takai. 1^4. Growth site localization of Rholp small GTP-binding
protein and its involvement in bud formation in Saccharomyces
cerevisiae. J. Cell Biol. 125:1077-1093.
143.
Yang, S., K. R. A3rscough, and D. G. Drubin. 1997. A role for actin
cytoskeleton of Saccharomyces cerevisiae in bipolar bud-site selection. J,
CeU Biol. 136:111-1123.
144.
Zahner, J. E., H. A. Harkins, and J. R. Pringle. 1996. Genetic analysis of
the bipolar pattern of bud site selection in the yeast Saccharomyces
cerevisiae. Mol. Cell. Biol. 16:1857-1870.
145.
Zheng, Y., A. Bender, and R. A. Cerione. 1995. hiteractions among
proteins involved in bud-site selection and bud-site assembly in
Saccharomyces cerevisiae. J. Biol. Chem. 270:626-630.
215
ACKNOWLEDGMENTS
I would like to thank Dr. Alan Myers for the enlivened discussions,
editing, guidance, and for his excellent taste in music. I would like to thank
all of the members of the Myers laboratory for great conversations regarding a
number of scientific and nonsdentific issues, and their willingness to offer
assistance, especially Dr. Melissa Blacketer, Dr. Carla Koehler, Tracy
Bierwagen, and Dr. Martha James. I thank my family for their emotional, and
sometimes fiscal, support during my extended education.
Finally, I would like to thank my wife Nancy for her immense capacity
for understanding £md patience.