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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 Follow this and additional works at: http://lib.dr.iastate.edu/rtd Part of the Cell Biology Commons, Genetics Commons, and the Molecular Biology Commons Recommended Citation 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. This Dissertation is brought to you for free and open access by Digital Repository @ Iowa State University. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Digital Repository @ Iowa State University. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI fibns the text direct^ fhmi the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter ii^e others may be fix)m any type of computo- printer. The qualiQr of thb reproduction is depcodent upon the quality of the copy submitted. Broken or indistinct prmt, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversety affect reproduction. &i the unlikely event that the author did not send UMI a complete manuscript and there are missmg pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, b^inning at the upper left-hand comer and continuing from left to right in equal sections with small overiaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographicalfy in this copy. Kgher quality fTx 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Iflformation Compai^ 300 North Zed> Road, Ann Atbor MI 48106-1346 USA 313n61-4700 800/521-0600 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 UMI Number: 9814639 UMI Microform 9814639 Copyrigiit 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copyii>8 under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ii Graduate College Iowa State University This is to certify that the Doctoral dissertation of Nicholas Patrick Edgington has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Major Professor Signature was redacted for privacy. For the Major Program Signature was redacted for privacy. Forme Graduate College 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. 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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. 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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. 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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. 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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.