Download Schizosaccharomyces pombe Rgf3p is a specific Rho1 GEF that

Research Article
6163
Schizosaccharomyces pombe Rgf3p is a specific
Rho1 GEF that regulates cell wall β-glucan
biosynthesis through the GTPase Rho1p
Virginia Tajadura, Blanca García, Ignacio García, Patricia García and Yolanda Sánchez*
Instituto de Microbiología Bioquímica, CSIC/Universidad de Salamanca, and Departamento de Microbiología y Genética, Universidad de
Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
*Author for correspondence (e-mail: [email protected])
Accepted 10 September 2004
Journal of Cell Science 117, 6163-6174 Published by The Company of Biologists 2004
doi:10.1242/jcs.01530
Summary
Rho1p regulates cell integrity by controlling the actin
cytoskeleton and cell-wall synthesis. Here, we describe the
cloning and characterization of rgf3+, a member of the Rho
family of guanine nucleotide exchange factors (Rho GEFs).
The rgf3+ gene was cloned by complementation of a mutant
(ehs2-1) hypersensitive to drugs that interfere with cell-wall
biosynthesis. The rgf3+ gene was found to be essential for
cell viability and depletion of Rgf3p afforded phenotypes
similar to those obtained following depletion of Rho1p.
However, the cell death caused by Rgf3p depletion could be
rescued by the presence of 1.2 M sorbitol, whereas
depletion of Rho1 was lethal under the same conditions.
We show that Rgf3p is a specific Rho1-GEF. The
hypersensitivity to drugs affecting the cell wall of the ehs21 mutant was suppressed by overexpression of rho1+ but
Key words: Cell-wall mutants, Rho GEF family, Rho1, Fission yeast,
Cytokinesis
Introduction
The fungal cell wall is the essential cellular boundary,
controlling many transport processes, cellular metabolism
and, indeed, all communications with the extracellular world.
Because of its mechanical strength, it allows cells to withstand
turgor pressure and consequently prevents cell lysis. In the
fission yeast Schizosaccharomyces pombe, the cell wall
mainly consists of three polysaccharides, β-1,3-glucan, α-1,3glucan and galactomannoproteins, all of which form a large
complex (for a review, see Duran and Perez, 2004). Their
coordinated synthesis represents an essential step for the
assembly of a functional cell wall to ensure cell integrity
(Ishiguro, 1998).
We have used the biosynthesis of β-1,3-glucan as a model
to study morphogenesis. It has been suggested that β-1,3glucan is the first polymer to be synthesized (Osumi et al.,
1989; Roh et al., 2002) and that the regulation of this
polysaccharide might be a key step in the sequential assembly
of the other cell wall components. β-1,3-Glucan comprises
~45% of the cell wall and is the major structural component,
as seen by the fact that its enzymatic degradation leads to the
solubilization of the other components. The enzymatic system
that catalyses the synthesis of this polysaccharide is β-1,3glucan synthase (GS). GS is composed of at least two fractions:
a catalytic moiety of the enzyme and a regulatory component.
In fission yeast, the catalytic subunit of GS is encoded by at
least four genes: cps1+/bgs1+ (Le Goff et al., 1999; Cortes et
al., 2002; Liu et al., 2000b; Liu et al., 2002), bgs2+ (Martin et
al., 2000; Liu et al., 2000a), bgs3+ (Martin et al., 2003) and
bgs4+ (Cortés et al., 2005). All of them code for essential
proteins. In addition to the catalytic subunit, the small GTPbinding protein Rho1p is an essential regulatory subunit
(Arellano et al., 1996). Rho1 acts as a binary switch by cycling
between an inactive GDP-bound and an active GTP-bound
conformational state. Rho1p stimulates GS in its GTP-bound
prenylated form, providing a rationale for the understanding of
the mechanism by which the cell can switch β-1,3-glucan
synthesis on and off by interconverting the GDP and GTP
forms of Rho1p.
The Rho1p of fission yeast is a functional homologue of
budding yeast Rho1p (Nakano et al., 1997), and belongs to a
family of small GTPases that are key regulators in polarity
processes (for reviews, see Mackay and Hall, 1998; Takai et
al., 2001; Burridge and Wennerberger, 2004). The fission-yeast
Rho family includes Cdc42p and Rho1p-Rho5p. The cdc42+
gene is essential and is involved in the establishment of cell
polarity (Miller and Johnson, 1994). The rho2+ gene has been
shown to be involved in the control of cell morphogenesis,
probably by regulating the synthesis of Mok1p, the α-1,3glucan synthase, via a Pck2p pathway (Hirata et al., 1998;
not by any of the other GTPases of the Rho family. Rgf3p
interacted with the GDP-bound form of Rho1p and
promoted the GDP-GTP exchange. In addition, we show
that overexpression of Rgf3p produces multiseptated cells
and increases β-1,3-glucan synthase activity and the
amount of cell wall β-1,3-glucan. Rgf3p localized to the
septum and the mRNA level was regulated in a cell-cycledependent manner peaking during septation. Our results
suggest that Rgf3p acts as a positive activator of Rho1p,
probably activating the Rho functions that coordinate
cell-wall biosynthesis to maintain cell integrity during
septation.
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Journal of Cell Science 117 (25)
Calonge et al., 2000). The rho3+ and rho4+ genes are nonessential and are both involved in cell separation processes.
Rho3p interacts with the formin For3p and modulates exocyst
function (Nakano et al., 2002; Wang et al., 2003). Rho4p might
be involved in septum degradation during cytokinesis (Santos
et al., 2003; Nakano et al., 2003).
Fission-yeast Rho1 localizes to sites of polarized growth,
the cell poles and the septum (Arellano et al., 1997).
Expression of a dominant-active Rho1 mutant (Rho1G15V
or Rho1Q64L) produces swollen cells, branched cells and
multiseptated cells, whereas that of a dominant-negative Rho1
mutant (Rho1T20N) produces shrunken or dumpy cells
(Arellano et al., 1996; Nakano et al., 1997). These cells have
defects in the organization of the actin cytoskeleton, cell
polarity and cell integrity. Rho1p seems to play several
functional roles upon interacting with its targets: it activates
GS (Arellano et al., 1996); it binds directly to the protein
kinase C (PKC) family of protein kinases Pck1p and Pck2p;
and it is a positive regulator of these kinases (Arellano et al.,
1999; Sayers et al., 2000). In addition, Rho1 regulates the
localization of F-actin patches (Arellano et al., 1997; Sayers
et al., 2000). However, little is known about the proteins that
turn Rho1p on and off in the cell. These proteins might play
important roles in the specificity of Rho functions. There are
at least nine proteins that belong to the Rho GTPase activating
protein (RhoGAP) family in the S. pombe genome. Three of
these – Rga1, Rga5 and Rga8 – function as GAPs for Rho1p.
None of them is essential for cell viability, although
deletion of rga1+ causes a slow-growth defect and severe
morphological abnormalities (Nakano et al., 2001). Rga5p is
involved in the regulation of GS activity and cell integrity
(Calonge et al., 2003) and Rga8p is a Shk1p (Cdc42/p21activated kinase) substrate that negatively regulates Shk1pdependent growth control pathways, potentially through
interaction with Rho1p GTPase (Yang et al., 2003). Regarding
the role of RhoGEFs as direct activators of Rho GTPases in
fission yeast, it has been reported that Scd1p and Gef1p could
activate Cdc42p (Chang et al., 1994; Coll et al., 2003; Hirota
et al., 2003). Recently, in a search for genomic sequences
bearing a Rho GEF domain, five new genes (rgf1+-rgf5+, for
RhoGEF 1-5) have been described and reported to be involved
in the regulation of cell morphology (Iwaki et al., 2003).
However, it has not yet been shown whether any of these
factors act specifically on Rho1p.
Our approach to the study of cell-wall biosynthesis and
regulation was to obtain mutants hypersensitive to the cell-wall
antifungal drugs Echinocandin (Ech) and Calcofluor White
(Cfw) (ehs mutants). In the present work, we cloned the rgf3+
gene as the structural gene that complements the ehs2-1
mutation. Genetic and biochemical studies have indicated that
Rgf3p is a Rho1p-specific GEF in S. pombe. Moreover, our
data suggest that, among the different Rho1p essential
functions, Rgf3p could specifically regulate β-1,3-glucan
biosynthesis and cell integrity during septation.
Materials and Methods
Media, reagents and genetics
The genotypes of the S. pombe strains used in this study are listed in
Table 1. Complete yeast growth medium (YES), selective medium
(MM) supplemented with the appropriate requirements and
sporulation medium (MEA) have been described elsewhere (Moreno
et al., 1991). Ech B (LY280949; LILLY Company) (Radding et al.,
1998) was stored at –20°C in a stock solution (2.5 mg ml–1) in 50%
ethanol and was added to the media at the corresponding final
concentration after autoclaving. Cfw was prepared (15 mg ml–1) in
water with a few drops of 10 N KOH, filter sterilized and added as
above to EMM or YES medium, the latter previously buffered with
50 mM potassium hydrogen phthalate, pH 6.1. Crosses were
performed by mixing appropriate strains directly on MEA plates.
Recombinant strains were obtained by tetrad analysis. For
overexpression experiments using the nmt1+ promoter, cells were
grown in EMM containing 15 µM thiamine up to the logarithmic
phase. Then, the cells were harvested, washed three times with MM
and inoculated into fresh medium (without thiamine) at an optical
density at 600 nm of 0.01.
Mapping of the ehs2-1 mutant
Genetic mapping was carried out by measuring genetic linkage in a
swi5 mutant background (Schmidt, 1993) (swi5 strains were a gift
from H. Schmidt, Institut für Genetik, Braunschweig, Germany).
First, the ehs2-1 mutant was shown to map to chromosome III.
Second, the position of ehs2-1 on the chromosome was determined.
An ehs2-1 swi5-39 leu1-32 h– strain was constructed and crossed with
a ura4-294 tps14-5 ade5-36 swi5-39 h90 strain. Tetrad analysis of this
cross revealed that ehs2-1 is localized to the right arm of chromosome
III, closer to the tps14 gene. Third, the ehs2-1 mutation was mapped
by linkage analysis in a swi5+ background. An ehs2-1 leu1-32 h– strain
was crossed with mutants in genes that map to this chromosome arm
(tps14 ade6-250 arg1-230 h+). Tetrad analysis of the crosses was
performed and the ehs2-1 mutant was found to be closely linked to
the ade6 gene (1.5 cM, 45 tetrads analysed). Cosmids in the ade6
region were screened for genes that might be related to cell-wall
biosynthesis. We chose mok1+ (which encodes an α-glucan synthase)
in cosmid C17A7 and rgf3+ and rgf1+, both in cosmid C645 (genes
bearing homology with ROM1 from Saccharomyces cerevisiae, a GEF
for Rho1p). The mok1+ gene was kindly provided by T. Toda (Cancer
Research UK, London) and we found no complementation of the
ehs2-1 phenotypes.
Table 1. S. pombe strains used in this work
Strains
PN22
GI 1
MS38
MS75
YSM373
YSM654
YSM656
VT88
VT128
PPG217
PN35
Genotypes
h– leu1-32
h+ leu1-32, ehs2-1
h– leu1-32 ade6M210 ura4D-18 his3D1
h+/h– leu1-32/leu1-32, ade6M210/ade6M216, ura4D-18/ ura4D-18, his3D1/his3D1
h+/h– leu1-32/leu1-32 ade6M210/ade6M216, rgf3::ura4+/rgf3+ his3D1/his3D1 ura4D-18/ura4D-18
h+/h– leu1-32/leu1+ his3+/his3D1 rgf3::ura4+/rgf3+ wee1-50/wee1-50 ura4D-18/ura4D-18
h+/h– leu1-32/leu1+ his3D1/his3+ rgf3::ura4+/rgf3+ sid2-250/sid2-250 ura4D-18/ura4D-18
h– leu1-32 ade6M210 ura4D-18, his3DI, 81 nmt-rgf3+-ura4+
h– leu1-32 ade6M210 ura4D-18, his3DI leu1+::EGFP-rgf3+
h– leu1-32 ade6M210 ura4D-18 his3D1 rho1::ura4+ + pREP41X-rho1
h+ leu1-32, ura4D-18, cdc25-22
GEF for Rho1p in S. pombe
Plasmid and DNA manipulations
The rgf3 open reading frame (ORF) was obtained from cosmid C645
in two pieces. First, we cloned a 2.5 kb SalI-HindIII fragment in
pALKS and then we introduced a 3.5 kb HindIII-HindIII fragment
adjacent in the cosmid to obtain pYS10 bearing the entire rgf3 ORF.
The pYS8 plasmid, containing the rgf1 ORF, was obtained inserting
a 7 kb EcoRI fragment from cosmid C645 into pAUKS. To tag Rgf3p
at its N-terminus with enhanced green fluorescent protein (EGFP) and
with the triple repeat of the influenza-virus haemagglutinin epitope
(HA) (Craven et al., 1998), pYS10 was modified by site-directed
mutagenesis. We created a SalI site at position –2 (before the ATG),
a NotI site at position +1 (after the ATG) and a SmaI site at the Cterminus (after the termination codon) (pVT1). The HA and EGFP
epitopes were inserted in frame at the NotI site of pVT1. pVTGFPrgf3 and pVT-HArgf3 fully complemented the ehs2-1
phenotypes. Strain VT128, with the GFP-rgf3+ integrated under its
own promoter, was constructed by subcloning the rgf3+ tagged with
EGFP (from plasmid pVT-GFPrgf3) into the integrative vector pIJ148
(Keeney and Boeke, 1994), resulting in pIJ148-GFP-rgf3+. This
plasmid was cut with Eco47III and integrated into the leu1 locus of
strain MS38. The nmt1-promoter-containing vectors pREP3X and
pREP41X (Forsburg, 1993) were used to overexpress rho1+ to rho5+,
cdc42+ and rgf3+. All GTPases of the Rho family were tagged with
two HA epitopes at the 5′ end (Calonge et al., 2003). The
overexpression plasmids were kindly provided by P. Perez and P. M.
Coll (Instituto de Microbiología Bioquímica, Salamanca, Spain). To
overexpress rgf3+, a SalI-SmaI fragment containing the rgf3+ gene
tagged with the HA epitope from plasmid pVT-HArgf3 was ligated
into the SalI-SmaI sites of plasmid pREP3X or the Xho1-SmaI sites
of pREP41X and pREP81X. For shut-off, a ura4+-81 nmt1-rgf3+
strain (VT88) was constructed using pVT-HArgf3 and one-step gene
replacement. A SalI fragment containing the rgf3+ promoter in
plasmid pVT-GFPrgf3 was substituted by another fragment
containing 5′ rgf3+ sequences, the ura4+ marker (cloned in SmaI), and
the 81nmt1 promoter (cloned in PstI-SalI). An ApaI fragment
containing the regulatory sequences and a 1.5 kb fragment from the
rgf3+ ORF (up to the ApaI site) was used to transform a haploid strain
(MS38). We selected for haploids in MM without thiamine and uracil,
and correct integration was analysed by the polymerase chain reaction
(PCR).
Construction of rgf3 null mutants
The rgf3::ura4+ disruption construct was obtained in a two-step
process. The 5′ non-coding region of the rgf3+ ORF [nucleotides (nt)
–1303 to –9] was obtained by PCR, inserting the SalI and HindIII sites
(one at each end), and was ligated into the same sites of the SK-ura4
vector to yield pYS52. The 3′ flanking region of the rgf3+ ORF (nt
+3769 to +5405) was obtained by PCR, inserting the BamHI and NotI
sites as above, and was cloned into the same sites of pYS52 to yield
pYS53. Disruption of rgf3+ was accomplished using the 4.7 kb
fragment from pYS53 cut with XhoI and NotI, and transforming the
MS75 diploid strain. Transformants were replica-plated five times
consecutively on YES medium in order to eliminate the cells that had
not integrated the construct. Then, correct integration was analysed
by PCR using the following oligonucleotides: IPCR-b (5′-CACCATGCCAAAAATTACACAAGATAGAAT-3′) in the ura4+ gene; R13e (5′-GGCAGGATTCACCGGATC-3′) downstream from nucleotide
–5405 and therefore external to the disruption cassette; GEF-s (5′CTCTCGTAGAGTCGCGTC-3′) and R15-i (5′-GGCCTTAGCTTGCCTTG-3′) in the rgf3+ gene. Correct integrations were also
confirmed by genomic Southern blotting. Tetrad analysis of the
heterozygous diploid disclosed two viable (ura–) and two unviable
spores, indicating that rgf3+ is essential for viability.
The rgf3+ gene was isolated from the ehs2-1 mutant by gap repair
(Orr-Weaver et al., 1991). Upstream and downstream flanking
sequences from rgf3+ were subcloned in pALKS. The plasmid was
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linearized with the 5′ and 3′ fragments at the ends and used to
transform the ehs2-1 haploid strain (GI 1). The gap in the plasmid was
repaired using the chromosomal sequences and the plasmids were
recovered from yeast. Transformants were replica plated five times
consecutively on YES medium, and those able to lose the plasmid
were selected. Based on the rgf3+ sequence, we designed
oligonucleotides 400 bp apart and sequenced the entire ORF of four
different clones. In all four clones, there was only one change
(cytosine to thymine at position +1834).
Two-hybrid analyses
We performed yeast two-hybrid assays essentially as described by
Durfee et al. (Durfee et al., 1993). We created a restriction fragment
carrying the entire rgf3+ ORF by site-directed mutagenesis,
introducing SmaI and SalI sites at the start and termination codon of
rgf3+, respectively. Then, the 3.8 kb fragment was fused in frame to
the GAL4 activation domain of pACT2. GTPases rho1+ to rho5+ and
cdc42+ were cloned into pAS2 (Coll et al., 2003) and were used as
bait against rgf3+ cloned in the pACT2 plasmid. The S. cerevisiae
Y190 strain, which carries the GAL4 recognition sequence and the
lacZ and HIS3 reporter genes, was transformed with different
combinations of plasmids. Expression of the HIS3 reporter gene was
examined by growth of the host on a –His plate containing 40 mM 3aminotriazole (3AT).
Pull-down assay for GTP-bound Rho proteins
The expression vector pGEX-C21RBD (rhotekin-binding domain)
(Reid et al., 1996) was used to transform Escherichia coli. The fusion
protein was produced according to the manufacturer’s instructions
and immobilized on glutathione/Sepharose-4B beads (Amersham).
After incubation, the beads were washed several times and the bound
proteins were analysed by sodium-dodecyl-sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and Coomassie staining.
The amount of GTP-bound Rho proteins was analysed using the
Rho-GTP pull-down assay modified from Ren et al. (Ren et al., 1999).
Briefly, wild-type, rgf3+-overexpressing and rgf3-mutant cells (ehs21) were transformed with either pREP3X-HArho1+ or pREP3XHArho2+ and grown for 18 hours in minimal medium without
thiamine. Extracts from 108 cells were obtained as described
previously (Arellano et al., 1997) using 500 µl lysis buffer (50 mM
Tris, pH 7.5, 20 mM NaCl, 0.5% NP-40, 10% glycerol, 0.1 mM
dithiothreitol, 1 mM NaF, 2 mM MgCl2, containing 100 µM paminophenyl methanesulfonyl fluoride, leupeptin and aprotinin). 100
µg glutathione-S-transferase/RBD (GST-RBD) fusion protein coupled
to glutathione-agarose beads was used to immunoprecipitate 1.5 mg
of the cell lysates. The extracts were incubated with GST-RBD beads
for 2 hours. The beads were washed with lysis buffer four times and
bound proteins were blotted against 1:2000 diluted 12CA5
monoclonal antibody (mAb) as primary antibody to detect HA-Rho1p
or HA-Rho2p. The total amount of HA-Rho1p or HA-Rho2p levels
were monitored in whole-cell extracts (10 µg total protein), which
were used directly for western blot and were developed with 12CA5
mAb. Immunodetection was accomplished using the ECL detection
kit (Amersham Biosciences).
Cell wall analyses
Enzyme preparations and GS assays were performed basically as
described previously (Martin et al., 2000). One unit of activity was
measured as the amount that catalyses the incorporation of 1 µmol
substrate (UDP/D-glucose) per minute at 30°C. For labelling and
fractionation of cell polysaccharides, exponentially growing cultures
of S. pombe cells were supplemented with [U-14C]glucose (1 µCi
ml–1) and incubated for an additional 4-6 hours at either 28°C or
37°C (depending on the experimental conditions assayed). Cells
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Journal of Cell Science 117 (25)
were harvested and total glucose incorporation was monitored by
measuring the radioactivity in trichloroacetic-acid-insoluble
material. Mechanical breakage of cells was performed using
prechilled glass beads added to the cells and lysis was achieved in
a Fast-Prep System (Bio 101; Savant), using six 15-second intervals
at speed 6. Cell walls were pelleted at 1000 g for 5 minutes and
washed three times with 5% NaCl and three times with 1 mM EDTA.
Aliquots (100 µl) of total wall were incubated with 100 U
Zymolyase 100T or Quantazyme (Quantum Biotechnologies) for 36
hours at 30°C. Aliquots without enzyme were included as a control.
The samples were centrifuged, and the supernatant and washed
pellet were counted separately. The supernatants from the
Zymolyase 100T reaction were considered to contain β-glucan plus
galactomannan and the pellet was considered to hold α-glucan. The
supernatants from the Quantazyme reaction were considered to
harbour β-glucan and the pellet was considered to hold α-glucan
plus galactomannan.
Microscopy techniques
The localization of EGFP-Rgf3p was visualized in living cells. For
Cfw staining, exponentially growing S. pombe cells were harvested,
washed once and resuspended in water with Cfw at a final
concentration 20 µg ml–1 for 5 minutes at room temperature. After
washing with water, cells were observed under a DMRXA microscope
(Leica, Wetzlar, Germany). Formaldehyde fixation was used before
visualization of F-actin using rhodamine-conjugated phalloidin as
described previously (Balasubramanian et al., 1997).
Results
Temperature-sensitive ehs2-1 mutant has a defect in GS
To identify the fission-yeast genes involved in glucan
biosynthesis, we searched for mutants hypersensitive to the
cell-wall inhibitors Cfw and Ech (Carnero et al., 2000). The
rationale behind this approach is that mutants with a weakened
cell wall are unable to withstand the additional disturbance
caused by these drugs and die at concentrations of the
antifungal agents that are not lethal for cells with a normal wall
(Klis, 1994).
The ehs2-1 mutant (for Ech hypersensitive) was unable to
grow at 1 µg ml–1 Ech or 0.1 mg ml–1 Cfw, whereas the wildtype strain was able to withstand concentrations of 7.5 µg ml–1
Ech and 1.5 mg ml–1 Cfw. In addition, the mutant cells showed
a lytic thermosensitive phenotype at 37°C, which was
suppressed when an osmotic stabilizer (1.2 M sorbitol) was
added to the medium (Fig. 1A). All these phenotypes cosegregated as a single Mendelian character in tetrad analysis,
and they were found to be recessive by diploid analysis (data
not shown). Some of the ehs2-1 mutant cells were lysed cells
and we found that lysis occurred mainly after cytokinesis. At
28°C, the proportion of lysis was less than 10% but, after 6
hours at 37°C, more than 60% of the cells showed that
phenotype. To examine the viability of the ehs2-1 mutants,
cells from cultures incubated at 28°C or 37°C, or at 37°C
supplemented with sorbitol were counted and plated in rich
medium at different times of growth. A rapid loss in viability
was observed in the cells growing at 37°C without osmotic
support (Fig. 1B). The hypersensitivity of the mutant cells to
cell-wall-specific drugs (Carnero et al., 2000) and the fact that
the lytic phenotype (observed at 37°C) could be suppressed by
an osmotic stabilizer suggest a defect in cell-wall architecture.
To test this possibility, GS activity was measured in ehs2-1 and
Fig. 1. Growth phenotypes of ehs2-1 mutant cells. (A) Morphology
of ehs2-1 mutant cells grown at different temperatures. Differential
interference-contrast micrographs of S. pombe wild-type (PN22) and
ehs2-1 (GI 1) grown in YES liquid medium at 28°C or 37°C for 6
hours in the presence or absence of 1.2 M sorbitol (S).
(B) Proportion of viable cells of the ehs2-1 mutant grown at different
temperatures for the times indicated (with or without 1.2 M sorbitol)
and plated on rich medium at 28°C.
Table 2. β-1,3-Glucan synthase activities from S. pombe
wild-type (PN22) and mutant (ehs2-1) strains
Temperature
28°C
37°C
Strain
Specific activity (%)
Wild type
ehs2-1
Wild type
ehs2-1
9.23±1.11 (100)
6.92±1.11 (75)
5.35±0.75 (100)
2.98±0.48 (56)
S. pombe wild-type (PN22) and mutant (ehs2-1) strains grown at 28°C and
37°C. The 37°C extracts were prepared from cells grown overnight at 28°C
and then for 2 hours at 37°C in rich medium. The strain-specific activity is
expressed as milliunits per mg protein. GTP was added to the assay. Values
are means±s.d. calculated from at least three independent experiments.
wild-type strains grown at 28°C and further incubated for 2
hours at either the permissive (28°C) or the restrictive
temperature (37°C). As shown in Table 2, the GS activity of
mutant cells after 2 hours at the restrictive temperature was
55%, compared with 100% in the wild-type strain. Even at
28°C, the GS activity in the mutant was diminished to 75%.
GEF for Rho1p in S. pombe
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Fig. 2. Complementation of the ehs2-1 thermosensitive and hypersensitive phenotypes by plasmids pAL-rgf3 (pYS10) and pAL-rgf1 (pYS8).
GI1 (h+ leu1-32, ehs2-1) cells were transformed with pAL-rgf3, pAL-rgf1 or pAL (empty plasmid). (A) Transformants were selected in MM
and the temperature-sensitive phenotype was scored by incubating the cultures for 4 hours at 37°C. Differential-interference-contrast images are
shown. (B) Transformants were streaked out on YES plates in the presence or absence of echinocandin (Ech) (1 µg ml–1) or Calcofluor White
(Cfw) (1 mg ml–1). Plates were incubated at 28°C for 4 days. (C) Schematic illustration of structural features analysed by the SMART program
(Letunic, 2002) (http://smart.embl-heidelberg.de/). Domains are indicated: CNH, citron homology domain (this acts as a regulatory domain and
could be involved in macromolecular interactions); DEP, domain of unknown function present in signalling proteins that contain PH, RasGEF,
RhoGEF, RhoGAP, RGS or PDZ domains; PH, pleckstrin-homology domain; RhoGEF, domain conserved among GEFs for Rho/Rac/Cdc42like GTPases. (D) Alignment of predicted amino acid sequence of ehs2-1 with the corresponding region of known GEF proteins from different
organisms (S. pombe Scd1, Caenorhabditis elegans unc-73, human Dbl, human Abr, human Bcr, mouse Vav and S. cerevisiae Cdc24). Multiple
sequence alignments were performed using the ClustalW program. The site of mutation is located within the RhoGEF domain in a highly
conserved region called CR3 and is marked with ‘611’ over the predicted amino-acid sequence of Ehs2-1p. Asterisks indicate identical amino
acids among all identified gene products. (.) and (:) indicate well-conserved and highly conserved amino acids, respectively.
Cloning of the ehs2-1 gene (rgf3)
In the process of cloning the ehs2-1 gene by complementation,
we isolated a plasmid from a S. pombe genomic library that
was able to suppress the hypersensitivity of ehs2-1 cells to Ech
and Cfw. Sequencing of the insert revealed that it contained the
bgs3+ gene, encoding one of the bgs family components in
S. pombe (Martin et al., 2003). The bgs3+ gene failed to
complement the lytic phenotype at 37°C of the ehs2-1 mutant,
in support of the notion that it was acting as a multicopy
suppressor (Martin et al., 2003). Accordingly, we used
positional cloning as an alternative method to clone the ehs2+
gene. The ehs2-1 mutation mapped very close to the ade6 gene.
Cosmids that spanned the region around the ade6 gene were
selected and screened for genes that could be related to cellwall biosynthesis. We first chose mok1+ (α-glucan synthase) in
cosmid C17A7, but found no complementation of the ehs2-1
phenotypes. Next, we tested two ORFs coding for proteins
containing Rho-GEF domains, rgf1+ and rgf3+, in cosmids
SPCC645.07C and SPCC645.06c, respectively. The name rgf
stands for RhoGEF (http://www.genedb.org/genedb/pombe/
index.jsp). The two ORFs are consecutive, with divergent
promoters. The rgf3+ gene completely rescued all phenotypes
of the ehs2-1 mutant, whereas rgf1+ partially complemented
the hypersensitivity to Cfw and Ech but did not rescue lysis at
37°C (Fig. 2A,B). To determine whether rgf3+ was the true
ehs2+ gene or whether it was acting as an extragenic multicopy
suppressor, we subcloned the rgf3+ ORF and flanking
sequences in the integrative vector pJK148 (Keeney and
Boeke, 1994). The construct was integrated into the genome of
a ehs2-1 mutant at the leu1 locus. The strain created behaved
like the wild type for hypersensitivity to antifungal drugs and
heat sensitivity, suggesting that rgf3+ is the structural gene that
complements the ehs2-1 mutation (data not shown).
The rgf3+ gene encodes a protein of 1275 amino acids with
a predicted molecular size of ~144 kDa. Structural analysis of
Rgf3p showed that it contains the putative Dbl homology (DH)
domain (amino acid residues 469-653) and a pleckstrin
homology (PH) domain (amino acids 693-855) adjacent to the
DH domain characteristic of most RhoGEFs (Fig. 2C) (for
reviews, see Zheng, 2001; Schmidt and Hall, 2002). There are
seven genes coding proteins with putative RhoGEF domains in
S. pombe – Scd1+ and gef1+ both encode GEFs for cdc42+ (Coll
et al., 2003; Hirota et al., 2003), and rgf1+, rgf2+, rgf3+, gef2+
and gef3+ have been shown to be involved in cell morphology
and the actin cytoskeleton (Iwaki et al., 2003). A comparison
of Rgf3p with Rgf1p and the S. cerevisiae Rom2p is shown in
Fig. 2C.
GEF domain is essential for Rgf3p function
We next examined which sort of mutation in the rgf3+ reading
frame was able to confer the ehs2-1 phenotype. The rgf3 ORF
6168
Journal of Cell Science 117 (25)
was rescued from the mutant strain GI 1 (ehs2-1) by gap repair,
and the sequences of four different clones were analysed. All
showed a cytosine-to-thymine change at position 1834. As a
control for the experiment, two different rescued ehs2-1 clones
were back-integrated into the leu1 locus of a ehs2-1 mutant
strain. We found that both integrants maintained the mutant
phenotype for Ech hypersensitivity and heat sensitivity (data
not shown). The mutation predicts that proline 611 (amino acid
numbering) in the wild-type Rgf3p is converted to a serine in
the mutant Rgf3p (ehs2-1). Proline 611 is located in the
RhoGEF domain that extends between amino acids 469 and
653, and is one of the few residues conserved in all the proteins
of the RhoGEF family in S. pombe as well as in DH domains
of other GEFs such as human Vav, Bcr, Dbl, Tiam1 and Unc73
(Fig. 2D) (Soisson et al., 1998). DH domains contain three
conserved blocks of sequences that have previously been
referred to as conserved regions 1-3 (CR1-CR3) (Boguski and
McCormick, 1993; Soisson et al., 1998). These three conserved
regions form three long helices (H1a, H2b and H8) that pack
together to form the core of the DH domain. Clustal alignment
of the DH domain of Rgf3p with DH domains of several GEFs
predicts that proline 611 is located on helix H8 (CR3), which
is the most highly conserved region of the DH domain and to
which many mutations that decrease nucleotide exchange
activity map (Soisson et al., 1998; Liu et al., 1998). This result
confirms that rgf3+ is the gene affected in the ehs2-1 mutant
and supports the hypothesis that Rgf3p may act as a GEF.
The rgf3+ gene is essential for cell viability and depletion
of Rgf3p leads to a lysis phenotype similar to the
depletion of Rho1p
Rgf3p displays limited homology to yeast Rom1p and Rom2p,
both GEFs of Rho1p in S. cerevisiae (Schmidt et al., 1997;
Ozaki et al., 1996). Moreover, a mutant in rgf3+ (ehs2-1) is
defective in cell-wall biosynthesis. We therefore attempted to
determine whether Rgf3p is a GEF for Rho1p in S. pombe. If
this were indeed the case then the rgf3∆ mutant would
presumably show phenotypes similar to those of the rho1∆
mutant. To investigate the phenotype resulting from complete
deletion of the rgf3+ gene, we constructed a diploid
strain of the genotype rgf3::ura4+/rgf3+ in which a
copy of rgf3+ had been deleted and replaced by ura4+.
Tetrad analysis revealed two viable and two nonviable
spores, and all the viable spores produced ura–e
colonies (Fig. 3A). The viability of the rgf3::ura4+
mutant spores was not rescued by the presence of 1.2
M sorbitol in the medium. Therefore, rgf3+ must be
essential for cell viability and must also be required
for germination. To further characterize the terminal
phenotype of the rgf3::ura4+ mutants, rgf3-null
Fig. 3. Rgf3p is essential for cell viability and depletion of
Rgf3p leads to a lysis phenotype similar to the depletion of
Rho1p. (A) Genomic organization of the rgf3+ and rgf1+
loci, and deletion strategy for rgf3+ disruption. The
direction of transcription is indicated by an arrow. Tetrads
from a rgf3::ura4+/rgf3+ strain dissected on YES medium
and incubated at 28°C for 4 days. (B) Terminal phenotype
of rgf3-null mutants. Spores prepared from the rgf3::ura4+
strain were inoculated in MM lacking uracil and
germinated for 18 hours. Cells were stained with
rhodamine-conjugated phalloidin and DAPI to visualize Factin and nuclei, respectively (top left) and with Calcofluor
White (Cfw) to visualize the cell-wall material (top right).
Spores with the wee1-50 rgf3∆ and sid2-250 rgf3∆ double
mutations (prepared from strains YSM654 and YSM656,
respectively) were inoculated in YES medium and
germinated for 14 hours at 25°C and then for 6 hours at
36°C. Cells were stained with Hoechst and Cfw (bottom).
(C) Lethal phenotype of the P81 nmt-rgf3 and P41 nmtrho1 shut-off mutants. Cells grown at 28°C in MM were
supplemented with thiamine to repress the nmt promoter.
Nomarsky micrographs were taken after 12 hours in MM
with or without thiamine. (D) Growth phenotypes of P81
nmt-rgf3 and P41 nmt-rho1mutants under different growth
conditions. Strains VT88 (81 nmt-rgf3+ + pREP81X) and
PPG217 (rho1∆ + pREP41X nmt-rho1+) were streaked
onto several plate media (YES, YES + Sorbitol and MMleu) and the plates were incubated for 3 days at 28°C. The
nmt promoter is off in rich medium (YES) and on in MM.
Strain VT88 carried pREP81X, an empty plasmid, to allow
cells to grow in MM-leu.
GEF for Rho1p in S. pombe
spores were germinated, fixed and stained to visualize F-actin,
nuclei and septa. Germinated rgf3::ura4+ spores were capable
of polarity establishment but appeared to be incapable of
finishing the division process, becoming spherical at one end
(Fig. 3B). Arrested cells showed two interphase nuclei and a
stable actomyosin ring, and most of them failed to assemble a
septum (Fig. 3B, Cfw-stained cells). This delay in cytokinesis
resembles what has been termed a ‘cytokinesis checkpoint’.
The cytokinesis checkpoint depends on a signalling pathway
called the septation initiation network (SIN) and the Wee1p
kinase (Simanis, 2003; Rajagopalan et al., 2003). We found
that, in both combinations of double mutants, sid2-250 rgf3∆
and wee1-50 rgf3∆, elongated cells with multiple nuclei and
multiple septa were seen frequently during spore germination
indicating a bypass of the checkpoint (Fig. 3B, bottom).
It has been shown previously that fission yeast rho1+ is an
essential gene and no conditional mutants are available
(Nakano et al., 1997). However, experiments in which the
Rho1p cellular pool was depleted ended with massive cell lysis
(shrinking cells) and actin depolymerization (Fig. 3C)
(Arellano et al., 1997). To investigate the lack of function of
Rgf3p during vegetative growth, we constructed a rgf3+ gene
under the control of the thiamine-regulatable and reducedexpression-rate nmt1 promoter P81nmt (Forsburg, 1993). This
construct was integrated into the genome of a wild-type
haploid strain (MS38), the endogenous rgf3+ promoter being
replaced by the P81nmt promoter. As shown in Fig. 3C, the
cells displayed a normal cell morphology when rgf3+ was
expressed in the absence of thiamine. 4 hours after the addition
of thiamine to repress rgf3+ expression, a large proportion of
cells had shrunk and, after 9 hours, the whole culture had lysed
(Fig. 3C). The phenotype of cells depleted for Rgf3p was very
similar to that observed in the ehs2-1 mutant at the restrictive
temperature (Fig. 1A) and in cells depleted for Rho1p (Fig.
3C). The same phenotype has also been reported (Nakano et
al., 1997) in cells expressing the dominant-negative mutant
Rho1T20N. We next examined whether the Rgf3p shut-off
phenotype could be rescued by osmotic support. As shown in
Fig. 3D, growth of the P81nmt-rgf3+ strain in rich medium
(promoter off) was dependent on the presence of 1.2 M
sorbitol, whereas Rho1p-depleted cells were unable to grow,
regardless of the presence or the absence of 1.2 M sorbitol
(Arellano et al., 1997). These results indicate that rgf3 mutant
phenotypes are very similar to those of the Rho1p-depleted
cells and suggest that Rgf3p and Rho1p function in the same
signal-transduction pathway. The fact that the Rgf3p switch off
could be rescued by sorbitol suggests that Rgf3p would control
a subset of the functions of Rho1p, probably those related to
cell-wall biosynthesis.
Hypersensitivity of the ehs2-1 mutant to cell-wall drugs
is suppressed by overexpression of rho1+ but not other
GTPases
If rgf3+ functions as an upstream regulator of rho1+,
overexpression of rho1+ would be expected to suppress the
hypersensitivity to Ech and Cfw as well as the temperaturesensitive growth phenotype of the ehs2-1 mutant. The GI 1
strain (ehs2-1, leu 1-32, h+) was transformed with plasmids
bearing rho1+, rho2+, rho3+, rho4+, rho5+ and cdc42+ under the
control of the nmt1 promoter or with an empty vector as a
6169
Fig. 4. Suppression of the echinocandin-hypersensensitive growth
phenotype of the ehs2-1 mutant by overexpression of rho1+. MS38
(rgf3+) was transformed with pREP3X (empty vector) and GI 1
(ehs2-1/rgf3) was transformed with pREP3X-rho1 (rho1+), pREP3Xrho2 (rho2+), pREP3X-rho3 (rho3+), pREP3X-rho4 (rho4+),
pREP3X-rho5 (rho5+), pREP3X-cdc42 (cdc42+) and pREP3X
(empty vector). Transformants were streaked onto MM, MM plus
thiamine, MM plus 1.5 µg ml–1 echinocandin and MM plus thiamine
and 1.5 µg ml–1 echinocandin plates, and incubated at 32°C for 4
days.
control. As shown in Fig. 4, the Ech hypersensitivity of the
ehs2-1 mutant was suppressed by rho1+ in minimal medium
without thiamine (promoter on). In medium with thiamine
(promoter off), no suppression was observed. The rho1+ gene
was also partially able to suppress the temperature-sensitive
phenotype and the hypersensitivity to Cfw (data not shown).
None of the other genes was able to suppress the phenotypes,
this being consistent with the idea that rgf3+ would act in the
same pathway as rho1+ (Fig. 4). Overexpression of rho2+ in
wild-type cells was lethal by itself, as well as in the ehs2-1
mutant background (Fig. 4). To avoid this problem, we used
rho2+ driven by the P41nmt promoter (medium level). This
construct produced viable cells. No complementation of the
hypersensitivity to Ech or Cfw was found either (data
not shown). Interestingly, overexpression of cdc42+ was
deleterious in a ehs2-1 background, whereas, in a wild-type
background, it was perfectly viable (Fig. 4) (Miller and
Johnson, 1994). It has recently been described that Rga8p, a
novel Rho1-GAP, is an effector of Cdc42p, providing a link
between the Cdc42p and Rho1p signalling pathways (Yang et
al., 2003).
Rgf3p specifically interacts with the GDP-bound form of
Rho1p and promotes GDP-GTP exchange
Using the yeast two-hybrid system, we investigated whether
Rgf3 interacts with Rho1 or any of the Rho-family proteins.
Plasmids for GTPases were kindly provided by P. Perez and
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Journal of Cell Science 117 (25)
Table 3. Two-hybrid analysis of the interactions between
different Rho GTPases (pAS2) and Rgf3p (pACT2) used
as bait
Gene (pAS2)*
Empty
rho1G15VC199S (GTP)
rho1T20NC199S (GDP)
rho2G17VC197S (GTP)
rho2T22NC197S (GDP)
rho3G24VC198S (GTP)
rho3T29NC198S (GDP)
rho4G15VC198S (GTP)
rho4T28NC198S (GDP)
rho5G15VC197S (GTP)
rho5T20NC197S (GDP)
cdc42G12V∆C (GTP)
cdc42T17N∆C (GDP)
rgf3 (pACT2)
Empty (pACT2)
+
+
+++
++
++
++
+
–
–
+
+++
–
+
–
+
+
++
++
++
+
–
–
+
+
–
+
* ‘GTP’ or ‘GDP’ indicates that this mutant emulates the GTP- or GDPbound form, respectively.
P. M. Coll. For each Rho protein, point mutations that trapped
the GTPase in the GTP-bound form (rho1-G15VC199S) or the
GDP-bound form (rho1-T20NC199S) fused to the DNAbinding domain were assayed. The entire ORF of rgf3+ was
fused to the transcriptional activation domain. The interaction
was examined by growth of the host on a –histidine plate
containing 40 mM 3-aminotriazole. As shown in Table 3 and
Fig. 5A, Rgf3p specifically interacted with the GDP-bound
form of Rho1p (rho1-T20NC199S) but not with GTP-bound
Fig. 5. Rgf3p is a specific Rho1-GEF. (A) Rgf3 binds directly to the
GDP-bound form of Rho1p (Rho1-T20N). Y190 cells expressing the
indicated proteins were cultured on a SD plate with histidine (left) or
without histidine plus 40 mM 3AT (right) at 30°C for 3 days. (B) The
Rgf3p level modulates the amount of GTP-bound Rho1p in vivo.
Wild-type (MS38) cells expressing pREP4X or pREP4X-rgf3, and
ehs2-1 (GI 1) mutant cells were transformed with either pREP3XHA-rho1 or pREP3X-HA-rho2. GTP-Rho1p or GTP-Rho2p were
pulled down from the cell extracts with GST-C21RBD and blotted
against 12CA5, anti-HA monoclonal antibody.
Rho1p (rho1-G15VC199S). There was also interaction with
Rho5p bound to GDP (rho5-TC199S) but not with any of the
other Rho proteins bound to GDP or GTP. The rho5+ gene is
a new member of the Rho family of unknown function and is
the closest homologue to rho1+.
To investigate further the possible role of Rgf3p as an Rho1
activator, we analysed the in vivo amount of GTP-bound Rho1p
in cells with different amounts of Rgf3p. Wild-type cells
carrying the control plasmid pREP4X, ehs2-1 mutant cells
(pREP4X) and wild-type cells overexpressing rgf3+ (carrying
pREP4X-rgf3+) were transformed with plasmid pREP3X-HArho1. After induction of the nmt1 promoter for 18 hours, the
amount of Rho1p bound to GTP was analysed by precipitation
with GST-C21RBD, the rhotekin-binding domain (which had
previously been obtained and purified from bacteria) and
blotting with anti-HA antibody (Fig. 5B). Western blots
of whole-cell extracts (10 µg protein) showed that the total
amount of Rho1p was similar in wild-type, mutant and cells
transformed with pREP3X-rgf3+ (Fig. 5B). The amount
of active Rho1p increased considerably in the strain
overexpressing rgf3+ compared with the control strain with
normal amounts of Rgf3p. No differences between the ehs2-1
mutant and the wild type were observed, possibly because under
the conditions assayed (32°C) the mutant phenotype was not as
strong as it was at 37°C. As a control, we also analysed the
amount of GTP-Rho2p in wild-type and ehs2-1 mutant cells and
in cells overexpressing rgf3+ (Fig. 5B). These cells were
transformed with the plasmid pREP3X-HA-rho2 and GTPbound Rho2p was pulled down from the extract by binding to
GST-C12RBD. No changes in the levels of Rho2p bound to
GTP were observed among the three strains (Fig. 5B). These
results indicate that Rgf3p acts as a specific Rho1p activator in
S. pombe.
Overexpression of Rgf3p interferes with septation and
increases cell-wall synthesis.
It has been shown that overexpression of rho1+ produces four
types of cell: swollen, branched, multiseptate and mixtures of
these phenotypes. It has also been reported that both the cell
wall and the septum are very thick in such cells (Arellano
et al., 1996; Nakano et al., 1997). We overproduced rgf3+
to determine whether the effect was similar to rho1+
overexpression or the overexpression of any of the other Rho
proteins. The rgf3+ gene was cloned under the thiaminerepressible nmt1 promoter in the pREP3X vector. After 20
hours of induction, overexpression of rgf3+ produced cells
containing multiple septa; the same phenotype has been
described before (Iwaki et al., 2003) (Fig. 6A). DAPI staining
revealed that, in most multiseptate cells, each compartment
contained one nucleus, indicative of a defect in cell separation
after septum assembly (not shown). Cfw mainly stains septa in
wild-type S. pombe. Cells overexpressing rgf3+ showed
a general increase in Cfw fluorescence, which was still
concentrated in the septum. Therefore, we analysed the
possible role of Rgf3p as an activator of cell-wall biosynthesis.
Because GS is one of the Rho1p effector proteins, we examined
the activity of the GS in cells that overexpressed Rgf3p. As
expected, an increase in enzymatic activity was detected in
cells overexpressing rgf3+ compared with the activity observed
in the wild-type strain (Fig. 6B). Consistently, rgf3 mutant cells
GEF for Rho1p in S. pombe
6171
We also analysed the cell-wall composition of cells that
overexpressed rgf3+, rho1+ or both. As shown in Fig. 6C, there
was an increase in the amount of β-glucan in cells that
overexpressed rgf3+ compared with wild-type cells (16% and
10%, respectively), and that increase was similar to that seen
in cells that overexpressed rho1+ (15%). There was also a
general increase in cell-wall biosynthesis in cells that
overexpresed rgf3+ compared with wild-type and rho1+ cells
(37%, 24.5% and 33%, respectively). In these cells, the ratio
between β- and α-glucan fractions was the same as that found
in the wild-type S. pombe cells, indicating a simultaneous
increase in both α- and β-glucan polymers. Additionally, the
amount of galactomannan was not significantly affected. Cells
that overexpressed rgf3+ and rho1+ at the same time did not
show any further increase in β-glucan biosynthesis with respect
to the overexpression of each gene separately. This could be
due to the limited amounts of other factors needed for cell-wall
assembly. These results suggested that Rgf3p was specifically
activating Rho1p, the GTPase that directly regulates the
biosynthesis of β-1,3-glucan and, through Pck2p, the
biosynthesis of the two main polymers α-1,3-glucan and β-1,3glucan.
Fig. 6. Phenotypes of Rgf3p overexpression. (A) Micrographs of
Calcofluor White (Cfw) stained wild-type cells transformed with
pREP3X (empty plasmid) or pREP3X-rgf3+ (rgf3+ overexpression)
grown without thiamine for 20 hours. (B) In vitro glucan synthase
(GS) activity assayed with the membrane fraction of wild-type cells
(MS38) transformed with pREP3X, pREP4X-rgf3 (rgf3+
overexpression), pREP3X-rho1 (rho1+ overexpression) or both
pREP4X-rgf3 and pREP3X-rho1 (rgf3+ and rho1+ overexpression).
Extracts were prepared from cells grown in MM without thiamine at
32°C for 18 hours. Specific activity is expressed as milliunits per mg
protein. Values are the means of at least three independent
experiments with duplicated samples, and error bars represent
standard deviations (s.d.). (C) Cell-wall composition in cells that
overexpress rgf3+. The relative levels of [14C]-glucose radioactivity
incorporated into each cell-wall polysaccharide are shown for the
same strains as above: wild-type (MS38) transformed with pREP3X,
pREP4X-rgf3 (rgf3+ overexpression), pREP3X-rho1 (rho1+
overexpression) or both at the same time. Cells were grown in the
absence of thiamine for 18 hours and then [14C]-glucose was added 6
hours before harvesting the cells. Values are the means of three
independent experiments with duplicate samples. Standard deviations
for total carbohydrate values are shown.
(ehs2-1) showed a severe reduction (50%) in GS enzymatic
activity (Table 2), indicating that changes in Rgf3p levels
caused changes in GS activity.
To corroborate these results, we also studied the activity in
cells that overexpressed rho1+ and rgf3+ at the same time
(transformed with the pREP3X-rho1 and pREP4X-rgf3
plasmids). As described previously (Arellano et al., 1996),
cells overexpressing rho1+ showed an increase in GS activity.
This increase was considerably (ten times) higher in cells that
overexpressed rgf3+ at the same time (Fig. 6B). These results
clearly indicate that Rgf3p is involved in the regulation of β1,3-glucan biosynthesis.
Rgf3p localizes to the septum
To gain further insight into the function of Rgf3p, we
determined its subcellular localization. We constructed a Rgf3EGFP fusion protein (at the 5′ end of the rgf3+ ORF) under the
control of the rgf3+ promoter (pVT-GFPrgf3). The GFP-Rgf3p
fusion was functional and restored the ability of the ehs2-1
mutant to grow in Ech and Cfw. Rgf3p localization was
examined in strains carrying a EGFP-rgf3+ gene integrated at
the leu1 locus of a wild-type strain. The staining pattern
observed was consistent with the localization of Rgf3p mainly
to the septum (Fig. 7A). EGFP-Rgf3p fluorescence appeared
in the medial region even before the septum was stained with
Cfw (see enlarged cells in Fig. 7A, bottom). This stage was
very transient. As the septum developed, the EGFP-Rgf3p
fluorescence extended further towards the centre of the cell
until it formed a band across the cell. This band was not
continuous (Fig. 7A), with dots of fluorescence being seen.
Finally, as cell separation began by digestion of the primary
septum, the EGFP-Rgf3p fluorescence began to disappear.
These observations indicate that Rgf3p is targeted to the
developing septum early in the septation process and persists
throughout cell separation. Some cells showed dots of green
fluorescence at one of the poles; this could reflect a small
amount of protein remaining there after cell separation. To test
the possibility that EGFP-Rgf3p concentrates at the cell ends
in interphase cells, we analysed the localization of the protein
in a cdc25-22 mutant strain carrying the pVT-GFPrgf3
plasmid. In cdc25-22 cells, which arrested in G2 phase at high
temperature with both ends growing, no Rgf3 fluorescence was
present at the poles. The cells were then released (at 25°C)
from the block (at 37°C) and the signal appeared in septating
cells (not shown). We also examined cells overexpressing
EGFP-rgf3 (from the nmt1 promoter) to see whether the fusion
was also localized to other weakly stained structures but we
failed to detect any other cellular area to which it was localized
(not shown).
Rgf3p was visualized only in cells with a developing
6172
Journal of Cell Science 117 (25)
septum. We therefore considered the
possibility that rgf3+ levels might be
regulated in a cell-cycle-dependent
manner. Thus, we determined the levels of
rgf3+ mRNA in a synchronous culture,
using a cdc25-22 strain. Cells were
synchronized as described above. We
found that rgf3+ mRNA levels were
sharply periodic, rising to a peak before
septation at 100 minutes, and decreasing
when most of the cells had a septum (Fig.
7B). Recently, a wide-ranging analysis of
cell-cycle periodic expression in S. pombe
has shown that rgf3+ mRNA is periodically
transcribed and its expression is dependent
on Ace2p, a transcription factor that also
controls other genes with predicted roles
in cell division (Rustici et al., 2004)
(http://www.sanger.ac.uk/). The results of
these experiments show that both the
localization of Rgf3p and the mRNA levels
fluctuate during the cell cycle, peaking
during septation.
Discussion
Yeast morphogenesis and cell growth
are coupled to the biosynthesis and
degradation of the cell wall. Therefore, all
these processes must be strictly controlled
by, and linked to, general signaltransduction pathways (Ishiguro, 1998;
Rajagopalan et al., 2003). Here, we used a
classical genetic approach to identify new
S. pombe genes involved in maintaining
cell-wall integrity. The rgf3+ gene was
cloned by complementation of the ehs2-1
mutant phenotypes. The ehs2-1 mutant
Fig. 7. Rgf3p localizes to the septum. (A) Wild-type cells were transformed with an
cells were hypersensitive to the cell-wall
integrative plasmid expressing EGFP-rgf3+ under its own promoter (strain VT128). Cells
inhibitors Ech and Cfw, and also displayed
were grown at 28°C. (Top) EGFP-Rgf3p localization in a population of living cells at
a thermosensitive lytic phenotype that
different stages of the cell cycle is shown in the centre. The corresponding differential
could be suppressed by an osmotic
interference contrast images are shown on the left and Calcofluor White (Cfw) staining is
stabilizer.
shown on the right. (Bottom) A square with cells that had not yet developed a visible
The predicted Rgf3p and Rgf1p (located
septum is enlarged. These cells already showed EGFP-Rgf3p dots of fluorescence. (B) The
adjacently in the chromosome) proteins
rgf3 mRNA levels were followed by northern-blot analyses in a cdc25-22 block-release
show a RhoGEF domain characteristic of
experiment. RNA samples were collected every 20 minutes along two consecutive cell
proteins that act as GDP-GTP exchange
cycles after release to 25°C. The blot was probed for rgf3 and for act1, the latter as an
factors for Rho GTPases (Zheng, 2001;
mRNA loading control.
Schmidt and Hall, 2002; Hoffman and
Cerione, 2002). Genetic and biochemical
evidence reported here support the notion that Rgf3p is a GEF
overexpression of rho1+ but not of any other of the Rho genes.
+
+
for Rho1p. Disruption of rgf3 and deletion of rho1 were
Interestingly, overexpression of cdc42+ was lethal in an ehs2+
unviable. Germinating spores with the rgf3::ura4 double
1 mutant background.
mutation arrested as single elongated cells with no visible
Our biochemical data strongly support the view that Rgf3p
septum, and lethality was not rescued in the presence of 1.2 M
acts as a specific positive regulator of Rho1p. The full-length
sorbitol. Rgf3p depletion in vegetative cells caused cell lysis,
Rgf3p interacted specifically with Rho1p in its GDP-bound
with a morphology very similar to those of cells devoid of
state but not with other Rho proteins (except for Rho5p), and
Rho1 or Pck1/2 activity. This suggests that the main function
a high level of Rgf3p increased the level of GTP-Rho1p in
of Rgf3p would be regulation of the Rho1p GTPase. Consistent
vivo. Moreover, the phenotype seen in the ehs2-1 mutant cells
with this idea, the hypersensitivity to Ech (Fig. 4) and
at the restrictive temperature (almost identical to the lack of
the temperature-sensitive phenotype were suppressed by
function of Rho1p) was due to a mutation located on helix H8
GEF for Rho1p in S. pombe
(CR3), which is the most highly conserved region of the DH
domain and to which many mutations that decrease nucleotide
exchange activity are mapped. Additionally, overexpression of
rgf3+ and rho1+ at the same time produces very refringent cells
with a phenotype similar to that of the constitutively active
allele Rho1G15V (Arellano et al., 1996) (data not shown).
The experiments reported in this study indicate that rgf3+ is
involved in the regulation of cell wall biosynthesis and cell
integrity. The rgf3 mutant cells (ehs2-1 mutant) were
hypersensitive to cell-wall antifungal drugs and showed a
temperature-sensitive lytic phenotype that could be rescued by
the presence of 1.2 M sorbitol. Cells with a mutation in the
rgf3 gene (ehs2-1) were defective in GS activity at 37°C and
cells that overexpressed rgf3+ showed a GS activity that was
twice that of wild-type cells and similar to the GS activity in
cells overexpressing Rho1p (Calonge et al., 2003).
Furthermore, cells overexpressing rgf3+ together with rho1+
showed a huge increase in GS activity (approximately sevento tenfold) compared with the wild-type level. Even without
GTP added to the reaction, the GS activity was seven times
higher than in the wild type, indicating that an excess of Rgf3p
had raised the intracellular pool of GTP-bound Rho1p (already
activated). Regarding cell-wall composition, an increase in the
amount of Rgf3p simultaneously increased the amount of αand β-glucan polymers. This is consistent with the notion that
Rho1p binds and activates Pck2p, which in turn activates the
synthesis of the two main structural polymers of the cell wall:
α- and β-glucans (Arellano et al., 1999; Calonge et al., 2000).
In previous work, we reported that the rgf3 mutation (ehs21 mutation) was suppressed by bgs3+, a putative β-1,3-GS
subunit. Multiple copies of bgs3+ complemented the
hypersensitivity to Ech and Cfw but not the temperaturesensitive phenotype (Martin et al., 2003). The cps1+/bgs1+ and
bgs2+ gene, which encode the other GS subunit homologues,
also suppressed the Ech hypersensitivity but not the Cfw or
temperature-sensitive phenotypes. These genes, which act
downstream from rgf3+, are key components in the final steps
of β-glucan biosynthesis, and their suppression provides
evidence that one of the main functions of rgf3+ is β-1,3-glucan
synthesis activation. The fact that a high dose of Mok1p (the
α-glucan synthase) was not able to complement any of the
ehs2-1 mutant phenotypes supports the hypothesis that β-1,3glucan is the most important polymer affected in the cell wall
of the rgf3 mutants.
Previous studies have shown that Rho1p depletion causes
cell death concomitant with a decrease in β-1,3-GS activity.
Lysis is not prevented by an osmotic stabilizer and occurs
mainly after cytokinesis (Arellano et al., 1997), probably
because correct cell-wall assembly is essential at that point of
the cell cycle. Here, we found that the cell-lysis phenotype
produced by Rgf3p depletion was prevented by 1.2 M sorbitol.
Furthermore, the protein localized to the septum region in the
early stages of cytokinesis and remained there until cell
separation. The model that we propose considers that Rgf3p
would activate Rho1p during cytokinesis, when cell-wall
integrity is compromised. It is possible that a spatial or
temporal localization of Rgf3p might be necessary for the local
production of an active (GTP-bound) form of Rho1p. In the
absence of Rgf3p, but in the presence of osmotic support,
Rho1p could be activated in some other ways.
GS activity must be strictly regulated in time and in
6173
synchrony with the cell cycle, and Rho1p might be the final
component of a GTPase cascade linking cell-cycle controls to
cell-wall biosynthesis. Our data support this hypothesis. The
rgf3+ mRNA levels peaked during septation and the protein
accumulated at the contractile ring. It is known that Bgs1p is
the GS involved in primary septum assembly and that mutants
in bgs1 can engage the cytokinetic checkpoint. Several mutants
in bgs1+ (cps1-12, drc1-191) arrest with two interphase nuclei
and a stable actomyosin ring (Le Goff et al., 1999; Liu et al.,
1999). We found that germinating spores lacking Rgf3p
showed a similar phenotype with stable actomyosin rings and
most of them lack a septum (Fig. 3B), suggesting that the lack
of Rgf3p activates the cytokinetic checkpoint. In fact, spores
from a double mutant such as wee1-50 ∆rgf3 or sid2-250 ∆rgf3
formed several nuclei and septa, suggesting that they can
bypass the septation checkpoint (Fig. 3B) (Simanis, 2003;
Rajagopalan et al., 2003). Identification of upstream regulators
of rgf3+ will be necessary to understand how Rho1p regulates
cell-wall integrity during cytokinesis in fission yeast.
We thank P. Perez, P. Coll and V. Martin for plasmids, strains and
all types of help throughout the work, and T. Toda and H. Schmidt
for strains. A. Durán, H. Valdivieso, J. C. Ribas and C. Roncero are
acknowledged for helpful discussions. V. Tajadura acknowledges
support from a fellowship granted by the MEC, Spain, and I. García
was supported by a fellowship from the Junta de Castilla y León. This
work was supported by grants BIO2001-1663 from the Comisión
Interministerial de Ciencia y Tecnología, Spain (CSI7/01) from the
Junta de Castilla y León.
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