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 ehs2-1 mutant was suppressed by overexpression of rho1+ but 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-cycle-dependent 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.
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-1,3-glucan 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-1,3-glucan 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-1,3-glucan 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 GTP-binding 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-1,3-glucan synthase, via a Pck2p pathway (Hirata et al., 1998; Calonge et al., 2000). The rho3+ and rho4+ genes are non-essential 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/p21-activated kinase) substrate that negatively regulates Shk1p-dependent 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.
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 C-terminus (after the termination codon) (pVT1). The HA and EGFP epitopes were inserted in frame at the NotI site of pVT1. pVT-GFPrgf3 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; R13-e (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 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).
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 3-aminotriazole (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 (ehs2-1) were transformed with either pREP3X-HArho1+ or pREP3X-HArho2+ 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 p-aminophenyl 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 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.
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).
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 wild-type 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 co-segregated 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 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%.
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 cell-wall 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 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 rgf3Δ mutant would presumably show phenotypes similar to those of the rho1Δ 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 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 rgf3Δ and wee1-50 rgf3Δ, 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 reduced-expression-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 temperature-sensitive 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 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 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 DNA-binding 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 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-HA-rho1. 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 GTP-bound 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 (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 β-1,3-glucan biosynthesis.
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-1,3-glucan.
Rgf3p localizes to the septum
To gain further insight into the function of Rgf3p, we determined its subcellular localization. We constructed a Rgf3-EGFP 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 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.
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 signal-transduction 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 cells were hypersensitive to the cell-wall inhibitors Ech and Cfw, and also displayed a thermosensitive lytic phenotype that could be suppressed by an osmotic stabilizer.
The predicted Rgf3p and Rgf1p (located adjacently in the chromosome) proteins show a RhoGEF domain characteristic of proteins that act as GDP-GTP exchange factors for Rho GTPases (Zheng, 2001; Schmidt and Hall, 2002; Hoffman and Cerione, 2002). Genetic and biochemical evidence reported here support the notion that Rgf3p is a GEF for Rho1p. Disruption of rgf3+ and deletion of rho1+ were unviable. Germinating spores with the rgf3::ura4+ double mutation arrested as single elongated cells with no visible septum, and lethality was not rescued in the presence of 1.2 M sorbitol. Rgf3p depletion in vegetative cells caused cell lysis, with a morphology very similar to those of cells devoid of Rho1 or Pck1/2 activity. This suggests that the main function of Rgf3p would be regulation of the Rho1p GTPase. Consistent with this idea, the hypersensitivity to Ech (Fig. 4) and the temperature-sensitive phenotype were suppressed by overexpression of rho1+ but not of any other of the Rho genes. Interestingly, overexpression of cdc42+ was lethal in an ehs2-1 mutant background.
Our biochemical data strongly support the view that Rgf3p acts as a specific positive regulator of Rho1p. The full-length Rgf3p interacted specifically with Rho1p in its GDP-bound state but not with other Rho proteins (except for Rho5p), and a high level of Rgf3p increased the level of GTP-Rho1p in vivo. Moreover, the phenotype seen in the ehs2-1 mutant cells at the restrictive temperature (almost identical to the lack of function of Rho1p) was due to a mutation 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 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 seven- to 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 α- and 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 (ehs2-1 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 temperature-sensitive 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-1,3-glucan 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 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 Δrgf3 or sid2-250 Δrgf3 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.
- Accepted September 10, 2004.
- © The Company of Biologists Limited