Novel Rho GTPase involved in cytokinesis and cell wall integrity in the fission yeast Schizosaccharomyces pombe

The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in Schizosaccharomyces pombe is composed of beta-glucan, alpha-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-beta-D-glucan by activation of the 1,3-beta-D-glucan synthase, and Rho2 regulates the synthesis of alpha-glucan by the 1,3-alpha-D-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4. rho4Delta cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of rho4(+) causes lysis and morphological defects. Several lines of evidence indicate that both rho4(+) deletion or rho4(+) overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity. Rho4Delta cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.     

Role of septins and the exocyst complex in the function of hydrolytic enzymes responsible for fission yeast cell separation

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-beta-1,3-glucanase and the Agn1 endo-alpha-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70delta mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disk-like structure that spanned the septum, rather than in a ring. Even though septin and mid2delta mutants have a cell separation defect, the septum and the distribution of linear beta-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.     

A Highlights from MBoC Selection: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.     

Rho3p regulates cell separation by modulating exocyst function in Schizosaccharomyces pombe

Cytokinesis is the final stage of the cell division cycle in which the mother cell is physically divided into two daughters. In recent years the fission yeast Schizosaccharomyces pombe has emerged as an attractive model organism for the study of cytokinesis, since it divides using an actomyosin ring whose constriction is coordinated with the centripetal deposition of new membranes and a division septum. The final step of cytokinesis in S. pombe requires the digestion of the primary septum to liberate two daughters. We have previously shown that the multiprotein exocyst complex is essential for this process. Here we report the isolation of rho3(+), encoding a Rho family GTPase, as a high-copy suppressor of an exocyst mutant, sec8-1. Overproduction of Rho3p also suppressed the temperature-sensitive growth phenotype observed in cells lacking Exo70p, another conserved component of the S. pombe exocyst complex. Cells deleted for rho3 arrest at higher growth temperatures with two or more nuclei and uncleaved division septa between pairs of nuclei. rho3Delta cells accumulate approximately 100-nm vesicle-like structures. These phenotypes are all similar to those observed in exocyst component mutants, consistent with a role for Rho3p in modulation of exocyst function. Taken together, our results suggest the possibility that S. pombe Rho3p regulates cell separation by modulation of exocyst function.     

Candida albicans Rho-type GTPase-encoding genes required for polarized cell growth and cell separation

Rho proteins are essential regulators of morphogenesis in eukaryotic cells. In this report, we investigate the role of two previously uncharacterized Rho proteins, encoded by the Candida albicans RHO3 (CaRHO3) and CaCRL1/CaRHO4 genes. The CaRHO3 gene was found to contain one intron. Promoter shutdown experiments using a MET3 promoter-controlled RHO3 revealed a strong cell polarity defect and a partially depolarized actin cytoskeleton. Hyphal growth after promoter shutdown was abolished in rho3 mutants even in the presence of a constitutively active ras1(G13V) allele, and existing germ tubes became swollen. Deletion of C. albicans RHO4 indicated that it is a nonessential gene and that rho4 mutants were phenotypically different from rho3. Two distinct phenotypes of rho4 cells were elongated cell morphology and an unexpected cell separation defect generating chains of cells. Colony morphology of crl1/rho4 resulted in a growth-dependent smooth (long cell cycle length) or wrinkled (short cell cycle length) phenotype. This phenotype was additionally dependent on the rho4 cell separation defect and was also found in a Cacht3 chitinase mutant that shows a strong cytokinesis defect. The overexpression of the endoglucanase encoding the ENG1 gene, but not CHT3, suppressed the cell separation defect of crl1/rho4 but could not suppress the cell elongation phenotype. C. albicans Crl1/Rho4 and Bnr1 both localize to septal sites in yeast and hyphal cells but not to the hyphal tip. Deletion of RHO4 and BNR1 produced similar morphological phenotypes. Based on the localization of Rho4 and on the rho4 mutant phenotype, we propose a model in which Rho4p may function as a regulator of cell polarity, breaking the initial axis of polarity found during early bud growth to promote the construction of a septum.     

Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.     

The alpha-glucanase Agn1p is required for cell separation in Schizosaccharomyces pombe

BACKGROUND INFORMATION: In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast, ring constriction is followed by deposition of a multilayered division septum that must be cleaved to release the two daughter cells. Although many studies have focused on the actomyosin ring and septum assembly, little is known about the later steps involving the cleavage of the cell wall. RESULTS: We identified a novel gene in Schizosaccharomyces pombe, namely the agn1(+) gene that has homology to fungal 1,3-alpha-glucanases (mutanases). Disruption of the agn1(+) gene is not lethal to the cells, but does interfere with their separation, whereas overexpression of Agn1p is toxic and causes cell lysis. Agn1p levels reach a peak during septation and the protein localizes to the septum region before cell separation. Moreover, agn1(+) is responsible for the 1,3-alpha-glucanase activity, which shows a maximum at the end of septation. CONCLUSIONS: Our results clearly suggest the existence of a relationship between agn1(+), 1,3-alpha-glucanase activity and the completion of septation in S. pombe. Agn1p could be involved in the cleavage of the cylinder of the old wall that surrounds the primary septum, a region rich in alpha-glucans.     

Rga5p is a specific Rho1p GTPase-activating protein that regulates cell integrity in Schizosaccharomyces pombe

Schizosaccharomyces pombe Rho1p regulates (1,3)beta-d-glucan synthesis and is required for cell integrity maintenance and actin cytoskeleton organization, but nothing is known about the regulation of this protein. At least nine different S. pombe genes code for proteins predicted to act as Rho GTPase-activating proteins (GAPs). The results shown in this paper demonstrate that the protein encoded by the gene named rga5+ is a GAP specific for Rho1p. rga5+ overexpression is lethal and causes morphological alterations similar to those reported for Rho1p inactivation. rga5+ deletion is not lethal and causes a mild general increase in cell wall biosynthesis and morphological alterations when cells are grown at 37 degrees C. Upon mild overexpression, Rga5p localizes to growth areas and possesses both in vivo and in vitro GAP activity specific for Rho1p. Overexpression of rho1+ in rga5Delta cells is lethal, with a morphological phenotype resembling that of the overexpression of the constitutively active allele rho1G15V. In addition (1,3)beta-d-glucan synthase activity, regulated by Rho1p, is increased in rga5Delta cells and decreased in rga5-overexpressing cells. Moreover, the increase in (1,3)beta-d-glucan synthase activity caused by rho1+ overexpression is considerably higher in rga5Delta than in wild-type cells. Genetic interactions suggest that Rga5p is also important for the regulation of the other known Rho1p effectors, Pck1p and Pck2p.     

Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae

The Rho small GTP-binding protein family regulates various actomyosin- dependent cell functions, such as cell morphology, locomotion, cytokinesis, membrane ruffling, and smooth muscle contraction. In the yeast Saccharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore the function of the RHO1 gene, we isolated a recessive temperature-sensitive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino acid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strains bearing the rho1-104 mutation accumulated tiny- or small- budded cells in which cortical actin patches were clustered to buds at the restrictive temperature. Cell lysis and cell death were also seen with the rho1-104 mutant. Indirect immunofluorescence microscopic study demonstrated that Rho1p was concentrated to the periphery of the cells where cortical actin patches were clustered, including the site of bud emergence, the tip of the growing buds, and the mother-bud neck region of cells prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturable. A mutant Rho1p with an amino acid substitution at the lipid modification site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth region of cells, where Rho1p exerts its function in controlling cell growth.     

The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.

Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.     

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.     

Rgf1p is a specific Rho1-GEF that coordinates cell polarization with cell wall biogenesis in fission yeast

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1(+) deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1(+), but none of the other GTPases of the Rho-family, suppressed the rgf1Delta phenotypes. Moreover, deletion of rgf1(+) suppressed the severe growth defect in rga1(+) null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1(+) specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in beta(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (beta[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Delta mutants. Rgf1p localized to the growing ends and the septum, where Rho1, Pck1p, and Bgs4p are known to function. Our results suggest that Rgf1p probably activates the Rho functions necessary for coordinating actin deposition with cell wall biosynthesis during bipolar growth, allowing the cells to remodel their wall without risk of rupture.     

Fission yeast Rho5p GTPase is a functional paralogue of Rho1p that plays a role in survival of spores and stationary-phase cells

The Rho GTPase family and their effectors are key regulators involved in many eukaryotic cell functions related to actin organization and polarity establishment. Schizosaccharomyces pombe Rho1p is essential, directly activates the (1,3)-beta-d-glucan synthase, and participates in regulation of cell wall growth and morphogenesis. Here we describe the characterization of the fission yeast Rho5p GTPase, highly homologous to Rho1p, sharing 86% identity and 95% similarity. Overexpression of the hyperactive allele rho5-G15V causes a morphological effect similar to that of rho1-G15V, but the penetrance is significantly lower, and overexpression of the dominant-negative allele rho5-T20N causes lysis like that of rho1-T20N. Importantly, overexpression of rho5(+) but no other rho genes is able to rescue the lethality of rho1Delta cells. Shutoff experiments indicated that Rho5p can replace Rho1p, but it is not as effective in maintaining cell wall integrity or actin organization. rho5(+) expression is hardly detected during log-phase growth but is induced under nutritional starvation conditions. rho5Delta cells are viable and do not display any defects during logarithmic growth. However, when rho1(+) expression is repressed during stationary phase, rho5Delta cells display reduced viability. Ascospores lacking Rho5p are less resistant to heat or lytic enzymes than wild-type spores. Moreover, h(90) mutant strains carrying the hyperactive rho5-G15V or the dominant-negative rho5-T20N alleles display severe ascospore formation defects. These results suggest that Rho5p functions in a way similar to, but less efficient than, Rho1p, plays a nonessential role during stationary phase, and participates in the spore wall formation.     

Regulation of cell separation in the dimorphic fungus Ustilago maydis

During its haploid phase the dimorphic fungus Ustilago maydis grows vegetatively by budding. We have identified two genes, don1 and don3, which control the separation of mother and daughter cells. Mutant cells form tree-like clusters in liquid culture and grow as ring-like (donut-shaped) colonies on solid medium. In wild-type U. maydis cells, two distinct septa are formed during cytokinesis and delimit a fragmentation zone. Cells defective for either don1 or don3 display only a single septum and fail to complete cell separation. don1 encodes a guanine nucleotide exchange factor (GEF) of the Dbl family specific for Rho/Rac GTPases. Don3 belongs to the germinal-centre-kinase (GC) subfamily of Ste20-like protein kinases. We have isolated the U. maydis homologues of the small GTP binding proteins Rho2, Rho3, Rac1 and Cdc42. Out of these, only Cdc42 interacts specifically with Don1 and Don3 in the yeast two-hybrid system. We propose that Don1 and Don3 regulate the initiation of the secondary septum, which is required for proper cell separation.     

Yeast src homology region 3 domain-binding proteins involved in bud formation

The yeast protein Bem1p, which bears two src homology region 3 (SH3) domains, is involved in cell polarization. A Rho-type GTPase, Rho3p, is involved in the maintenance of cell polarity for bud formation, and the rho3 defect is suppressed by a high dose of BEM1. Mutational analysis revealed that the second SH3 domain from the NH2 terminus (SH3-2) of Bem1p is important for the functions of Bem1p in bud formation and in the suppression of the rho3 defect. Boi2p, which bound to SH3-2 Bem1p, was identified using the two-hybrid system. Boi2p has a proline-rich sequence that is critical for displaying the Boi2p-Bem1p two-hybrid interaction, an SH3 domain in its NH2-terminal half, and a pleckstrin homology domain in its COOH-terminal half. A BOI2 homologue, BOI1, was identified as a gene whose overexpression inhibited cell growth. Cells overexpressing either BOI1 or BOI2 were arrested as large, round, and unbudded cells, indicating that the Boi proteins affect cell polarization. Genetic analysis revealed that BOI1 and BOI2 are functionally redundant and important for cell growth. delta boi1 delta boi2 cells became large round cells or lysed with buds, displaying defects in bud formation and in the maintenance of cell polarity. Analysis using several truncated versions of BOI2 revealed that the COOH-terminal half, which contains the pleckstrin homology domain is essential for the function of Boi2p in cell growth, while the NH2- terminal half is not, and the NH2-terminal half might be required for modulating the function of Bem1p. Overproduction of either Rho3p or the Rho3p-related GTPase Rho4p suppressed the boi defect. These results demonstrate that Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation.     

Regulation of phosphoinositide levels by the phospholipid transfer protein Sec14p controls Cdc42p/p21-activated kinase-mediated cell cycle progression at cytokinesis

Sec14p is an essential phosphatidylcholine/phosphatidylinositol transfer protein with a well-described role in the regulation of Golgi apparatus-derived vesicular transport in yeast. Inactivation of the CDP-choline pathway for phosphatidylcholine synthesis allows cells to survive in the absence of Sec14p function through restoration of Golgi vesicular transport capability. In this study, Saccharomyces cerevisiae cells containing a SEC14 temperature-sensitive allele along with an inactivated CDP-choline pathway were transformed with a high-copy-number yeast genomic library. Genes whose increased expression inhibited cell growth in the absence of Sec14p function were identified. Increasing levels of the Rho GTPase Cdc42p and its direct effector kinases Cla4p and Ste20p prevented the growth of cells lacking Sec14p and CDP-choline pathway function. Growth suppression was accompanied by an increase in large and multiply budded cells. This effect on polarized cell growth did not appear to be due to an inability to establish cell polarity, since both the actin cytoskeleton and localization of the septin Cdc12p were unaffected by increased expression of Cdc42p, Cla4p, or Ste20p. Nuclei were present in both the mother cell and the emerging bud, consistent with Sec14p regulation of the cell cycle subsequent to anaphase but prior to cytokinesis/septum breakdown. Increased expression of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate 5-kinase prevented growth arrest by CDC42, CLA4, or STE20 upon inactivation of Sec14p function. Sec14p regulation of phosphoinositide levels affects cytokinesis at the level of the Cdc42p/Cla4p/Ste20p signaling cascade.     

Suppression of the profilin-deficient phenotype by the RHO2 signaling pathway in Saccharomyces cerevisiae

Profilin plays an important role in actin organization in all eukaryotic cells through mechanisms that are still poorly understood. We had previously shown that Mid2p, a transmembrane protein and a potential cell wall sensor, is an effective multicopy suppressor of the profilin-deficient phenotype in Saccharomyces cerevisiae. To better understand the role of Mid2p in the organization of the actin cytoskeleton, we isolated five additional multicopy suppressors of pfy1Delta cells that are Rom1p, Rom2p, Rho2p, Smy1p, and the previously uncharacterized protein Syp1p. The problems of caffeine and NaCl sensitivity, growth defects at 30 degrees and 37 degrees, the accumulation of intracellular vesicular structures, and a random budding pattern in pfy1Delta cells are corrected by all the suppressors tested. This is accompanied by a partial repolarization of the cortical actin patches without the formation of visible actin cables. The overexpression of Mid2p, Rom2p, and Syp1p, but not the overexpression of Rho2p and Smy1p, results in an abnormally thick cell wall in wild-type and pfy1Delta cells. Since none of the suppressors, except Rho2p, can correct the phenotype of the pfy1-111/rho2Delta strain, we propose a model in which the suppressors act through the Rho2p signaling pathway to repolarize cortical actin patches.