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.     

The novel Rho GTPase-activating protein family protein, Rga8, provides a potential link between Cdc42/p21-activated kinase and Rho signaling pathways in the fission yeast, Schizosaccharomyces pombe

The PAK family kinase, Shk1, is an essential regulator of polarized growth in the fission yeast, Schizosaccharomyces pombe. Here we describe the characterization of a novel member of the RhoGAP family, Rga8, identified from a two-hybrid screen for proteins that interact with the Shk1 kinase domain. Although deletion of the rga8 gene in wild type S. pombe cells results in no obvious phenotypic defects under normal growth conditions, it partially suppresses the cold-sensitive growth and morphological defects of S. pombe cells carrying a hypomorphic allele of the shk1 gene. By contrast, overexpression of rga8 is lethal to shk1-defective cells and causes morphological and cytokinesis defects in wild type S. pombe cells. Consistent with a role for Rga8 as a downstream target of Shk1, we show that the Rga8 protein is directly phosphorylated by Shk1 in vitro and phosphorylated in a Shk1-dependent fashion in S. pombe cells. Fluorescence photomicroscopy of the GFP-Rga8 fusion protein indicates that Rga8 is localized to the cell ends during interphase and to the septum-forming region during cytokinesis. In S. pombe cells carrying the orb2-34 allele of shk1, Rga8 exhibits a monopolar pattern of localization, providing evidence that Shk1 contributes to the regulation of Rga8 localization. Although molecular analyses suggest that Rga8 functions as a GAP for the S. pombe Rho1 GTPase, genetic experiments suggest that Rga8 and Rho1 have a positive functional interaction and that gain of Rho1 function, like gain of Rga8 function, is lethal to Shk1-defective cells. Our results suggest that Rga8 is a Shk1 substrate that negatively regulates Shk1-dependent growth control pathway(s) in S. pombe, potentially through interaction with the Rho1 GTPase.     

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 GTPase-Activating Protein Rga1 Interacts with Rho3 GTPase and May Regulate Its Function in Polarized Growth in Budding Yeast

In budding yeast, Rga1 negatively regulates the Rho GTPase Cdc42 by acting as a GTPase-activating protein (GAP) for Cdc42. To gain insight into the function and regulation of Rga1, we overexpressed Rga1 and an N-terminally truncated Rga1-C538 (a.a. 538-1007) segment. Overexpression of Rga1-C538 but not full-length Rga1 severely impaired growth and cell morphology in wild-type cells. We show that Rga1 is phosphorylated during the cell cycle. The lack of phenotype for full-length Rga1 upon overexpression may result from a negative regulation by G1-specific Pho85, a cyclin-dependent kinase (CDK). From a high-copy suppressor screen, we isolated RHO3, SEC9, SEC1, SSO1, SSO2, and SRO7, genes involved in exocytosis, as suppressors of the growth defect caused by Rga1-C538 overexpression. Moreover, we detected that Rga1 interacts with Rho3 in two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Rga1 preferentially interacts with the GTP-bound form of Rho3 and the interaction requires the GAP domain and additional sequence upstream of the GAP domain. Our data suggest that the interaction of Rga1 with Rho3 may regulate Rho3’s function in polarized bud growth.     

Cdc42 GTPase-activating proteins (GAPs) regulate generational inheritance of cell polarity and cell shape in fission yeast

The highly conserved small GTPase Cdc42 regulates polarized cell growth and morphogenesis from yeast to humans. We previously reported that Cdc42 activation exhibits oscillatory dynamics at cell tips of Schizosaccharomyces pombe cells. Mathematical modeling suggests that this dynamic behavior enables a variety of symmetric and asymmetric Cdc42 activation distributions to coexist in cell populations. For individual wild-type cells, however, Cdc42 distribution is initially asymmetrical and becomes more symmetrical as cell volume increases, enabling bipolar growth activation. To explore whether different patterns of Cdc42 activation are possible in vivo, we examined S. pombe rga4∆ mutant cells, lacking the Cdc42 GTPase-activating protein (GAP) Rga4. We found that monopolar rga4∆ mother cells divide asymmetrically leading to the emergence of both symmetric and asymmetric Cdc42 distributions in rga4∆ daughter cells. Motivated by different hypotheses that can mathematically reproduce the unequal fate of daughter cells, we used genetic screening to identify mutants that alter the rga4∆ phenotype. We found that the unequal distribution of active Cdc42 GTPase is consistent with an unequal inheritance of another Cdc42 GAP, Rga6, in the two daughter cells. Our findings highlight the crucial role of Cdc42 GAP localization in maintaining consistent Cdc42 activation and growth patterns across generations.     

Regulation of cell diameter, For3p localization, and cell symmetry by fission yeast Rho-GAP Rga4p

Control of cellular dimensions and cell symmetry are critical for development and differentiation. Here we provide evidence that the putative Rho-GAP Rga4p of Schizosaccharomyces pombe controls cellular dimensions. rga4 Delta cells are wider in diameter and shorter in length, whereas Rga4p overexpression leads to reduced diameter of the growing cell tip. Consistent with a negative role in cell growth control, Rga4p protein localizes to the cell sides in a "corset" pattern, and to the nongrowing cell tips. Additionally, rga4 Delta cells show an altered growth pattern similar to that observed in mutants of the formin homology protein For3p. Consistent with these observations, Rga4p is required for normal localization of For3p and for normal distribution of the actin cytoskeleton. We show that different domains of the Rga4p protein mediate diverse morphological functions. The C-terminal GAP domain mediates For3p localization to the cell tips and maintains cell diameter. Conversely, overexpression of the N-terminal LIM homology domain of Rga4p promotes actin cable formation in a For3p-dependent manner. Our studies indicate that Rga4p functionally interacts with For3p and has a novel function in the control of cell diameter and cell growth.     

Spatial control of Cdc42 activation determines cell width in fission yeast

The fission yeast Schizosaccharomyces pombe is a rod-shaped cell that grows by linear extension at the cell tips, with a nearly constant width throughout the cell cycle. This simple geometry makes it an ideal system for studying the control of cellular dimensions. In this study, we carried out a near-genome-wide screen for mutants wider than wild-type cells. We found 11 deletion mutants that were wider; seven of the deleted genes are implicated in the control of the small GTPase Cdc42, including the Cdc42 guanine nucleotide exchange factor (GEF) Scd1 and the Cdc42 GTPase-activating protein (GAP) Rga4. Deletions of rga4 and scd1 had additive effects on cell width, and the proteins localized independently of one another, with Rga4 located at the cell sides and Scd1 at the cell tips. Activated Cdc42 localization is altered in rga4Δ, scd1Δ, and scd2Δ mutants. Delocalization and ectopic retargeting experiments showed that the localizations of Rga4 and Scd1 are crucial for their roles in determining cell width. We propose that the GAP Rga4 and the GEF Scd1 establish a gradient of activated Cdc42 within the cellular tip plasma membrane, and it is this gradient that determines cell growth-zone size and normal cell width.     

GTPase-activating proteins for Cdc42

The Rho-type GTPase, Cdc42, has been implicated in a variety of functions in the yeast life cycle, including septin organization for cytokinesis, pheromone response, and haploid invasive growth. A group of proteins called GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, thereby inactivating Cdc42. At the time this study began, there was one known GAP, Bem3, and one putative GAP, Rga1, for Cdc42. We identified another putative GAP for Cdc42 and named it Rga2 (Rho GTPase-activating protein 2). We confirmed by genetic and biochemical criteria that Rga1, Rga2, and Bem3 act as GAPs for Cdc42. A detailed characterization of Rga1, Rga2, and Bem3 suggested that they regulate different subsets of Cdc42 function. In particular, deletion of the individual GAPs conferred different phenotypes. For example, deletion of RGA1, but not RGA2 or BEM3, caused hyperinvasive growth. Furthermore, overproduction or loss of Rga1 and Rga2, but not Bem3, affected the two-hybrid interaction of Cdc42 with Ste20, a p21-activated kinase (PAK) kinase required for haploid invasive growth. These results suggest Rga1, and possibly Rga2, facilitate the interaction of Cdc42 with Ste20 to mediate signaling in the haploid invasive growth pathway. Deletion of BEM3 resulted in cells with severe morphological defects not observed in rga1Δ or rga2Δ strains. These data suggest that Bem3 and, to a lesser extent, Rga1 and Rga2 facilitate the role of Cdc42 in septin organization. Thus, it appears that the GAPs play a role in modulating specific aspects of Cdc42 function. Alternatively, the different phenotypes could reflect quantitative rather than qualitative differences in GAP activity in the mutant strains.     

A novel connection between the yeast Cdc42 GTPase and the Slt2-mediated cell integrity pathway identified through the effect of secreted Salmonella GTPase modulators

Modulation of host cellular GTPases through the injection of the effector proteins SopE2 and SptP is essential for Salmonella typhimurium to enter into non-phagocytic cells. Here we show that expression of the guanine nucleotide exchange factor for Cdc42 SopE2 in Saccharomyces cerevisiae leads to the activation of Fus3 and Kss1 MAPKs, which operate in the mating and filamentation pathways, causing filamentous growth in haploid yeast cells. Furthermore, it promotes the activation of the cell integrity MAPK Slt2. Cdc42 activation by removal of its putative intrinsic GTPase-activating proteins (GAPs), Rga1, Rga2, and Bem3, also results in the phosphorylation of Kss1, Fus3, and Slt2 MAPKs. These data support the role of these GAP proteins as negative regulators of Cdc42, confirm the modulating effect of this GTPase on the filamentation and mating pathways and point to a novel connection between Cdc42 and the cell integrity pathway. Cdc42-induced activation of Slt2 occurs in a mating and filamentation pathway-dependent manner, but it does not require the function of Rho1, which is the GTPase that operates in the cell integrity pathway. Moreover, we report that Salmonella SptP can act as a GAP for Cdc42 in S. cerevisiae, down-regulating MAPK-mediated signaling. Thus, yeast provides a useful system to study the interaction of bacterial pathogenic proteins with eukaryotic signaling pathways. Furthermore, these proteins can be used as a tool to gain insight into the mechanisms that regulate MAPK-mediated signaling in eukaryotes.     

Up-regulation of the Cdc42 GTPase limits the replicative life span of budding yeast

Cdc42, a conserved Rho GTPase, plays a central role in polarity establishment in yeast and animals. Cell polarity is critical for asymmetric cell division, and asymmetric cell division underlies replicative aging of budding yeast. Yet how Cdc42 and other polarity factors impact life span is largely unknown. Here we show by live-cell imaging that the active Cdc42 level is sporadically elevated in wild type during repeated cell divisions but rarely in the long-lived bud8 deletion cells. We find a novel Bud8 localization with cytokinesis remnants, which also recruit Rga1, a Cdc42 GTPase activating protein. Genetic analyses and live-cell imaging suggest that Rga1 and Bud8 oppositely impact life span likely by modulating active Cdc42 levels. An rga1 mutant, which has a shorter life span, dies at the unbudded state with a defect in polarity establishment. Remarkably, Cdc42 accumulates in old cells, and its mild overexpression accelerates aging with frequent symmetric cell divisions, despite no harmful effects on young cells. Our findings implicate that the interplay among these positive and negative polarity factors limits the life span of budding yeast.     

A Highlights from MBoC Selection: Rga6 is a fission yeast Rho GAP involved in Cdc42 regulation of polarized growth

Active Cdc42 is essential for the establishment of polarized growth. This GTPase is negatively regulated by the GTPase-activating proteins (GAPs), which are important for the spatial specificity of Cdc42 function. Rga4 is the only GAP described as negative regulator of fission yeast Cdc42. We report here that Rga6, another fission yeast Cdc42 GAP, shares some functions with Rga4. Cells lacking Rga6 are viable but slightly shorter and broader than wild type, and cells lacking Rga6 and Rga4 simultaneously are rounded. In these cells, active Cdc42 is observed all around the membrane. These additive effects indicate that both GAPs collaborate in the spatial regulation of active Cdc42. Rga6 localizes to the plasma membrane, forming clusters different from those formed by Rga4. A polybasic region at the Rga6 C-terminus is responsible for its membrane localization. Rga6-GFP fluorescence decreases considerably at the growing tips, and this decrease is dependent on the actin cables. Of note, in the absence of Rga6, the amplitude of active Cdc42 oscillations at the tips decreases, and less GTP-Cdc42 accumulates at the new end of the cells. We propose that Rga6 collaborates with Rga4 to spatially restrict active Cdc42 at the cell tips and maintain cell dimensions.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Pom1 DYRK regulates localization of the Rga4 GAP to ensure bipolar activation of Cdc42 in fission yeast

BACKGROUND: In the fission yeast Schizosaccharomyces pombe, cell growth takes place exclusively at both ends of the cylindrical cell. During this highly polarized growth, microtubules are responsible for the placement of the cell-end marker proteins, the Tea1-Tea4/Wsh3 complex, which recruits the Pom1 DYRK-family protein kinase. Pom1 is required for proper positioning of growth sites, and the Deltapom1 mutation brings about monopolar cell growth. RESULTS: Pom1 kinase physically interacts with Rga4, which has a GAP (GTPase-activating protein) domain for Rho-family GTPase. Genetic and biochemical evidence indicates that Rga4 functions as GAP for the Cdc42 GTPase, an evolutionarily conserved regulator of F-actin. CRIB (Cdc42/Rac interactive binding)-GFP microscopy has revealed that GTP-bound, active Cdc42 is concentrated to growing cell ends accompanied by developed F-actin structures, where the Rga4 GAP is excluded. The monopolar Deltapom1 mutant fails to eliminate Rga4 from the nongrowing cell end, resulting in monopolar distribution of GTP-Cdc42 to the growing cell end. However, mutational inactivation of Rga4 allows Cdc42 to be active at both ends of Deltapom1 cells, suggesting that mislocalization of Rga4 in the Deltapom1 mutant contributes to its monopolar phenotype. CONCLUSIONS: Pom1 kinase recruited to cell ends by the Tea1-Tea4/Wsh3 complex is essential for proper localization of a GAP for Cdc42, Rga4, which ensures bipolar localization of GTP-bound, active Cdc42. Because of the established role of Cdc42 in F-actin formation, these observations provide a new insight into how the microtubule system achieves localized formation of F-actin to generate cell polarity.     

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.     

Zds1/Zds2–PP2ACdc55 complex specifies signaling output from Rho1 GTPase

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1–mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2–protein phosphatase 2A^Cdc55 (PP2A^Cdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2–PP2A^Cdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1–Mpk1 activity inhibits cortical PP2A^Cdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2A^Cdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1–PP2A^Cdc55 and Rho1–Pkc1 explains how only one signaling pathway is robustly activated at a time.     

Phosphatidylinositol-4-phosphate 5-kinase regulates fission yeast cell integrity through a phospholipase C-mediated protein kinase C-independent pathway

Fission yeast its3-1 mutant is an allele of the essential gene its3+ that encodes a phosphatidylinositol-4-phosphate 5-kinase (PIP5K) that produces phosphatidylinositol 4,5-bisphosphate. We found that the its3-1 mutant is sensitive to micafungin, a (1,3)-beta-D-glucan synthase inhibitor, suggesting a cell wall integrity defect. Consistently, its3-1 mutation caused synthetic lethality with a (1,3)-beta-D-glucan synthase mutant, bgs1-i2, and its3-1 mutant cells showed aberrant localization of green fluorescent protein-Bgs1. Similar aberrant localization of green fluorescent protein-tagged Rgf1, a putative phosphatidylinositol 4,5-bisphosphate-binding guanine nucleotide exchange factor for Rho protein, in its3-1 mutants was observed, suggesting a defective Rgf1/Rho pathway. To unravel the molecular mechanism(s), putative downstream components of PIP5K signaling were analyzed. Unexpectedly, overexpression of phospholipase C (Plc1), but not that of protein kinase C (PKC; Pck1 and Pck2), suppressed the phenotypes of the its3-1 mutant. These findings indicate that PKCs are not involved in the suppression, and further analysis revealed that PKCs are not downstream of Plc1 in fission yeast. Also, the enzymatic activity of Plc1 is essential for the suppression of the phenotypes and for the viability of the its3-1 mutant. These findings suggest that Its3 PIP5K regulates cell integrity through a Plc1-mediated PKC-independent pathway, in addition to the Rho/PKC pathway.     

E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens

In simple polarized epithelial cells, the Rho GTPase commonly localizes to E-cadherin-based cell–cell junctions, such as the zonula adherens (ZA), where it regulates the actomyosin cytoskeleton to support junctional integrity and tension. An important question is how E-cadherin contributes to Rho signaling, notably whether junctional Rho may depend on cadherin adhesion. We sought to investigate this by assessing Rho localization and activity in epithelial monolayers depleted of E-cadherin by RNAi. We report that E-cadherin depletion reduced both Rho and Rho-GTP at the ZA, an effect that was rescued by expressing a RNAi-resistant full-length E-cadherin transgene. This impact on Rho signaling was accompanied by reduced junctional localization of the Rho GEF ECT2 and the centralspindlin complex that recruits ECT2. Further, the Rho signaling pathway contributes to the selective stabilization of E-cadherin molecules in the apical zone of the cells compared with E-cadherin at the lateral surface, thereby creating a more defined and restricted pool of E-cadherin that forms the ZA. Thus, E-cadherin and Rho signaling cooperate to ensure proper ZA architecture and function.     

Activation of the Cdc42p GTPase by cyclin-dependent protein kinases in budding yeast

Cyclin-dependent kinases (CDKs) trigger essential cell cycle processes including critical events in G1 phase that culminate in bud emergence, spindle pole body duplication, and DNA replication. Localized activation of the Rho-type GTPase Cdc42p is crucial for establishment of cell polarity during G1, but CDK targets that link the Cdc42p module with cell growth and cell cycle commitment have remained largely elusive. Here, we identify the GTPase-activating protein (GAP) Rga2p as an important substrate related to the cell polarity function of G1 CDKs. Overexpression of RGA2 in the absence of functional Pho85p or Cdc28p CDK complexes is toxic, due to an inability to polarize growth. Mutation of CDK consensus sites in Rga2p that are phosphorylated both in vivo and in vitro by Pho85p and Cdc28p CDKs results in a loss of G1 phase-specific phosphorylation. A failure to phosphorylate Rga2p leads to defects in localization and impaired polarized growth, in a manner dependent on Rga2p GAP function. Taken together, our data suggest that CDK-dependent phosphorylation restrains Rga2p activity to ensure appropriate activation of Cdc42p during cell polarity establishment. Inhibition of GAPs by CDK phosphorylation may be a general mechanism to promote proper G1-phase progression.     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.     

RNA-binding protein Khd1 and Ccr4 deadenylase play overlapping roles in the cell wall integrity pathway in Saccharomyces cerevisiae

The Saccharomyces cerevisiae RNA-binding protein Khd1/Hek2 associates with hundreds of potential mRNA targets preferentially, including the mRNAs encoding proteins localized to the cell wall and plasma membrane. We have previously revealed that Khd1 positively regulates expression of MTL1 mRNA encoding a membrane sensor in the cell wall integrity (CWI) pathway. However, a khd1Δ mutation has no detectable phenotype on cell wall synthesis. Here we show that the khd1Δ mutation causes a severe cell lysis when combined with the deletion of the CCR4 gene encoding a cytoplasmic deadenylase. We identified the ROM2 mRNA, encoding a guanine nucleotide exchange factor (GEF) for Rho1, as a target for Khd1 and Ccr4. The ROM2 mRNA level was decreased in the khd1Δ ccr4Δ mutant, and ROM2 overexpression suppressed the cell lysis of the khd1Δ ccr4Δ mutant. We also found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding a GTPase-activating protein (GAP) for Rho1. The LRG1 mRNA level was increased in the ccr4Δ and khd1Δ ccr4Δ mutants, and deletion of LRG1 suppressed the cell lysis of the khd1Δ ccr4Δ mutant. Our results presented here suggest that Khd1 and Ccr4 modulate a signal from Rho1 in the CWI pathway by regulating the expression of RhoGEF and RhoGAP.