Phosphorylation of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candida albicans hyphal growth

Cyclin-dependent kinases (CDKs) control yeast morphogenesis, although how they regulate the polarity machinery remains unclear. The dimorphic fungus Candida albicans uses Cdc28/Hgc1, a CDK/cyclin complex, to promote persistent actin polarization for hyphal growth. Here, we report that Rga2, a GTPase-activating protein (GAP) of the central polarity regulator Cdc42, undergoes Hgc1-dependent hyperphosphorylation. Using the analog-sensitive Cdc28as mutant, we confirmed that Cdc28 controls Rga2 phosphorylation in vitro and in vivo. Deleting RGA2 produced elongated yeast cells without apparent effect on hyphal morphogenesis. However, deleting it or inactivating its GAP activity restored hyphal growth in hgc1Delta mutants, suggesting that Rga2 represses hyphal development and Cdc28/Hgc1 inactivates it upon hyphal induction. We provide evidence that Cdc28/Hgc1 may act to prevent Rga2 from localizing to hyphal tips, leading to localized Cdc42 activation for hyphal extension. Rga2 also undergoes transient Cdc28-dependent hyperphosphorylation at bud emergence, suggesting that regulating a GAP(s) of Cdc42 by CDKs may play an important role in governing different forms of polarized morphogenesis in yeast. This study reveals a direct molecular link between CDKs and the polarity machinery.     

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.     

Rga2 is a Rho2 GAP that regulates morphogenesis and cell integrity in S. pombe

Schizosaccharomyces pombe Rho2 GTPase regulates alpha-D-glucan synthesis and acts upstream of Pck2 to activate the MAP kinase pathway for cell integrity. However, little is known about its regulation. Here we describe Rga2 as a Rho2 GTPase-activating protein (GAP) that regulates cell morphology. rga2+ gene is not essential for growth but its deletion causes longer and thinner cells whereas rga2+ overexpression causes shorter and broader cells. rga2+ overexpression also causes abnormal accumulation of Calcofluor-stained material and cell lysis, suggesting that it also participates in cell wall integrity. Rga2 localizes to growth tips and septum region. The N-terminal region of the protein is required for its correct localization whereas the PH domain is necessary exclusively for Rga2 localization to the division area. Also, Rga2 localization depends on polarity markers and on actin polymerization. Rga2 interacts with Rho2 and possesses in vitro and in vivo GAP activity for this GTPase. Accordingly, rga2Delta cells contain more alpha-D-glucan and therefore partially suppress the thermosensitivity of mok1-664 cells, which have a defective alpha-D-glucan synthase. Additionally, genetic interactions and biochemical analysis suggest that Rga2 regulates Rho2-Pck2 interaction and might participate in the regulation of the MAPK cell integrity pathway.     

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.     

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 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.     

The Ustilago maydis a2 mating-type locus genes lga2 and rga2 compromise pathogenicity in the absence of the mitochondrial p32 family protein Mrb1

The Ustilago maydis mrb1 gene specifies a mitochondrial matrix protein with significant similarity to mitochondrial p32 family proteins known from human and many other eukaryotic species. Compatible mrb1 mutant strains were able to mate and form dikaryotic hyphae; however, proliferation within infected tissue and the ability to induce tumor development of infected maize (Zea mays) plants were drastically impaired. Surprisingly, manifestation of the mrb1 mutant phenotype selectively depended on the a2 mating type locus. The a2 locus contains, in addition to pheromone signaling components, the genes lga2 and rga2 of unknown function. Deletion of lga2 in an a2Deltamrb1 strain fully restored pathogenicity, whereas pathogenicity was partially regained in an a2Deltamrb1Deltarga2 strain, implicating a concerted action between Lga2 and Rga2 in compromising pathogenicity in Deltamrb1 strains. Lga2 and Rga2 localized to mitochondria and Mrb1 interacted with Rga2 in the yeast two-hybrid system. Conditional expression of lga2 in haploid cells reduced vegetative growth, conferred mitochondrial fragmentation and mitochondrial DNA degradation, and interfered with respiratory activity. The consequences of lga2 overexpression depended on the expression strength and were greatly exacerbated in Deltamrb1 mutants. We propose that Lga2 interferes with mitochondrial fusion and that Mrb1 controls this activity, emphasizing a critical link between mitochondrial morphology and pathogenicity.     

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.     

Targeted disruption of a G protein α subunit gene results in reduced growth and pathogenicity in <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Cloning and disruption of Rga1, the gene encoding the G protein α subunit in the rice sheath blight fungus Rhizoctonia solani, was investigated. The deduced primary structure of the Rga1-encoded protein showed high identity to those of Gα subunits from other filamentous fungi. Disruption of Rga1 led to decreased vegetative growth and pathogenicity. The Rga1 disruptant showed altered colony morphology. In addition, the sclerotia formation ability of the disruptant was completely lost. These results suggest that the Gα subunit encoded by Rga1 is involved in a signal transduction pathway in R. solani that controls growth, development and pathogenicity.     

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.     

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.