Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.     

Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae

The non-essential RGD1 gene from Saccharomyces cerevisiae encodes a protein that has been characterized in vitro as a Rho GTPase activating protein (RhoGAP) for the Rho3 and Rho4 proteins. Rgd1p, which displays a conserved FCH-coiled coil-Rho-GAP domain organization, showed a patch-like distribution in the cell, including a localization in growing buds. Using a genetic screen, we found that rgd1delta and vrp1alpha mutations exhibited a synthetic lethality, thus revealing an interaction between these genes. The VRP1 product is an actin and myosin interacting protein involved in polarized growth. Using mutant forms of both Rho3 and Rho4 proteins, we provide evidence for the involvement of these two GTPases in RGD1-VRP1 co-lethality. In addition, these results strongly argue in favour of Rho3p and Rho4p being the targets of Rgd1p RhoGAP activity in vivo. Genetic relationships between either VRP1 or RGD1 and actin cytoskeleton-linked genes were also studied. These and other well-established data support the idea that Vrp1, Las17, Rvs167 proteins belong to the same complex. This protein structure might act with myosins in various actin cytoskeleton-based activities, in co-operation with a Rho3p/Rho4p signalling pathway that is negatively regulated by Rgd1p GAP activity.     

The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins.

The RGD1 gene, identified during sequencing of the Saccharomyces cerevisiae genome, encodes a protein with a Rho-GTPase activating protein (GAP) domain at the carboxy-terminal end. The Rgd1 protein showed two-hybrid interactions with the activated forms of Rho2p, Rho3p and Rho4p. Using in vitro assays, we demonstrated that Rgd1p stimulated the GTPase activity of both Rho3p and Rho4p; no stimulation was observed on Rho2p. In addition, the rho3Deltargd1Delta double mutant exhibited a dramatic growth defect compared to the single mutants, suggesting that Rgd1p has a GAP activity in vivo. The present study allowed the identification of the first GAP of Rho3p and Rho4p.     

Aberrant processing of the WSC family and Mid2p cell surface sensors results in cell death of Saccharomyces cerevisiae O-mannosylation mutants

Protein O mannosylation is a crucial protein modification in uni- and multicellular eukaryotes. In humans, a lack of O-mannosyl glycans causes congenital muscular dystrophies that are associated with brain abnormalities. In yeast, protein O mannosylation is vital; however, it is not known why impaired O mannosylation results in cell death. To address this question, we analyzed the conditionally lethal Saccharomyces cerevisiae protein O-mannosyltransferase pmt2 pmt4Delta mutant. We found that pmt2 pmt4Delta cells lyse as small-budded cells in the absence of osmotic stabilization and that treatment with mating pheromone causes pheromone-induced cell death. These phenotypes are partially suppressed by overexpression of upstream elements of the protein kinase C (PKC1) cell integrity pathway, suggesting that the PKC1 pathway is defective in pmt2 pmt4Delta mutants. Congruently, induction of Mpk1p/Slt2p tyrosine phosphorylation does not occur in pmt2 pmt4Delta mutants during exposure to mating pheromone or elevated temperature. Detailed analyses of the plasma membrane sensors of the PKC1 pathway revealed that Wsc1p, Wsc2p, and Mid2p are aberrantly processed in pmt mutants. Our data suggest that in yeast, O mannosylation increases the activity of Wsc1p, Wsc2p, and Mid2p by enhancing their stability. Reduced O mannosylation leads to incorrect proteolytic processing of these proteins, which in turn results in impaired activation of the PKC1 pathway and finally causes cell death in the absence of osmotic stabilization.     

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites

Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.     

Phosphoinositides affect both the cellular distribution and activity of the F-BAR-containing RhoGAP Rgd1p in yeast

Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth.     

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.     

Hog1p mitogen-activated protein kinase determines acetic acid resistance in Saccharomyces cerevisiae

When glucose-repressed, Saccharomyces cerevisiae cannot use acetic acid as a carbon source and is inhibited in growth by high levels of this compound, especially at low pH. Cultures exposed to a 100 mM acetate stress activate both the Hog1p and Slt2p stress-activated MAP kinases. Nevertheless, only active Hog1p, not Slt2p, is needed for the acquisition of acetate resistance. Hog1p undergoes more rapid activation by acetate in pH 4.5, than in pH 6.8 cultures, an indication that the acid may have to enter the cells in order to generate the Hog1p activatory signal. Acetate activation of Hog1p is absent in the ssk1Delta and pbs2Delta mutants, but is present in sho1Delta and ste11Delta, showing that it involves the Sln1p branch of the high-osmolarity glycerol (HOG) pathway signaling to Pbs2p. In low-pH (pH 4.5) cultures, the acetate-activated Hog1p, although conferring acetate resistance, does not generate the GPD1 gene or intracellular glycerol inductions that are hallmarks of activation of the HOG pathway by hyperosmotic stress.     

Yeast Nap1-binding protein Nbp2p is required for mitotic growth at high temperatures and for cell wall integrity

Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Delta mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.     

Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.     

Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae

Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7 and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression of MSG5 in a sac7Delta mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway.     

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.     

Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae

Rho proteins are down-regulated in vivo by specific GTPase activating proteins (RhoGAP). We have functionally studied three Saccharomyces cerevisiae putative RhoGAP. By first identifying Rho partners with a systematic two-hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p. In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac-like GTPase Rho5.     

Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Delta. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Delta cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Delta and rvs161Delta. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Delta. Furthermore, lrg1Delta caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of beta(1-3)-glucan synthase in vitro. beta(1-3)-glucan deposition was increased in lrg1Delta strains and mislocalized to the tip of the mating projection in fus2Delta strains. High-copy LRG1 suppressed the mislocalization of beta(1-3) glucan in fus2Delta strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.