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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article proposes that Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae, participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the protein kinase C-mitogen-activated protein kinase pathway. It details the yeast strains and genetic procedures. The article also discusses the production of Rho3 and Rho4 GTPases.