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.
The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the -glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl ؊ homeostasis; rgf1⌬ cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and -glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1⌬ cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.
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