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Eukaryotic Cell doi:10.1128/EC.00120-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Rho5p is involved in mediating the osmotic stress response in S. cerevisiae and its activity is regulated via Msi1p and Npr1p by phosphorylation and ubiquitination

Department of Biochemistry, McGill University, Montreal, QC, H3G 1Y6, Canada; Biotechnology Research Institute, National Research Council, Montreal, QC H4P 2R2, Canada; Division of Experimental Medicine, McGill University, Montreal, QC, H3A 1A3, Canada; Department of Biology, McGill University, Montreal, QC, H3A 1B1, Canada

^* To whom correspondence should be addressed. Email: robert.annan{at}mail.mcgill.ca.

	Abstract

Small GTPases of the Rho family act as molecular switches, and modulating the GTP-bound state of Rho proteins is a well-characterized means of regulating their signaling activity in vivo. In contrast, regulation of Rho-type GTPases by post-translational modifications is poorly understood. Here we present evidence of control of the Saccharomyces cerevisiae Rho-type GTPase Rho5p by phosphorylation and ubiquitination. Rho5p binds to Ste50p, and the expression of the activated RHO5^Q91H allele in a ste50 strain is lethal under conditions of osmotic stress. An overexpression screen identified RGD2 and MSI1 as high-copy suppressors of the osmotic sensitivity of this lethality. Rgd2p had been identified as a possible Rho5p-GAP based on an in vitro assay; this result supports its function as a regulator of Rho5p activity in vivo. MSI1 has previously been identified as a suppressor of hyperactive Ras/cAMP signaling, where it antagonizes Npr1p kinase activity and promotes ubiquitination. Here we show that Msi1p also acts via Npr1p to suppress activated Rho5p signaling. Rho5p is ubiquitinated and its expression is lethal in a strain that is compromised for proteasome activity. These data identify Rho5p as a target of Msi1p/Npr1p regulation and describe a regulatory circuit involving phosphorylation and ubiquitination.