Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein Chaperones and the MAPK Pathway Scaffold Bem4

Abstract

All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be reorganized during development and in response to extrinsic cues to produce new cell types. Rho GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome. Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that Cdc42pQ61L, which mimics the active (GTP-bound) conformation of the protein, was turned over at elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42pQ61L led to multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p, stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another. Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis. Significance StatementRho GTPases are switch-like proteins that govern major decisions in cell polarity and signaling in eukaryotes. We elucidate here a pathway that turns over the yeast Rho GTPase Cdc42p, which is mediated by the heat-shock family of proteins (HSPs) and the NEDD4-type E3 ubiquitin ligase Rsp5p. This finding provides a way for HSPs to exert their widespread effects on morphogenetic responses, phenotypic plasticity, and signaling pathways. We also found that turnover of an active version of Cdc42p is critical for modulating cell polarity. Cdc42p turnover also impacted its function in a pathway specific setting, as stabilization of Cdc42p by Bem4p (SmgGDS-type scaffold) influenced the activity of a specific MAPK pathway. HSPs may regulate Rho GTPase turnover in many systems.