Exploring the Regulation of Cdc42 Stability and Turnover in Yeast

Abstract

Rho GTPases govern many cellular processes, including actin cytoskeleton dynamics and signal transduction pathways. Rho GTPase levels can be regulated by stability and turnover, yet many aspects of this type of regulation remain largely unexplored. We report here a new environmental stress, high temperature (37C), that stimulates yeast Cdc42p turnover to impact its biological functions. At 37C, Cdc42p turnover required the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones. Specific lysine residues promoted Cdc42p degradation at 37C [K166; and residues in the Poly-Basic (PB) domain: K183, K184, K186, K187], which occurred in both the 26S proteosome and ESCRT-to-vacuole pathway. Degradation of Cdc42p at 37C reduced the sensitivity to mating pheromone, demonstrating biological role for Cdc42p turnover in this context. Stabilization of Cdc42p at high temperatures restored pheromone sensitivity but caused growth and polarity defects, suggesting a tradeoff between sexual propagation and cellular fitness. One lysine residue (K16) in the P-loop of the protein was critical for stability. Overproduction of the protein, expression of Cdc42pK16R in a mutant where the protein accumulates, and other types of proteostatic stress led to the formation of Cdc42p aggregates in aging mother cells. These new aspects of Cdc42p protein quality control may extend to other members of the Rho GTPase family of proteins.