Exploring the Regulation of Cdc42 Stability and Turnover in Yeast

Abstract

ABSTRACTRho 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 (37°C), that stimulates yeast Cdc42p turnover to impact its biological functions. At 37°C, Cdc42p turnover required the NEDD4 ubiquitin ligase Rsp5p and HSP40/HSP70 chaperones. Specific lysine residues promoted Cdc42p degradation at 37°C [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 37°C 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.Summary statementRho GTPases regulate cell polarity and signaling (e.g. MAPK) pathways. Here, we discovered that yeast Cdc42p is targeted for degradation at 37°C by a NEDD4 ubiquitin ligase and HSP40 and HSP70 chaperones through lysine residues in the C-terminus of the protein. At 37°C, Cdc42p was degraded both by the 26S proteasome and in an ESCRT-dependent manner in the vacuole. Preventing Cdc42p turnover at 37°C resulted in improved mating sensitivity but also viability and polarity defects, suggesting a tradeoff between sexual responses and fitness. In addition, one residue (K16) was critical for Cdc42p stability. Cdc42pK16R formed aggregates in aging mother cells, and aggregates were also observed in cells undergoing proteostatic stress. Protein quality control regulation of a Rho-type GTPase therefore has ramification in the regulation of cellular responses, evolutionary tradeoffs, and protein aggregation in ways that might impact aging.HIGHLIGHTSHigh temperatures (37°C) induce turnover of the Rho GTPase Cdc42pTurnover of Cdc42p at 37°C requires the HSP40/HSP70 proteins and the NEDD4-type E3 ubiquitin ligase Rsp5p.K166 and four lysines at the extreme C-terminus [poly-basic (PB: K183, K184, K186, K187] promote turnover of Cdc42p at 37°CCdc42p is degraded at 37°C by the proteosome and the ESCRT-to-vacuole pathways.GTP-Cdc42p does not accumulate in ESCRT mutants and is not turned over in the vacuole.Turnover of Cdc42p at 37°C inhibits sensitivity to mating pheromone Preventing Cdc42p turnover restores pheromone sensitivity at the cost of cell viability and proper cell polarity. These results reveal a tradeoff between sexual responses and overall cellular fitness.An internal lysine residue (K16) is required for Cdc42p stability.verproduction of the protein, or accumulation of Cdc42pK16R in certain mutants induces protein aggregation in aging mother cells.