Cdc42p Is Activated during Vacuole Membrane Fusion in a Sterol-dependent Subreaction of Priming

Abstract

Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP-or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion.

Wsp1 Is Downstream of Cin1 and Regulates Vesicle Transport and Actin Cytoskeleton as an Effector of Cdc42 and Rac1 in Cryptococcus neoformans

“…To induce vacuole fusion in C. neoformans, we adapted the hypotonic stress method used for S. cerevisiae (20,48). Cells grown in yeast extract-peptone-dextrose (YPD) were stained with FM4-64 either directly or after 20 min of hypotonic treatment (10-fold dilution of YPD in water) and were examined microscopically at the time points indicated in the figure legends.…”

Cdc42 regulation of polarized traffic in fission yeast

“…In S. cerevisiae it has been shown that actin disassembly is an early subreaction of vacuole membrane fusion, whereas the formation of F-actin is needed for fusion and is likely catalyzed by components activated by Cdc42 at the membrane surface 22 , 23 . In this yeast, overexpression of either constitutively active or dominant-negative Cdc42 resulted in vacuole fragmentation 21 . It is likely that multiple vesicle transport and fusion processes include a Cdc42-regulated actin remodeling subreaction, and the hyperactive Cdc42L160S concentrated in endomembranes might block these processes.…”

Yeast Translation Elongation Factor-1A Binds Vacuole-localized Rho1p to Facilitate Membrane Integrity through F-actin Remodeling

“…Previously, we have shown the requirement for actin dynamics and turnover in membrane fusion (10), and we have linked fusiondependent actin dynamics on vacuoles with two Rho GTPases, Rho1p and Cdc42p (11)(12)(13). Cdc42p is rapidly activated during in vitro fusion reactions and stimulates the rapid polymerization of actin (11,14).…”