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Cdc42p is activated during vacuole membrane fusion in a sterol-dependent subreaction of priming.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2010 Feb 12; Vol. 285 (7), pp. 4298-306. Date of Electronic Publication: 2009 Dec 10. - Publication Year :
- 2010
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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.
- Subjects :
- Membrane Fusion genetics
Microscopy
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae Proteins genetics
Vacuoles genetics
Vacuoles metabolism
rho GTP-Binding Proteins genetics
Membrane Fusion physiology
Saccharomyces cerevisiae cytology
Saccharomyces cerevisiae metabolism
Saccharomyces cerevisiae Proteins metabolism
Vacuoles physiology
rho GTP-Binding Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 285
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 20007700
- Full Text :
- https://doi.org/10.1074/jbc.M109.074609