Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

Abstract

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Membrane Organization and Dynamics in Cell Polarity

“…Another piece of evidence for the role of Cdc42 in exocytosis came from the analysis of a specific allele of cdc42 mutant, cdc42-6 (Adamo et al 2001). In this mutant, postGolgi secretory vesicles accumulate in the cytoplasm at the restrictive temperature.…”

RhoGTPase-binding proteins, the exocyst complex and polarized vesicle trafficking

“…26 Moreover, overexpression of wild-type versions of each protein was capable of suppressing mutant phenotypes of the other. 25 Indeed, several late-acting secretory proteins of the exocyst family (most notably Sec9) also suppressed phenotypes of both cdc42-6 and rho3-V51, confirming that the exocyst functions downstream of Cdc42 and Rho3 to efficiently polarize secretory traffic in budding yeast. 25 Another small GTPase regulator of the exocyst is Ral, 27 which associates with secretory granules 28 and vesicles destined to traffic to the basolateral membrane of differentiated epithelial cells 29 and the growth cones of maturing neurons.…”

“…24 A particularly interesting temperature sensitive mutant of Cdc42 (cdc42-6) has identified a specific role of Cdc42 in secretion. 25 At the restrictive temperature, cdc42-6 led to an accumulation of secretory vesicles in the bud, although the actin cytoskeleton was appropriately polarized. Strikingly, this defect was observed only in small budded cells, a stage at which rates of membrane expansion in the bud are at its highest.…”

“…Importantly, cdc42-6 genetically interacted with Rho3. 25 A temperature-sensitive mutant of Rho3 (rho3-V51) shared many phenotypes with cdc42-6. 26 Moreover, overexpression of wild-type versions of each protein was capable of suppressing mutant phenotypes of the other.…”

Regulation of polarised growth in fungi

“…In S. cerevisiae, Rho3p promotes the localisation of the exocyst component Exo70p and the fusion of secretory vesicles with the exocyst, a role it shares with Cdc42p (Adamo et al, 1999;Adamo et al, 2001). Rho3p, together with Rho4p also acts on the formins to polarise the actin cytoskeleton (Dong et al 2003).…”