SUBCELLULAR-LOCALIZATION OF CDC42P, A SACCHAROMYCES-CEREVISIAE GTP-BINDING PROTEIN INVOLVED IN THE CONTROL OF CELL POLARITY

The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superf amily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein ha s a consensus sequence (CAAX) for geranylgeranyl modification and is l ikely to be associated, at least in part, with cell membranes. Using c ell fractionation and immunolocalization techniques, we have investiga ted the subcellular localization of Cdc42p. Cdc42p was found in both s oluble and particulate pools, and neither its abundance nor its distri bution varied through the cell cycle. The particulate form of Cdc42p c ould be solubilized with detergents but not with NaCl or urea, suggest ing that it is tightly associated with membranes. An increase in solub le Cdc42p was observed in a geranylgeranyltransferase mutant strain (c dc43-2(ts)) grown at the restrictive temperature. In addition, Cdc42p from a cdc42(C188S) mutant strain (that has an alteration at the preny lation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microsc opy experiments demonstrated that Cdc42p localizes to the plasma membr ane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in ct-factor-arrested cells. These res ults indicate that Cdc42p is localized to the bud site early in the ce ll cycle and suggest that this localization is critical for the select ion of the proper site for bud emergence and for polarized cell growth .