The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of lowmolecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2') grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane 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 a-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecularweight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Gin to and Asp to in the putative GTP-binding and hydrolysis domains and one mutation (Cys to in the putative isoprenylation site. The incapable of complementing the cdc42-l' mutation and was recessive to both wild-type and cdc42-1'. In double-mutant alleles, the cdc42srel188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.The Saccharomyces cerevisiae CDC42 gene product is involved in the control of several morphogenetic events during the cell cycle, including the generation of cell polarity, development of normal cell shape, localization of secretion, and deposition of cell-surface material (1,2,20). Its predicted amino acid sequence (20) is similar to that of members of the ras superfamily of low-molecular-weight GTP-binding proteins from S. cerevisiae and higher eukaryotes (16), especially in those domains that have been implicated in the binding and hydrolysis of GTP and in carboxylterminal modifications. CDC42 shows the greatest degree of sequence similarity to the products of two related human cDNAs, CDC42Hs (39) and G25K (27), which encode isoforms of the low-molecular-weight GTP-binding protein previously referred to as Gp or G25K (11, 33). Both of these human gene products are 80% identical (88% related amino acids) in predicted amino acid sequence to the S. cerevisiae CDC42 gene product, which we have redesignated CDC42Sc, and they are 95% identical to each other. These human genes, when expressed in S. cerevisiae under the control of a yeast promoter on a multicopy plasmid, can functionally complement the cdc42-1's mutation (27, 39). The Gp/G25K protein is an excellent in vitro phosphosubstrate for the epidermal growth factor receptor tyrosine kinase (17), but its in vivo function is unknown.In order to further elucidate the role of the CDC42Sc gene product in the control of cell polarity, we have generated new mutations in CDC42Sc by using site-directed mutagenesis (see Fig. 1). A carboxyl-terminal mutation predicted to * Corresponding author.eliminate isoprenylation results in a nonfunctional product. Mutations in the putative guanine-nucleotide-binding domain lead to a lethal phenotype that can be suppressed by combination with the carboxyl-terminal mutation. This lethality is manifested as several different and unique morphological abnormalities, which have provided clues to the function of the CDC42Sc gene product in controlling cell polarity. MATERIALS AND METHODSReagents. Enzymes, M13 dideoxy sequencing kit, mutagenesis kit, and other reagents were obtained from standard commercial sources and used according to the suppliers' Media, growth conditions, and strains. Conditions for...

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

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The Saccharomyces cerevisiae CDC43 gene product is involved in establishing cell polarity during the cell-division cycle. When grown at restrictive temperatures, temperature-sensitive cdc43 mutants are unable to form buds and display delocalized cell-surface deposition [Adams et al., J. Cell Biol. (1990) in press]. We have isolated a cdc43-complementing plasmid from a yeast genomic-DNA library and localized the CDC43 gene, by subcloning and transposon-mutagenesis experiments, to a 1.2-kb region of DNA that contained only one significant ATG-initiated open reading frame of 213 codons. The putative CDC43 gene product contains a possible nuclear-localization signal sequence, a cysteine-rich domain and a histidine-rich domain, and a region that is similar in structure to alpha-helix-turn-alpha-helix structural domains present in some prokaryotic and eukaryotic DNA-binding proteins.