Abstract. Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindies), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.A important class of questions about the cell division cycle concerns the dependency relationships or other coordinating mechanisms that ensure that cell-cycle events occur in an appropriate sequence. Such questions have been investigated in the yeast Saccharomyces cerevisiae by using mutations and inhibitors that block specific cellcycle events (Hartwell et al., 1974;Pringle, 1978;Pringle and Hartwell, 1981; Moir and Botstein, 1982;Wood and Hartwell, 1982;Jacobs et al., 1988; Hartwell and Weinert, 1989). In this context, temperature-sensitive (Ts-) ~ mutants defective in gene CDC24 have been of great interest. The observation that such mutants can continue DNA synthesis and nuclear division while bud emergence is blocked (Hartwell et al., 1973(Hartwell et al., , 1974 suggests that the nuclear cycle is not dependent on the cytoplasmic processes involved in budding. Conversely, experiments with a variety of other mutations and inhibitors suggest that bud emergence is not dependent on the nuclear cycle (Hartwell et al., 1974;Pringle and Hartwell, 1981). Thus, it appears that many of the events of the yeast cell cycle are organized into two parallel and indepen-A. E. M. Adams' present address is

Abstract. The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. E Sloat, and J. R. Pringle. 1990. J. Cell Biol. 111:131-142). To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library. The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential. Overexpression of CDC42 under control of the GALIO promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites. Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R. Axel, and A. M. Myers. 1987. Proc. Natl. Acad. Sci. 84:779-783) families of gene products. The similarities to ras proteins (,,~40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties. The similarities to the rho proteins (~60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.

SUMMARY Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway. The 11 current members ofthe Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs. Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth. In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus. Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand. In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors. While much is known about Cdc42p sturcture and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell. Future research sould focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors.

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.

Cdc42p is a highly conserved low-molecular-weight GTPase that is involved in controlling cellular morphogenesis. We have isolated the Cdc42p homolog from the fission yeast Schizosaccharomyces pombe by its ability to complement the Saccharomyces cerevisiae cdc42-l' mutation. S. pombe Cdc42p is 85% identical in predicted amino acid sequence to S. cerevisiae Cdc42p and 83% identical to the human Cdc42p homolog. The Cdc42p protein fractionates to both soluble and particulate fractions, suggesting that it exists in two cellular pools. We have disrupted the cdc42+ gene and shown that it is essential for growth. The cdc42 null phenotype is an arrest as small, round, dense cells. In addition, we have generated three site-specific mutations, G12V, Q61L, and D118A, in the Cdc42p GTP-binding domains that correspond to dominant-lethal mutations in S. cerevisiae CDC42. In contrast to the S. cerevisiae cdc42 mutations, the S. pombe cdc42 mutant alleles were not lethal when overexpressed. However, the cdc42 mutants did exhibit an abnormal morphological phenotype of large, misshapen cells, suggesting that S. pombe Cdc42p is involved in controlling polarized cell growth.Cells generate and maintain characteristic shapes as they grow and divide (25). The rod-shaped fission yeast cell and the ellipsoidal budding yeast cell accomplish this by directing the insertion of new material to specific regions of their cell surfaces in a spatial and temporal pattern of growth that is precisely coordinated with the cell division cycle. Although a switch point for polarized growth is present in both yeasts, the manifestations of polarized growth are different between these distantly related yeasts. In the budding yeast Saccharomyces cerevisiae, growth is unidirectional during the cell cycle, with the majority of growth being directed from the mother cell into the emerging daughter cell (6,7,28). After cytokinesis and cell septation, however, the undersized new daughter cell switches to isotropic growth in order to attain the proper size to initiate the next round of cell division. Over 20 genes that are involved in producing a daughter cell by budding have been identified (6,7,25 Rho/Rac subgroup of the Ras superfamily of GTPases (17). These proteins are believed to act as molecular switches by virtue of their ability to exist in two forms, an active GTP-bound form and an inactive GDP-bound form (4, 11). The human Cdc42p homolog, which is 80% identical to S. cerevisiae Cdc42p in predicted amino acid sequence, is able to complement the S. cerevisiae cdc42-1ts mutation, indicating both functional and structural homology (24, 36). We previously generated site-specific mutations in the GTPbinding domains of S. cerevisiae Cdc42p that were analogous to dominant transforming mutations in ras (37). These cdc42 mutations gave a dominant-lethal phenotype in S. cerevisiae, resulting in cells with abnormal growth and morphological properties.To study the control of polarized cell growth in S. pombe, we have isolated the S. pombe cdc42+ homolog by function...