Genes CDC24 and CDC42 are required for the establishment of cell polarity and for bud formation in Saccharomyces cerevisiae. Temperature-sensitive (Ts-) mutations in either of these genes cause arrest as large, unbudded cells in which the nuclear cycle continues. MSB1 was identified previously as a multicopy suppressor of Ts-cdc24 and cdc42 mutations. We have now sequenced MSBI and constructed a deletion of this gene Bud formation in the yeast Saccharomyces cerevisiae is coordinated with the nuclear division cycle such that a single bud emerges at a particular time during each nuclear cycle (35). Bud formation is also spatially regulated: cells that express only a or a mating-type information generally choose budding sites in a cluster at one pole, whereas cells that express both a and a information utilize both poles for budding (reviewed in reference 46). During bud emergence, secretion and cell wall deposition become localized to the bud (10, 36). Thus, bud formation is a cell cycle-regulated event that involves the establishment and maintenance of cell polarity.Several genes are known to be involved in polarity establishment and bud emergence. Notably, genes CDC24, CDC42, and CDC43 were identified in the course of extensive screens for temperature-sensitive (Ts-) mutations that cause uniform cell cycle arrest (1,18,19,46). Although unable to form buds, cdc24, cdc42, and cdc43 mutants continue growth and the nuclear cycle (often becoming multinucleate) at restrictive temperatures. Thus, the normal coordination between the nuclear division cycle and the budding cycle and between polarized and nonpolarized growth is perturbed in these mutants.For several reasons, the screens for Ts-cell division cycle mutants have probably missed many genes involved in the establishment of cell polarity and bud emergence. First, it is likely that some genes essential for these processes will not * Corresponding author. t Present address: Department of Biology, Indiana University, Bloomington, IN 47405. be easily mutable to Ts-alleles that give a clear-cut cell cycle arrest (16,23,34). Second, it is likely that some genes involved in these processes are nonessential (14,15,34). As another strategy to search for genes involved in polarity establishment and bud formation, we screened a high-copynumber yeast genomic library for genes whose overexpression could suppress a Ts-cdc24 mutation (5). This screen yielded CDC42, the ras-related gene RSRJ, and two additional genes called MSBI and MSB2 (multicopy suppression of a budding defect). Overexpression of MSBJ could also suppress a Ts-cdc42 mutation, suggesting strongly that MSBI plays a role in polarity establishment and bud formation.

Genes CDC24, CDC42, and CDC43 are required for the establishment of cell polarity and the localization of secretion in Saccharomyces cerevisiae; mutants defective in these genes fail to form buds and display isotropic expansion of the cell surface. To Johnson, and J.R.P., unpublished data), and overproduction of the CDC42 product can produce a mislocalization of budding sites like that seen in some cdc24 mutants (D. Johnson and J.R.P., unpublished data). Sequencing of CDC42 (D. Johnson and J.R.P., unpublished data) revealed that it is a member of the rho family (11) of ras oncogenerelated genes and encodes typical domains for GTP binding and hydrolysis. Moreover, its C-terminal sequence suggests that the CDC42 product, like the ras products, may be modified and thence membrane-associated. The available observations suggest a tentative model in which the products of CDC24, CDC42, and related genes may mark the budding site and provide orientational signals to elements of the cytoskeleton.Testing and amplifying this model will require identification of other genes whose products interact with those of CDC24 and CDC42. We report here the identification of four genes* that, when overexpressed, can suppress a Ts-mutation in CDC24. This approach was suggested by the observation that such "multicopy suppression" is observed not infrequently during attempts to clone genes by complementation. In at least some cases, it seems clear that the genes so identified are indeed related in function to the gene harboring the original mutation [AAS3/aasl or aas2 (12), STE5/ste4 (13), CDCII/cdcl2 (2), SIR31sir4 (14), suc1+/cdc2-(15), SCGJ/sst2 (16) high-copy-number plasmid containing the URA3 selectable marker and the 2-,m-plasmid origin of replication) and YCp5O (a low-copy-number plasmid containing URA3, CEN4, and the ARSI origin of replication). pSL113 is YEp13 [a high-copy-number plasmid containing the LEU2 selectable marker and the 2-,m-plasmid origin of replication (20)] with an inversion of the Xho I-Sal

Abstract. The SH3 domain-containing protein Bemlp is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae.

Bud formation in yeast involves the actions of the Ras-type GTPase Rsr1, which is required for the proper selection of the bud site, and the Rho-type GTPase Cdc42, which is necessary for the assembly of cytoskeletal structures at that site. The Cdc24 protein is required both for proper bud-site selection and bud-site assembly and has been recently shown to display guanine-nucleotide-exchange factor (GEF) activity toward Cdc42. Here, we demonstrate, using recombinant proteins, that Cdc24 can also bind directly to Rsr1. This binding has no effect on the ability of Rsr1 to undergo intrinsic or GEF-stimulated GDP-GTP exchange. However, Cdc24 can inhibit both the intrinsic and GTPase-activating protein-stimulated GTPase activity of Rsr1 and thereby acts as a GTPase-inhibitor protein for Rsr1. Cdc24 thus appears to bind preferentially to the activated form of Rsr1. The SH3 domain-containing bud-site assembly protein Bem1 also binds directly to Cdc24, and we show here that this interaction is inhibited by Ca2+. Neither Bem1 nor Cdc42 affects the GTPase-inhibitor protein activity of Cdc24 toward Rsr1, and neither Bem1 nor Rsr1 affects the GEF activity of Cdc24 toward Cdc42. Taken together, these results suggest that Cdc24 enables the direct convergence of a Ras-like protein (Rsr1) and a Rho-like protein (Cdc42) with the SH3-domain-containing protein (Bem1) and that independent domains of Cdc24 are responsible for these different interactions. These results also suggest that rather than directly controlling the GEF activity of Cdc24, the primary roles of Rsr1 and Bem1 might be to control the positioning of Cdc24 within the cell.

Cell polarization requires that a cellular axis or cell-surface site be chosen and that the cytoskeleton be organized with respect to it. Details of the link between the cytoskeleton and the chosen axis or site are not clear. Cells of the yeast Saccharomyces cerevisiae exhibit cell polarization in two phases of their life cycle, during vegetative growth and during mating, which reflects responses to intracellular and extracellular signals, respectively. Here we describe the isolation of two mutants defective specifically in cell polarization in response to peptide mating pheromones. The mutants carry special alleles (denoted bem1-s) of the BEM1 gene required for cell polarization during vegetative growth. Unlike other bem1 mutants, the bem1-s mutants are normal for vegetative growth. Complete deletion of BEM1 leads to the defect in polarization of vegetative cells seen in bem1 mutants. The predicted sequence of the BEM1 protein (Bem1p) reveals two copies of a domain (denoted SH3) that is found in many proteins associated with the cortical cytoskeleton and which may mediate binding to actin or some other component of the cell cortex. The sequence of Bem1p and the properties of mutants defective in this protein indicate that it may link the cytoskeleton to morphogenetic determinants on the cell surface.

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Abstract. The src homology region 3 (SH3) domainbearing protein Bemlp and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cerevisiae. Here, we present evidence that through its second SH3 domain, Bemlp binds to the structurally and functionally similar proteins Boilp and Boi2p, each of which contain an SH3 and a pleckstrin homology (PH) domain. Deletion of BOll and BOI2 together leads to impaired morphogenesis and poor viability. A PH domain-bearing segment of Boilp that lacks the Bemlp-binding site is necessary and sufficient for function. This segment of Boilp displays a twohybrid interaction with Cdc42p, suggesting that Boilp either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boilp inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth, suppresses the growth defects of boil boi2 mutants, suggesting that Boilp and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boilp and Boi2p play a role in linking the actions of Cdc42p and Rho3p.T HE src homology region 3 (SH3) 1 and pleckstrin homology (PH) domains are present in many proteins that are involved in signal transduction and the organization of the cortical cytoskeleton (Pawson, 1995). The binding sites for SH3 domains generally appear to be short, proline-rich sequences . Less is known about the binding sites for PH domains. The findings that the PH domains in some proteins overlap with sequences that can bind to the 13y subunit of some trimeric G proteins (Touhara et al., 1994) and that some PH domains can bind PIP2 (Harlan et al., 1994), however, raise the possibility that a general role of PH domains might be to target proteins to membranes.Rho-type GTPases also are involved in signal transduction and the organization of the cortical cytoskeleton

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