Role in Bud Formation

RHO? encodes a Rho-type small GTPase of the yeast Saccharomyces cereuisiae. We isolated temperaturesensitive alleles and a dominant active allele of RH03. Tsrho? cells lost cell polarity during bud formation and grew more isotropically than wild-type cells at nonpermissive temperatures. In contrast, cells carrying a dominant active mutant RHO? displayed cold sensitivity, and the cells became elongated and bent, often at the position where actin patches were concentrated. These phenotypes of the rho3 mutants strongly suggest that RHO? is involved in directing the growing points during bud formation. In addition, we found that SR06, previously isolated as a multicopy suppressor of rho?, is the same as SEW. The sec4-2 mutation was synthetic lethal with temperature-sensitive rho3 mutations and suppressed the cold sensitivity caused by a dominant active mutant RH03. The genetic interactions between RHO? and SEC4, taken together with the fact that the Rab-type GTPase Sec4p is required to fuse secretory vesicles together with plasma membrane for exocytosis, support a model in which the Rhosp pathway modulates morphogenesis during bud growth via directing organization of the actin cytoskeleton and the position of the secretory machinery for exocytosis. S MALL GTPases of the Ras superfamily act as molecular switches through their conformational change between the GTP-bound active form and GDP-bound inactive form of the proteins (BARBACID 1987; BOURNE et al. 1991; BOGUSKI and MCCORMICK 1993). In the yeast Saccharomyces cereuisiae, three Rho-type GTPases of the Ras superfamily, Cdc42p, Rho3p, and Rho4p, participate in the morphogenetic event of bud formation (ADAMS et al. 1990; JOHNSON and PRINGLE 1990; MATSUI and TOH-E 1992a,b). In bud formation, cell polarity is established and the cytoskeleton is reorganized. Patches of actin filaments become concentrated at the bud site, to which transport of secretory vesicles is directed for the construction of the daughter cell surface (TKACZ and LAMPEN 1972; FIELD and SCHEKMAN 1980; PRINGLE and HARTWELL 1981; WIB et al. 1982; ~ A M S and PRINGLE 1984; KILMARTIN and ADAMS 1984; NOVICK and BOTSTEIN 1985; PRINGLE et al. 1986; DRUBIN 1991; JOHNSTON et al. 1991). Loss of Cdc42p function disrupts the asymmetric localization of actin filaments and causes cells to become unbudded, large, and round (ADAMS et al. 1990; JOHNSON and PRINGLE 1990; ZIMAN andJOHNSON 1994). Cells carrying a dominant active CDC42 mutation (e.g., CDC42v""'2) initiate bud emergence independent of the cell cycle and cells with multiple buds are accumulated (ZIMAN et al. 1991). These facts suggest that Cdc42p is required for cell polarity establishment at the initiation of bud emergence. Cell deleted for R H 0 3 grow very slowly, and simultaneous disruption of R H 0 4 enhances the growth defect, whereas disruption of R H 0 4 alone does not inhibit cell Corresponding authur; Yasushi Matsui, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. Genetics 142: 359-369 (February, 1996) growth. In addition, RH04 is a multicopy suppressor of rho3 (MATSUI and TOH-E 1992a), suggesting that Rho4p possesses a Rho3prelated function. Arho3 Arho4 cells, where both Rho3p and Rho4p are depleted using a conditionally expressed promoter, undergo lysis with small buds (MATSUI and TOH-E 199213). These facts suggest that Rho3p and Rho4p are required for bud growth. The genes SROl SR09 were previously isolated as multicopy suppressors of rho3. SR02 is the same as CDC42 and SROl is identical to BEMl (MATSUI and TOH-E 1992b). BEMl encodes a SH3 domain-bearing protein required for cell polarization both during bud formation and during the formation of mating projections (BENDER and PRINGLE 1991; CHANT et al. 1991; CHENEVERT et al. 1992). The genetic interactions of R H 0 3 with BEMl and CDC42 suggest that R H 0 3 is involved in development or maintenance of cell polariza-