Homology Region 3 Domain-binding Proteins Involved in Bud Formation

The yeast protein Bemlp, which bears two src homology region 3 (SH3) domains, is involved in cell polarization. A Rho-type GTPase, Rho3p, is involved in the maintenance of cell polarity for bud formation, and the rho3 defect is suppressed by a high dose of BEM1. Mutational analysis revealed that the second SH3 domain from the NH2 terminus (SH3-2) of Bemlp is important for the functions of Bemlp in bud formation and in the suppression of the rho3 defect. Boi2p, which bound to SH3-2 of Bemlp, was identified using the two-hybrid system. Boi2p has a proline-rich sequence that is critical for displaying the Boi2pBemlp two-hybrid interaction, an SH3 domain in its NH2-terminal half, and a pleckstrin homology domain in its COOH-terminal half. A BOI2 homologue, BOIl , was identified as a gene whose overexpression inhibited cell growth. Cells overexpressing either BOIl or BOI2 were arrested as large, round, and unbudded cells, indicating that the Boi proteins affect cell polarization. Genetic analysis revealed that BOl l and BOI2 are functionally redundant and important for cell growth, zlboil Aboi2 cells became large round cells or lysed with buds, displaying defects in bud formation and in the maintenance of cell polarity. Analysis using several truncated versions of BOI2 revealed that the COOH-terminal half, which contains the pleckstrin homology domain, is essential for the function of Boi2p in cell growth, while the NH2-terminal half is not, and the NH2-terminal half might be required for modulating the function of Bemlp. Overproduction of either Rho3p or the Rho3prelated GTPase Rho4p suppressed the boi defect. These results demonstrate that Rho3p GTPases and Boi proteins function in the maintenance of cell polarity for bud formation. URING bud formation in the yeast Saccharomyces cerevisiae, cell polarity is established for the initiation of bud emergence and it is maintained during bud growth. Patches of actin filaments become concentrated at the bud site, towards which the transport of secretory vesicles is directed for the construction of the daughter cell (Tkacz and Lampen, 1972; Field and Schekman, 1980; Pringle and Hartwell, 1981; Cabib et al., 1982; Adams and Pringle, 1984; Kilmartin and Adams, 1984; Novick and Botstein, 1985; Pringle et al., 1986; Drubin, 1991). The establishment and maintenance of cell polarity require the functions of Rho-type GTPases Cdc42p, Rho3p, and Rho4p, which belong to the Ras superfamily (Johnson and Pringle, 1990; Matsui and Toh-e, 1992a, b; Imai et al., 1996). GTPases of the Ras superfamily act as molecular switches through their conformational change between the GTP-bound active form and GDP-bound inactive form (Barbacid, 1987; Bourne et al., 1991; Boguski Please address all correspondence to Yasushi Matsui, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel. and Fax: 81 3 5684 9420; e-mail: [email protected] and McCormick, 1993). Defects in either CDC42 or CDC24, which encodes a GTP-GDP exchange factor for Cdc42p, disrupt the asymmetric localization of actin filaments and cause cells to become unbudded, large, and round, an indication that Cdc42p and Cdc24p are essential for the establishment of cell polarity (Sloat and Pringle, 1978; Sloat et al., 1981; Adams et al., 1990; Johnson and Pringle, 1990; Zheng et al., 1994). Defects in RH03 cause severe growth defects. Disruption of RH04, which encodes a Rho3p-related GTPase, enhances the growth defect of Arho3 ceils (Matsui and Toh-e, 1992a). Temperature-sensitive rho3 mutant cells lose cell polarity at nonpermissive temperatures: the asymmetric localization of actin filaments is disrupted in the rho3 cells, and the rho3 cells are arrested as large, round cells, although, in contrast to cdc42 mutant cells, not all of these cells are arrested as unbudded cells (Imai et al., 1996). Depletion of both Rho3p and Rho4p results in lysis of cells that have small buds (Matsui and Toh-e, 1992b). These observations strongly suggest that Rho3p is required for the maintenance of cell polarity for bud growth. The rho3 defect is suppressed by the overexpression of BEM1, an indication that Bemlp has functions that affect © The Rockefeller Umversity Press, 0021-9525/96/05/865/14 $2.00 The Journal of Cell Biology, Volume 133, Number 4, May 1996 865-878 865 on D ecem er 7, 2012 jcb.rress.org D ow nladed fom Published May 15, 1996