Interaction of Bnr1p with a Novel Src Homology 3 Domain-containing Hof1p

Abstract

Proteins containing the formin homology (FH) domains FH1 and FH2 are involved in cytokinesis or establishment of cell polarity in a variety of organisms. We have shown that the FH proteins Bni1p and Bnr1p are potential targets of the Rho family small GTP-binding proteins and bind to an actin-binding protein, profilin, at their proline-rich FH1 domains to regulate reorganization of the actin cytoskeleton in the yeast Saccharomyces cerevisiae. We found here that a novel Src homology 3 (SH3) domain-containing protein, encoded by YMR032w, interacted with Bnr1p in a GTP-Rho4p-dependent manner through the FH1 domain of Bnr1p and the SH3 domain of Ymr032wp. Ymr032wp weakly bound to Bni1p. Ymr032wp was homologous to cdc15p, which is involved in cytokinesis in Schizosaccharomyces pombe, and we named this gene HOF1 (homolog of cdc 15). Both Bnr1p and Hof1p were localized at the bud neck, and both the bnr1 and hof1 mutations showed synthetic lethal interactions with the bni1 mutation. The hof1 mutant cells showed phenotypes similar to those of the septin mutants, indicating that HOF1 is involved in cytokinesis. These results indicate that Bnr1p directly interacts with Hof1p as well as with profilin to regulate cytoskeletal functions in S. cerevisiae.The Rho family belongs to the small G protein superfamily and regulates various cell functions through reorganization of the actin cytoskeleton (for reviews, see Refs. 1 and 2). Many potential targets of Rho have been identified (for a review, see Ref.3), but it has not yet been thoroughly clarified how Rho regulates reorganization of the actin cytoskeleton through these targets.The actin cytoskeleton plays a pivotal role in the budding processes in the yeast Saccharomyces cerevisiae (for a review, see Ref. 4). This yeast has the Rho family members, including RHO1, RHO2, RHO3, RHO4, and CDC42, which are involved in the budding processes (for reviews, see Refs. 4 and 5). We have isolated BNI1 as a potential target of RHO1, which links RHO1 with the actin cytoskeleton (6). BNI1 has subsequently been shown to be a potential target of CDC42, RHO3, and RHO4 (7). BNR1 is a BNI1-related gene and is a potential target of RHO4 (8). Bni1p and Bnr1p are members of the FH 1 family of proteins, which are defined by the presence of two formin homology domains, the proline-rich FH1 domain and the FH2 domain. The FH proteins play an important role in the actin cytoskeleton-dependent processes, including cytokinesis and establishment of cell polarity (for reviews, see Refs. 9 and 10). We have recently shown that Bni1p interacts with elongation factor 1␣, which binds to and bundles actin filaments (11), and that Spa2p is required for the localization of Bni1p at the bud tip (12). Bni1p and Bnr1p, at their FH1 domains, bind to an actin monomer-binding protein, profilin, which is implicated in actin polymerization (7,8). A proline-rich sequence also interacts with an SH3 domain, which is found in a wide variety of proteins, ranging from cytoskeletal components to signal transducing enzymes (for a ...

Stable and Dynamic Axes of Polarity Use Distinct Formin Isoforms in Budding Yeast

“…A COOHterminal GFP-fusion of full-length Bnr1p expressed from the BNR1 locus also displayed a localization in agreement with previous overexpression studies ( Kamei et al, 1998), showing a polarized distribution in very few unbudded cells (5% of unbudded cells), but a ring of fluorescence around the neck of cells with buds of all sizes ( Figures 1C and S1A). In yeast, formin-dependent actin filaments can be distinguished from the Arp2/3 complex-dependent filaments by their association with the F-actin binding protein tropomyosin, encoded by the TPM1 and TPM2 genes (Liu and Bretscher, 1989;Pruyne et al, 1998;Evangelista et al, 2002;Sagot et al, 2002a;Tolliday et al, 2002).…”

“…A COOHterminal GFP-fusion of full-length Bnr1p expressed from the BNR1 locus also displayed a localization in agreement with previous overexpression studies ( Kamei et al, 1998), showing a polarized distribution in very few unbudded cells (5% of unbudded cells), but a ring of fluorescence around the neck of cells with buds of all sizes ( Figures 1C and S1A).…”

“…As has been seen for similar Bni1p-derived constructs, this construct stimulated the polymerization of actin from monomers, whereas GST did not. Thus, the conserved COOH-terminal regions of the two yeast formins stimulate actin assembly.Based on studies of overexpressed Bni1p and Bnr1p, the NH 2 termini of these formins help confer distinct localizations on the two (Jansen et al, 1996;Evangelista et al, 1997;Fujiwara et al, 1998;Kamei et al, 1998;Ozaki-Kuroda et al, 2001). We wished to examine whether these localizations impart differences in the distribution of the actin filaments assembled by the two isoforms.…”

“…We wished to examine whether these localizations impart differences in the distribution of the actin filaments assembled by the two isoforms. However, overexpression of either formin alters actin organization and cell morphology (Kamei et al, 1998;Evangelista et al, 2002;Sagot et al, 2002a). To avoid this complication, we observed the distribution of the formins and formin-dependent actin filaments in cells that expressed Bni1p and Bnr1p at endogenous levels.…”

From Function to Shape: A Novel Role of a Formin in Morphogenesis of the FungusAshbya gossypii

“…In S. cerevisiae, the two formins Bni1p and Bnr1p are required for cell polarity and cytokinesis with some overlapping and some different functions (Zahner et al, 1996;Evangelista et al, 1997;Imamura et al, 1997;Kamei et al, 1998;Pruyne et al, 2004). In Schizosaccharomyces pombe, three formins exist which are also involved in cell polarity and cytokinesis.…”