RHO1P-BNI1P-SPA2P INTERACTIONS - IMPLICATION IN LOCALIZATION OF BNI1PAT THE BUD SITE AND REGULATION OF THE ACTIN CYTOSKELETON IN SACCHAROMYCES-CEREVISIAE

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using th e yeast two-hybrid screening system, we cloned a gene encoding a prote in that interacted with Bni1p. This protein, Spa2p, was known to be lo calized at the bud tip and to be implicated in the establishment of ce ll polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213 -1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826- 987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encod ing components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microsco pic analysis showed that Bni1p was localized at the bud tip in wild-ty pe cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni 1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.