GROWTH SITE LOCALIZATION OF RHO1 SMALL GTP-BINDING PROTEIN AND ITS INVOLVEMENT IN BUD FORMATION IN SACCHAROMYCES-CEREVISIAE

The Rho small GTP-binding protein family regulates various actomyosin- dependent cell functions, such as cell morphology, locomotion, cytokin esis, membrane ruffling, and smooth muscle contraction. In the yeast S accharomyces cerevisiae, there is a homologue of mammalian RhoA, RHO1, which is essential for vegetative growth of yeast cells. To explore t he function of the RHO1 gene, we isolated a recessive temperature-sens itive mutation of RHO1, rho1-104. The rho1-104 mutation caused amino a cid substitutions of Asp 72 to Asn and Cys 164 to Tyr of Rho1p. Strain s bearing the rho1-104 mutation accumulated tiny- or small-budded cell s in which cortical actin patches were clustered to buds at the restri ctive temperature. Cell lysis and cell death were also seen with the r ho1-104 mutant. Indirect immunofluorescence microscopic study demonstr ated that Rho1p was concentrated to the periphery of the cells where c ortical actin patches were clustered, including the site of bud emerge nce, the tip of the growing buds, and the motherbud neck region of cel ls prior to cytokinesis. Indirect immunofluorescence study with cells overexpressing RHO1 suggested that the Rho1p-binding site was saturabl e. A mutant Rho1p with an amino acid substitution at the lipid modific ation site remained in the cytoplasm. These results suggest that Rho1 small GTP-binding protein binds to a specific site at the growth regio n of cells, where Rho1p exerts its function in controlling cell growth .