Rho1p-Bni1p-Spa2p interactions: implication in localization of Bni1p at 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 the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell 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 encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, 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.
Fujiwara
T
T
Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565-0871, Japan.
Tanaka
K
K
Mino
A
A
Kikyo
M
M
Takahashi
K
K
Shimizu
K
K
Takai
Y
Y
D000199:Actins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins; D011493:Protein Kinase C; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D020741:rho GTP-Binding Proteins
D000199:Actins; C105191:Bni1 protein, S cerevisiae; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C065889:SPA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C097993:Pkc1 protein, Trichoderma reesei; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins