ABSTRACTThe RHO1gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiaebut is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1mutation and isolated two genes. They were the previously identifiedPAC1and NIP100genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150Glued, a component of the dynein-activated dynactin complex. Disruption ofBNI1in either the pac1or nip100mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1mutant showed a synthetic lethal phenotype while the cin8 bni1mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1and nip100 bni1mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.