Mutations in the YRB1 gene encoding yeast Ran-binding-protein-1 that impair nucleocytoplasmic transport and suppress yeast mating defects

We identified two temperature-sensitive (ts) mutations in the essential gen e, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in sing le amino acid substitutions of evolutionarily conserved residues (A91D and R127K respectively) in the Ran-binding domain of Yrb1. The altered proteins have reduced affinity for Ran (Gsp1) in vivo. After shift to restrictive t emperature, both mutants display impaired nuclear protein import and one al so reduces poly(A)(+) RNA export, suggesting a primary defect in nucleocyto plasmic trafficking. Consistent with this conclusion, both yrb(ts) mutation s display deleterious genetic interactions with mutations in many other gen es involved in nucleocytoplasmic transport, including SRP1 (alpha -importin ) and several beta -importin family members. These yrb1(ts) alleles were is olated by their ability to suppress two different types of mating-defective mutants (respectively, fus1 Delta and ste5(ts)), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in bot h yrb1t(s) mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1(ts) mutations suppress the mating defect of a null mutation in M5N5, which encodes the receptor for pheromone-stimulated nuclear expor t of Ste5. Our results suggest that reimport of Ste5 into the nucleus is im portant in downregulating mating response.