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 gene, YRB1, which encodes the yeast homolog of Ran-binding-protein-1 (RanBP1), a known coregulator of the Ran GTPase cycle. Both mutations result in single 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 temperature, both mutants display impaired nuclear protein import and one also reduces poly(A)+ RNA export, suggesting a primary defect in nucleocytoplasmic trafficking. Consistent with this conclusion, both yrb1ts mutations display deleterious genetic interactions with mutations in many other genes involved in nucleocytoplasmic transport, including SRP1 (alpha-importin) and several beta-importin family members. These yrb1ts alleles were isolated by their ability to suppress two different types of mating-defective mutants (respectively, fus1Delta and ste5ts), indicating that reduction in nucleocytoplasmic transport enhances mating proficiency. Indeed, in both yrb1ts mutants, Ste5 (scaffold protein for the pheromone response MAPK cascade) is mislocalized to the cytosol, even in the absence of pheromone. Also, both yrb1ts mutations suppress the mating defect of a null mutation in MSN5, which encodes the receptor for pheromone-stimulated nuclear export of Ste5. Our results suggest that reimport of Ste5 into the nucleus is important in downregulating mating response.
Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202, USA. markus.kuenzler@urz.uni-heidelberg.de
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Künzler
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Trueheart
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Sette
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Hurt
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Thorner
D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D005656:Fungal Proteins / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020798:Two-Hybrid System Techniques; D020931:ran GTP-Binding Protein / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C091775:ran-binding protein 1; D020931:ran GTP-Binding Protein