Isolated mammalian and Schizosaccharomyces pombe ran-binding domains rescue S. pombe sbp1 (RanBP1) genomic mutants.
Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1(-) yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1(-) yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells.
Novoa
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Department of Biochemistry, New York University School of Medicine, New York, New York 10016, USA.
Rush
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MG
D'Eustachio
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002455:Cell Division / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D008322:Mammals; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009154:Mutation; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D020931:ran GTP-Binding Protein
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; C091775:ran-binding protein 1; C094493:ran-binding protein 2; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein