RanBP1 stabilizes the interaction of Ran with p97 nuclear protein import.
Three factors have been identified that reconstitute nuclear protein import in a permeabilized cell assay: the NLS receptor, p97, and Ran/TC4. Ran/TC4, in turn, interacts with a number of proteins that are involved in the regulation of GTP hydrolysis or are components of the nuclear pore. Two Ran-binding proteins, RanBP1 and RanBP2, form discrete complexes with p97 as demonstrated by immunoadsorption from HeLa cell extracts fractionated by gel filtration chromatography. A > 400-kD complex contains p97, Ran, and RanBP2. Another complex of 150-300 kD was comprised of p97, Ran, and RanBP1. This second trimeric complex could be reconstituted from recombinant proteins. In solution binding assays, Ran-GTP bound p97 with high affinity, but the binding of Ran-GDP to p97 was undetectable. The addition of RanBP1 with Ran-GDP or Ran-GTP increased the affinity of both forms of Ran for p97 to the same level. Binding of Ran-GTP to p97 dissociated p97 from immobilized NLS receptor while the Ran-GDP/RanBP1/p97 complex did not dissociate from the receptor. In a digitonin-permeabilized cell docking assay, RanBP1 stabilizes the receptor complex against temperature-dependent release from the pore. When added to an import assay with recombinant NLS receptor, p97 and Ran-GDP, RanBP1 significantly stimulates transport. These results suggest that RanBP1 promotes both the docking and translocation steps in nuclear protein import by stabilizing the interaction of Ran-GDP with p97.
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
N C
NC
Chi
E J
EJ
Adam
G D
GD
Visser
S A
SA
Adam
D001692:Biological Transport; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D009685:Nuclear Envelope / Q000737:chemistry; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D028901:alpha Karyopherins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; C091775:ran-binding protein 1; C094493:ran-binding protein 2; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein; D004072:Digitonin