Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.
The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.
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Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Adam
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D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism*; D003545:Cysteine / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D017354:Point Mutation; D028901:alpha Karyopherins; D020931:ran GTP-Binding Protein
D009687:Nuclear Proteins; D028901:alpha Karyopherins; C094680:nuclear protein import factor p97; D006160:Guanosine Triphosphate; D020931:ran GTP-Binding Protein; D003545:Cysteine