GTP hydrolysis links initiation and termination of nuclear import on the nucleoporin nup358.
Binding of GTP-bound Ran (RanGTP) to karyopherin beta1 (Kapbeta1) releases import cargo into the nucleus. Using an ultrastructural, biochemical, and functional approach, we have studied the mechanism by which Kapbeta1.RanGTP is recycled at the nuclear pore complex for repeated rounds of import. In vitro, Kapbeta1 bound to the RanBP1-homologous (RBH) domains of Nup358 in the presence of either RanGTP or RanGDP, forming trimeric complexes. The Kapbeta1.RanGTP. RBH complex resisted dissociation by RanBP1 and GTP hydrolysis by Ran GTPase activating protein 1. Ran-dependent binding of gold-conjugated Kapbeta1 to the cytoplasmic fibers of the nuclear pore complex in digitonin-permeabilized cells and RanBP1 competition confirmed the in vitro binding data. Interaction of karyopherin alpha and a classical nuclear localization sequence peptide with the Kapbeta1.RanGTP.RBH complex stimulated GTP hydrolysis by Ran GTPase activating protein 1 both in vitro and in permeabilized cells. This GTP hydrolysis was required for reinitiation of import of a nuclear localization sequence-bearing substrate in permeabilized cells. These data suggest that GTP hydrolysis on the RBH domains of Nup358 couples the termination of one cycle of nuclear import with the initiation of the next.
Yaseen
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Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA.
Blobel
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G
D001665:Binding Sites; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D028961:beta Karyopherins
D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D028961:beta Karyopherins; C094493:ran-binding protein 2; D006160:Guanosine Triphosphate