Abstract
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.
Highlights
Nuclear import of proteins bearing a classical nuclear localization sequence (NLS)1 has been recently reviewed in Refs. 1–3
We show that Nup358 is a site at which the first and the last steps of nuclear import are linked and that the resulting GTP hydrolysis by Ran is required for subsequent import
Trimeric complexes between RanBP1, karyopherin 1 (Kap1), and either RanGTP or RanGDP have been demonstrated in solution binding assays [34, 35]
Summary
Nuclear import of proteins bearing a classical nuclear localization sequence (NLS)1 has been recently reviewed in Refs. 1–3. Ultrastructural Analysis of Kap1 Docking at the NPC—To assess the significance of these in vitro binding data in the context of intact NPCs, Kap1 was coupled to 10-nm colloidal gold and incubated with digitonin-permeabilized cells, either alone or in the presence of RanGDP or RanGTP.
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