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The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear
protein import. Importin binds the nuclear localization signal (NLS).
Translocation of the resulting import ligand complex through the nuclear pore
complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its
disassembly. The principal GTP exchange factor for Ran is the nuclear protein
RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is
mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we
show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that
RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and
GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by
the Ran binding sites of importin-beta, suggesting that translocation is not
driven from these sites. Consistently, a mutant importin-beta deficient in Ran
binding can deliver its cargo up to the nucleoplasmic side of the NPC. However,
the mutant is unable to release the import substrate into the nucleoplasm. Thus,
binding of nucleoplasmic RanGTP to importin-beta probably triggers termination,
i.e. the dissociation of importin-alpha from importin-beta and the subsequent
release of the import substrate into the nucleoplasm.
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