IDENTIFICATION OF DIFFERENT ROLES FOR RANGDP AND RANGTP IN NUCLEAR-PROTEIN IMPORT

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization sig nal (NLS), Translocation of the resulting import ligand complex throug h the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly, The principal GTP exchange fac tor for Ran is the nuclear protein RCC1, whereas the major RanGAP is c ytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form, Here, we show that nuclear i mport depends on cytoplasmic RanGDP and free GTP, and that RanGDP bind s to the NPC, Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran, RanGDP binding to the NPC is not medi ated by the Ran binding sites of importin-beta, suggesting that transl ocation is not driven from these sites. Consistently, a mutant importi n-beta deficient in Ran binding can deliver its cargo up to the nucleo plasmic side of the NPC. However the mutant is unable to release the i mport substrate into the nucleoplasm. Thus, binding of nucleoplasmic R anGTP to importin-beta probably triggers termination, i.e. the dissoci ation of importin-alpha from inportin-beta and the subsequent release of the import substrate into the nucleoplasm.