Dominant‐negative mutants of importin‐β block multiple pathways of import and export through the nuclear pore complex

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin‐β mediates translocation via direct interaction with NPC components and carries importin‐α with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin‐β which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin‐β for its multiple partners. Ran and importin‐α respectively require residues 1–364 and 331–876 of importin‐β for binding. Thus, RanGTP‐mediated release of importin‐α from importin‐β is likely to be an active displacement rather than due to simple competition between Ran and importin‐α for a common binding site. Importin‐β has at least two non‐overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin‐β from the NPC. Importin‐β fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin‐β mutants are very efficient inhibitors of NLS‐dependent protein import. Surprisingly, however, they also inhibit M9 signal‐mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES‐containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.