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

Nuclear protein import proceeds through the nuclear pore complex (NPC), Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus, The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer, Here, we analyse the sites of interaction on importin-beta for its multiple partners, Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding, Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site, Importin-beta 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-beta from the NPC, Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism, As would be predicted from this, these importin-beta 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.