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.
Kutay
U
U
Zentrum für Molekulare Biologie der Universität Heidelberg, Germany.
Izaurralde
E
E
Bischoff
F R
FR
Mattaj
I W
IW
Görlich
D
D
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active; D005260:Female; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D015394:Molecular Structure; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism*; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein