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

EMBO J. 1997 Mar 17;16(6):1153-63. doi: 10.1093/emboj/16.6.1153.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites / genetics
  • Biological Transport, Active
  • Female
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Molecular Structure
  • Mutation*
  • Nuclear Envelope / metabolism*
  • Nuclear Localization Signals
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism*
  • Oocytes / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Binding
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Xenopus
  • beta Karyopherins
  • ran GTP-Binding Protein

Substances

  • Nuclear Localization Signals
  • Nuclear Proteins
  • Peptide Fragments
  • Recombinant Proteins
  • beta Karyopherins
  • ran GTP-Binding Protein