Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Bruno Catimel, Trazel Teh, Marcos R.M. Fontes, Ian G. Jennings, David A. Jans, Geoffrey J. Howlett, Edouard C. Nice, Bostjan Kobe

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Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; KD = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (KD = 3.5 × 10-8 M and 4.8 × 10-8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, KD = 1.7 × 10-8 M; nucleoplasmin NLS, KD = 1.4 × 10-8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Original languageEnglish
Pages (from-to)34189-34198
Number of pages10
JournalThe Journal of Biological Chemistry
Issue number36
Publication statusPublished - 7 Sep 2001
Externally publishedYes

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