Biophysical characterization of interactions involving importin-alpha during nuclear import.
Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.
Catimel
B
B
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria 3050, Australia.
Teh
T
T
Fontes
M R
MR
Jennings
I G
IG
Jans
D A
DA
Howlett
G J
GJ
Nice
E C
EC
Kobe
B
B
D021581:Active Transport, Cell Nucleus; D000818:Animals; D015374:Biosensing Techniques; D002467:Cell Nucleus / Q000378:metabolism; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D028884:Karyopherins; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology*; D056489:Nucleoplasmins; D010452:Peptide Biosynthesis; D010750:Phosphoproteins / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014461:Ultracentrifugation
D028884:Karyopherins; D008024:Ligands; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D020033:Protein Isoforms