Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.
Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.
Bayliss
R
R
MRC Laboratory of Molecular Biology, Hills Rd, Cambridge, CB2 2QH, England.
Ribbeck
K
K
Akin
D
D
Kent
H M
HM
Feldherr
C M
CM
Görlich
D
D
Stewart
M
M
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009685:Nuclear Envelope / Q000378:metabolism / Q000648:ultrastructure; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D009865:Oocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D029701:Saccharomyces cerevisiae Proteins; D012995:Solubility; D014982:Xenopus laevis; D020931:ran GTP-Binding Protein / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C062748:NSP1 protein, S cerevisiae; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503613:Nutf2 protein, mouse; C503612:Nutf2 protein, rat; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C049312:nuclear pore protein p62; D020931:ran GTP-Binding Protein