Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP

Nuclear transport factor (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1 p 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 t he-crystal structures of rat NTF2 and its complex with RanGDP to design:a m utant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is red uced while wild-type binding to RanGDP is retained. The 2.5 Angstrom resolu tion crystal structure of W7A-NTF2 is virtually superimposable upon the wil d-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the e xposed side-chain of residue 7 is crucial to the interaction between NTF2 a nd xFxFG repeat-containing nucleoporins. Consistent-with its reduced affini ty for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less stron gly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated wi th W7A-NTF2 binds less strongly to the central channel of nuclear pore comp lexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing dir ect evidence that an interaction between NTF2 and xFxFG repeat-containing n ucleoporins is required to mediate the nuclear import of RanGDP. (C) 1999 A cademic Press.