Clarkson W D, Corbett A H, Paschal B M, Kent H M, McCoy A J, Gerace L, Silver P A, Stewart M
MRC Laboratory of Molecular Biology, Cambridge, UK.
J Mol Biol. 1997 Oct 10;272(5):716-30. doi: 10.1006/jmbi.1997.1255.
Nuclear transport factor 2 (NTF2) is associated with the translocation stage of nuclear protein import and binds both to nuclear pore proteins (nucleoporins) containing phenylalanine-rich repeats and to the Ras family GTPase Ran. In this study we probed the role of the NTF2-Ran interaction in nuclear protein import using site-directed mutants of NTF2 that interfere with its interaction with GDP-Ran. The design of these mutants was based on the X-ray crystal structure of NTF2 and was concentrated on conserved residues in and around the molecule's hydrophobic cavity. The mutant NTF2 cDNAs were expressed in Escherichia coli. Purified mutant proteins retained the interaction with FxFG-repeat nucleoporins, but several mutants in the negatively charged residues that surround the NTF2 cavity or in residues in the cavity itself were unable to bind GDP-Ran in vitro. The crystal structure of the E42K mutant protein showed significant structural changes only in this side-chain, indicating that it participated directly in the interaction with GDP-Ran. In permeabilised cell nuclear protein import assays, only wild-type NTF2 and mutants that bound GDP-Ran were functional. Furthermore, when the NTF2 E42K and D92N/D94N NTF2 mutants that failed to bind GDP-Ran in vitro were substituted for the chromosomal yeast NTF2, the yeast cells became non-viable, whereas yeast substituted with wild-type human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient nuclear protein import.
核转运因子2（NTF2）与核蛋白输入的转运阶段相关，它既能与含有富含苯丙氨酸重复序列的核孔蛋白结合，也能与Ras家族的GTP酶Ran结合。在本研究中，我们使用干扰NTF2与GDP-Ran相互作用的定点突变体来探究NTF2-Ran相互作用在核蛋白输入中的作用。这些突变体的设计基于NTF2的X射线晶体结构，主要集中在分子疏水腔及其周围的保守残基上。突变的NTF2 cDNA在大肠杆菌中表达。纯化后的突变蛋白保留了与FxFG重复核孔蛋白的相互作用，但NTF2腔周围带负电荷残基或腔本身残基中的几个突变体在体外无法结合GDP-Ran。E42K突变蛋白的晶体结构仅在该侧链上显示出显著的结构变化，表明它直接参与了与GDP-Ran的相互作用。在通透细胞的核蛋白输入实验中，只有野生型NTF2和能结合GDP-Ran的突变体具有功能。此外，当在体外无法结合GDP-Ran的NTF2 E42K和D92N/D94N NTF2突变体替代染色体酵母NTF2时，酵母细胞无法存活，而用野生型人NTF2替代的酵母仍能存活。我们得出结论，NTF2与GDP-Ran之间的相互作用对于高效的核蛋白输入很重要。
