Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran
Nuclear protein import is decreased by engineered mutants of nuclear transport factor 2 (NTF2) that do not bind GDP-Ran
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DOI：
10.1006/jmbi.1997.1255
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发表时间：
1997-10-10
影响因子：
5.6
通讯作者：
Stewart, M
中科院分区：
文献类型：
--
作者：
Clarkson, WD;Corbett, AH;Stewart, M
核转运因子2（NTF 2）与核蛋白输入的易位阶段相关，并结合到含有富含苯丙氨酸的重复序列的核孔蛋白（核孔蛋白）和Ras家族的GTCRan Ran。在本研究中，我们使用NTF 2的定点突变体来干扰其与GDP-Ran的相互作用，从而探索NTF 2-Ran相互作用在核蛋白输入中的作用。这些突变体的设计是基于NTF 2的X射线晶体结构，并集中在分子疏水腔中和周围的保守残基上。在大肠杆菌中表达突变NTF 2 cDNA。纯化的突变体蛋白保留与FxFG重复核孔蛋白的相互作用，但在NTF 2腔周围的带负电荷的残基或在腔本身的残基中的几个突变体不能在体外结合GDP-Ran。E42 K突变蛋白的晶体结构仅在该侧链中显示出显著的结构变化，表明其直接参与了与GDP-Ran的相互作用。在透化细胞核蛋白导入测定中，仅野生型NTF 2和结合GDP-Ran的突变体是功能性的。此外，当体外不能结合GDP-Ran的NTF 2 E42 K和D92 N/D94 N NTF 2突变体取代染色体酵母NTF 2时，酵母细胞变得不能存活，而用人NTF 2取代的酵母保持存活。我们的结论是，NTF 2和GDP-Ran之间的相互作用是重要的有效的核蛋白输入。(C)出版社：Academic Press Limited。
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 tl-tis 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 in 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 human NTF2 remained viable. We conclude that interaction between NTF2 and GDP-Ran is important for efficient nuclear protein import. (C) 1997 Academic Press Limited.