Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97
Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97
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DOI：
10.1074/jbc.272.10.6818
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发表时间：
1997-03-07
影响因子：
4.8
通讯作者：
Adam, SA
中科院分区：
文献类型：
--
作者：
Chi, NC;Adam, EJH;Adam, SA
将核蛋白从细胞质转运到细胞核需要多种蛋白质，包括核定位序列受体（NLS 受体）、p97、小核 GTPase Ran/TC4 和几种核孔蛋白。 Ran 与 p97 的相互作用被认为可以调节这些运输成分的相互作用。 Ran-GTP 单独结合 p97，但 Ran-GDP 仅与 RanBP1 一起结合 p97。使用定点诱变和缺失分析，我们在 p97 上鉴定了 Ran-GTP 和 Ran-GDP/RanBP1 的两个不同但重叠的结合域。 p97 中的短酸性序列是 Ran-GDP/RanBP1 结合域的一部分，其功能可能与 Ran 中的 C 端酸性序列类似。 p97 中的保守半胱氨酸残基 Cys-158 是结合 Ran-GDP/RanBP1 所必需的，但不需要 Ran-GTP 与 p97 的结合。在透化细胞蛋白导入测定中，用丙氨酸取代 Cys-158 的突变体 p97 在 NLS 受体和 Ran 存在的情况下无法支持导入。这些结果支持 Ran-GDP 在受体复合物中的直接积极作用，并提供证据表明小 GTP 酶下游效应子的活性可能受到 GTP 和 GDP 结合形式的蛋白质的调节。
Several proteins are required for the transport of nuclear proteins from the cytoplasm to the nucleus, including the nuclear location sequence receptor (NLS receptor), p97, the small nuclear GTPase Ran/TC4, and several nucleoporins. The interaction of Ran with p97 is thought to regulate the interaction of these transport components. Ran-GTP alone binds p97, but Ran-GDP binds p97 only in conjunction with RanBP1. Using site-directed mutagenesis and deletion analysis, we have identified two distinct but overlapping binding domains for Ran-GTP and Ran-GDP/RanBP1 on p97. A short acidic sequence in p97 is part of the Ran-GDP/RanBP1 binding domain, possibly functioning in a similar manner as the C-terminal acidic sequence in Ran. A conserved cysteine residue in p97, Cys-158, is required for binding Ran-GDP/RanBP1, but not for binding of Ran-GTP to p97. In a permeabilized cell protein import assay, a mutant p97 with alanine substituted for Cys-158 is unable to support import in the presence of NLS receptor and Ran. These results support a direct active role for Ran-GDP in the receptor complex and provide evidence that the activity of downstream effecters of small GTPases may be regulated by both GTP- and GDP-bound forms of the protein.