Richards S A, Lounsbury K M, Macara I G
Department of Pathology, University of Vermont Medical College, Burlington 05405-0068, USA.
J Biol Chem. 1995 Jun 16;270(24):14405-11. doi: 10.1074/jbc.270.24.14405.
Ran/TC4 is a member of the Ras superfamily of GTPases. It is unusual in being predominantly nuclear and because it possesses an acidic -DEDDDL sequence instead of a consensus prenylation domain at the C terminus. Ran is required for nuclear protein import and cell cycle progression, and has been implicated in mRNA processing and export and DNA replication. The inhibition of cell cycle progression by a dominant gain-of-function mutant of Ran has been shown to be abrogated by removal of the -DEDDDL sequence, suggesting that this domain is essential for Ran function. We demonstrate here that the -DEDDDL sequence stabilizes GDP binding to Ran, and that the domain is required for high affinity interaction with a Ran-binding protein, HTF9A/RanBP1. HTF9A functions as a co-stimulator of Ran-GAP (GTPase activating protein) activity on wild-type Ran, but in the absence of the acidic C terminus of Ran, HTF9A behaves as a Ran-GAP inhibitor. An antibody directed against the C-terminal region preferentially recognizes the GTP-bound form of Ran, suggesting that this domain undergoes a nucleotide-dependent conformational change. The results suggest that the acidic C-terminal domain is important in modulating the interaction of Ran with regulatory factors, and implicate Ran-binding proteins in mediating the effects of Ran on cell cycle progression.
Ran/TC4是GTP酶Ras超家族的成员。它不同寻常之处在于主要定位于细胞核，并且在其C末端具有一个酸性的 -DEDDDL序列，而非共有异戊二烯化结构域。Ran是核蛋白导入和细胞周期进程所必需的，并且与mRNA加工、输出及DNA复制有关。已表明，去除 -DEDDDL序列可消除Ran的显性功能获得型突变体对细胞周期进程的抑制作用，这表明该结构域对Ran的功能至关重要。我们在此证明，-DEDDDL序列可稳定Ran与GDP的结合，并且该结构域是与Ran结合蛋白HTF9A/RanBP1进行高亲和力相互作用所必需的。HTF9A可作为野生型Ran的Ran-GAP（GTP酶激活蛋白）活性的共刺激因子，但在Ran缺乏酸性C末端时，HTF9A表现为Ran-GAP抑制剂。一种针对C末端区域的抗体优先识别Ran的GTP结合形式，这表明该结构域会发生依赖核苷酸的构象变化。结果表明，酸性C末端结构域在调节Ran与调节因子的相互作用中很重要，并且提示Ran结合蛋白在介导Ran对细胞周期进程的影响中发挥作用。
