Lounsbury K M, Macara I G
Department of Pathology, University of Vermont, and the Vermont Cancer Center, Burlington 05405, USA.
J Biol Chem. 1997 Jan 3;272(1):551-5. doi: 10.1074/jbc.272.1.551.
The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore. RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran. RanBP1 also promotes the binding of Ran to karyopherin beta (also called importin beta and p97) and is a co-stimulator of RanGAP activity. Yeast karyopherin beta inhibits the GTP hydrolysis by Ran catalyzed by RanGAP. To further define the roles of RanBP1 and karyopherin beta in Ran function, we explored the effects of RanBP1 and karyopherin beta on mammalian proteins known to regulate Ran. Like RanBP1, karyopherin beta prevented the release of GTP from Ran stimulated by RCC1 or EDTA. As with the yeast protein, mammalian karyopherin beta completely blocked RanGAP activity. However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity. Kinetic analysis of the effects on RanGAP activity by karyopherin beta and RanBP1 revealed a combination of competitive and noncompetitive interactions. Solution binding assays confirmed the ability of RanBP1 to associate with Ran and karyopherin beta in a ternary complex, and RanBP1 binding was not competed out by the addition of karyopherin beta. These results demonstrate that RanBP1 and karyopherin beta interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to karyopherin beta.
含有核定位信号的蛋白质的核积累需要Ran GTP酶以及在核孔处组装的蛋白质复合物。RanBP1是一种胞质Ran结合蛋白，可抑制RCC1刺激的Ran从GTP的释放。RanBP1还促进Ran与核转运蛋白β（也称为输入蛋白β和p97）的结合，并且是RanGAP活性的共刺激因子。酵母核转运蛋白β抑制RanGAP催化的Ran的GTP水解。为了进一步确定RanBP1和核转运蛋白β在Ran功能中的作用，我们研究了RanBP1和核转运蛋白β对已知调节Ran的哺乳动物蛋白的影响。与RanBP1一样，核转运蛋白β可阻止RCC1或EDTA刺激的Ran从GTP的释放。与酵母蛋白一样，哺乳动物核转运蛋白β完全阻断了RanGAP活性。然而，在此测定中加入RanBP1可部分挽救被抑制的RanGAP活性。对核转运蛋白β和RanBP1对RanGAP活性影响的动力学分析揭示了竞争性和非竞争性相互作用的组合。溶液结合试验证实了RanBP1在三元复合物中与Ran和核转运蛋白β结合的能力，并且加入核转运蛋白β不会竞争RanBP1的结合。这些结果表明RanBP1和核转运蛋白β与Ran的不同位点相互作用，并表明RanBP1通过允许RanGAP介导的与核转运蛋白β复合的Ran上的GTP水解在核运输中起重要作用。
