Plafker K, Macara I G
Markey Center for Cell Signaling and Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908, USA.
Mol Cell Biol. 2000 May;20(10):3510-21. doi: 10.1128/MCB.20.10.3510-3521.2000.
The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)-RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin beta. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.
Ran结合蛋白RanBP1定位于间期细胞的胞质溶胶中。RanBP1 C末端附近富含亮氨酸的核输出信号（NES）对于维持这种分布至关重要。我们现在表明，RanBP1在经输出抑制剂雷帕霉素B处理的细胞的细胞核中积累，并且核质Ran:GTP梯度的崩溃导致RanBP1在核膜两侧达到平衡。低温可阻止RanBP1的核积累，这表明其入核并非通过简单扩散发生。缺乏NES的谷胱甘肽S-转移酶（GST）-RanBP1(1-161)在细胞质显微注射后会在细胞核中积累。在通透细胞中，GST-RanBP1(1-161)的核积累需要核内Ran:GTP，但不受Ran的显性干扰G19V突变体的抑制。添加外源性核转运蛋白/输入蛋白或RCC1可增强核积累，这两者也会增强核内Ran的积累。入核与Ran浓度相关。值得注意的是，尽管RanBP1的E37K突变体能够与Ran和输入蛋白β形成三元复合物，但在任何测试条件下都不会进入细胞核。这些数据表明，RanBP1通过一种活跃的、非经典机制穿过核孔，并且核积累需要Ran:GTP。RanBP1的穿梭可能起到清除核孔中Ran:GTP的作用，以防止输入货物过早地从转运受体上释放。
