Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1
Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1
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DOI：
10.1128/mcb.20.10.3510-3521.2000
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发表时间：
2000-05-01
影响因子：
5.3
通讯作者：
Macara, IG
中科院分区：
文献类型：
--
作者：
Plafker, K;Macara, IG
Ran 结合蛋白 RanBP1 定位于间期细胞的胞浆。 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 在用输出抑制剂瘦霉素 B 处理的细胞核中积聚！核质 Ran:GTP 梯度的崩溃有助于 RanBP1 在核膜上的平衡。低温阻止 RanBP1 的核积累，表明输入不是通过简单扩散发生的。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积聚在细胞核中。在透化细胞中，GST-RanBP1 (1-161) 的核积累需要核 Ran:GTP，但不会被 Ran 的显性干扰 G19V 突变体抑制。添加外源核转运蛋白/导入蛋白或 RCC1 可以增强核积累，这两种物质也可以增强核 Ran 积累。进口与 Ran 浓度相关。值得注意的是，RanBP1 的 E37K：突变体在任何测试条件下都不会导入细胞核，尽管它可以与 Ran 和导入蛋白 beta 形成三元复合物。这些数据表明 RanBP1 通过主动的非经典机制通过孔易位，并且需要 Ran:GTP 进行核积累。 RanBP1 的穿梭可能起到清除 Ran:GTP 核孔的作用，以防止转运受体过早释放输入货物。
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 lends 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.