The Nup358-RanGAP complex is required for efficient importin α/β-dependent nuclear import
The Nup358-RanGAP complex is required for efficient importin α/β-dependent nuclear import
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DOI：
10.1091/mbc.e07-12-1279
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发表时间：
2008-05-01
影响因子：
3.3
通讯作者：
Kehlenbach, Ralph H.
中科院分区：
文献类型：
--
作者：
Hutten, Saskia;Flotho, Annette;Kehlenbach, Ralph H.
在脊椎动物细胞中，核孔蛋白Nup358/RanBP2是核孔复合物进入细胞质的丝的主要组成部分。Nup358与SUMOylated RanGAP1形成复合物，RanGAP1是Ran的GTPase激活蛋白。RanGAP1在RanGTP跨越核膜梯度的建立中起关键作用，因此在大多数核细胞质运输途径中起关键作用。在这里，我们结合体内和体外方法研究了Nup358-RanGAP1复合物和可溶性RanGAP1在核蛋白转运中的作用。RNA干扰导致Nup358缺失，导致各种报告蛋白的α / β依赖性核输入明显减少。在体外，通过在转运反应中加入进口蛋白β、RanBP1和/或RanGAP1，可以部分恢复转运。在完整的nup358缺失细胞中，输入蛋白β的过度表达强烈刺激核输入，这表明在nup358浓度降低时，转运受体是最限速的因素。作为一种替代方法，我们使用抗体抑制实验。抗RanGAP1的抗体抑制了可溶性和核孔相关的RanGAP1的酶活性，以及核进出口。虽然出口可以通过可溶性RanGAP完全恢复，但进口只能部分恢复。综上所述，这些数据表明Nup358-RanGAP1复合物具有双重功能，作为进口β循环和新型进口复合物改造的协调者。
In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.