Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import.
Evidence using a green fluorescent protein-glucocorticoid receptor chimera that the Ran/TC4 GTPase mediates an essential function independent of nuclear protein import.
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DOI：
10.1083/jcb.133.5.985
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发表时间：
1996-06
期刊：
影响因子：
--
通讯作者：
Macara IG
中科院分区：
文献类型：
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作者：
Carey KL;Richards SA;Lounsbury KM;Macara IG
Ran/TC4 GTPase 是透化细胞测定中人工亲核细胞核积累所必需的。为了使用哺乳动物细胞研究生理完整环境中的 Ran 功能，我们检查了几种 Ran 突变体对细胞生长和糖皮质激素受体-绿色荧光蛋白融合 (GR-GFP) 核转位的影响。糖皮质激素受体在没有配体的情况下位于胞质中，但在与激动剂地塞米松结合时易位至细胞核。转染幼仓鼠肾细胞 (BHK21) 后，通过直接荧光显微镜可在活细胞中检测到 GR-GFP。添加地塞米松导致嵌合蛋白从胞质溶胶快速易位到细胞核中（t1/2 大约 5 分钟）。与表位标记的野生型 Ran 共转染导致 HA1-Ran 的表达比内源蛋白的水平高约 1.6 倍，但它对 GR-GFP 的核输入没有有害影响。然而，Ran 突变体 G19V、T24N 或 COOH 末端缺失 (delta C) 突变体的表达显着减少了细胞核中 GR-GFP 的积累。 Ran 的 L43E 突变体对核 GR-GFP 导入没有显着影响。将重组 Ran 突变体显微注射到表达 GR-GFP 的细胞中后获得了相同的结果。值得注意的是，所有 Ran 突变体，包括 L43E，都强烈抑制细胞生长。这些结果证明了 GR-GFP 在核运输实时成像中的用途。他们还表明，多种类型的 Ran 突变体在此过程中发挥主导作用，并且正常的 Ran 功能需要在蛋白质的 GTP 和 GDP 结合状态之间循环。最重要的是，L43E Ran 突变体的结果提供了强有力的证据，证明 Ran 介导了独立于核蛋白输入的细胞活力所必需的功能。
The Ran/TC4 GTPase is required for the nuclear accumulation of artificial karyophiles in permeabilized cell assays. To investigate Ran function in a physiologically intact setting using mammalian cells, we examined the effects of several Ran mutants on cell growth and on the nuclear translocation of a glucocorticoid receptor-green fluorescent protein fusion (GR-GFP). Glucocorticoid receptor is cytosolic in the absence of ligand, but translocates to the nucleus on binding the agonist dexamethasone. After transfection into baby hamster kidney cells (BHK21), GR-GFP was detectable in living cells by direct fluorescence microscopy. Addition of dexamethasone caused a rapid translocation of the chimeric protein from the cytosol into the nucleus (t1/2 approximately 5 min). Cotransfection with epitope-tagged, wild- type Ran led to expression of HA1-Ran that was approximately 1.6-fold higher than the level of the endogenous protein, but it had no deleterious effect on nuclear import of the GR-GFP. However, expression of the Ran mutants G19V, T24N, or a COOH-terminal deletion (delta C) mutant dramatically reduced the accumulation of GR-GFP in the nuclei. An L43E mutant of Ran was without significant effect on nuclear GR-GFP import. Identical results were obtained following micro-injection of recombinant Ran mutants into cells expressing GR-GFP. Significantly, all of the Ran mutants, including L43E, strongly inhibited cell growth. These results demonstrate the use of GR-GFP in real-time imaging of nuclear transport. They also show that multiple types of Ran mutant exert dominant effects on this process, and that normal Ran function requires cycling between the GTP- and GDP-bound states of the protein. Most importantly, the results with the L43E Ran mutant provide strong evidence that Ran mediates a function essential to cell viability that is independent of nuclear protein import.