Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.
Rna1p, a Ran/TC4 GTPase activating protein, is required for nuclear import.
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DOI：
10.1083/jcb.130.5.1017
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发表时间：
1995-09
期刊：
影响因子：
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通讯作者：
Silver PA
中科院分区：
文献类型：
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作者：
Corbett AH;Koepp DM;Schlenstedt G;Lee MS;Hopper AK;Silver PA
酿酒酵母基因RNA1编码一种与哺乳动物RAN/TC4 GTP酶激活蛋白具有广泛同源性的蛋白。利用间接免疫荧光显微镜，我们已经证明了rNA1-1突变细胞在核输入几种蛋白质方面存在缺陷。当使用与天然绿色荧光蛋白融合的核蛋白在活细胞中检测核进口时，也得到了同样的结果。这些发现表明，RNA1p在蛋白质跨核膜运输中发挥了作用。为了更直接地研究这一作用，使用了一种体外导入试验，该试验监测了一种荧光标记的底物进入半完整酵母细胞细胞核的情况。进入细胞核需要外源胞浆的加入。结果表明，与野生型胞浆相比，从rNA1-1突变细胞中提取的提取物不能支持荧光标记的底物进入感受核。免疫印迹显示，这些突变型提取液中的RNA1p被耗尽。然而，当纯化的RNA1p被加回这些提取物中时，进口活性以剂量依赖的方式恢复。这些结果表明，RNA1p在蛋白质进入细胞核的过程中起着直接作用。
The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rna1-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rna1p in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semi- intact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rna1-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rna1p. However, when purified Rna1p is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rna1p plays a direct role in the import of proteins into the nucleus.