Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.
Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor.
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DOI：
10.1083/jcb.123.6.1649
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发表时间：
1993-12
期刊：
影响因子：
--
通讯作者：
Gerace L
中科院分区：
文献类型：
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作者：
Melchior F;Paschal B;Evans J;Gerace L
我们利用洋地黄素通透性细胞和外源胞浆的体外转运系统，研究了GTP酶在核蛋白输入中的可能参与。该系统中的转运是用一种新的基于ELISA法的检测方法来测量的，该检测方法允许快速定量分析。GTP、伽马、S等GTP的非水解性类似物能迅速抑制体外核进口速率。S的转运抑制作用依赖于通透性细胞和胞浆的浓度，并被胞浆因子(S)强烈增强。在分子筛层析中，20-30-kD组分是导致这种抑制作用的主要胞质组分。此外，这种20-30 kD馏分中的一种成分(S)本身就是有效的核进口所必需的。与细菌表达的蛋白质的生化互补表明，这个对S敏感的必需GTP运输因子是RAN/TC4，这是一个先前描述的Ras超家族的GTP酶，存在于细胞核和细胞质中。RAN/TC4及其鸟嘌呤核苷酸释放蛋白RCC1参与了DNA复制、细胞周期检查点控制以及RNA合成、加工和输出。我们的结果表明，RAN/TC4有助于将核蛋白输入与其他核活动整合在一起。
We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin- permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.