A MUTANT FORM OF THE RAN/TC4 PROTEIN DISRUPTS NUCLEAR FUNCTION IN XENOPUS-LAEVIS EGG EXTRACTS BY INHIBITING THE RCC1 PROTEIN, A REGULATOR OF CHROMOSOME CONDENSATION
A MUTANT FORM OF THE RAN/TC4 PROTEIN DISRUPTS NUCLEAR FUNCTION IN XENOPUS-LAEVIS EGG EXTRACTS BY INHIBITING THE RCC1 PROTEIN, A REGULATOR OF CHROMOSOME CONDENSATION
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DOI：
10.1002/j.1460-2075.1994.tb06911.x
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发表时间：
1994-12-01
期刊：
影响因子：
11.4
通讯作者：
NISHIMOTO, T
中科院分区：
文献类型：
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作者：
DASSO, M;SEKI, T;NISHIMOTO, T
Ran蛋白是一种小的GTPase，涉及大量的核过程，包括运输，RNA加工和细胞周期检查点控制，类似的核活动谱已被证明需要RCC1， Ran的鸟嘌呤核苷酸交换因子（GEF），我们使用非洲爪蟾卵提取系统和纯化蛋白的体外分析来研究Ran或RCC1如何参与这些众多的过程，在这些研究中，我们使用Ran突变蛋白来干扰核组装和功能，添加细菌表达的Ran突变形式（T24N-Ran），预计主要处于gdp结合状态，严重破坏了提取液中的核组装和DNA复制。我们进一步研究了T24N-Ran破坏正常核活性的分子机制，发现T24N-Ran与提取液中的RCC1蛋白紧密结合，导致其作为GEF失活。通过添加过量的RCC1， t24n -Ran阻断的间期提取物可以恢复从去膜精子染色质中组装细胞核并复制DNA的能力，从而提供过量的GEF活性。相反，在缺乏RCC1的提取物中，通过添加高水平的野生型gtp结合的Ran蛋白，核组装和DNA复制都得到了恢复。这表明RCC1除了在爪蟾间期提取物中作为GEF的作用外，没有其他重要功能。
The Ran protein is a small GTPase that has been implicated in a large number of nuclear processes including transport, RNA processing and cell cycle checkpoint control, A similar spectrum of nuclear activities has been shown to require RCC1, the guanine nucleotide exchange factor (GEF) for Ran, We have used the Xenopus laevis egg extract system and in vitro assays of purified proteins to examine how Ran or RCC1 could be involved in these numerous processes, In these studies, we employed mutant Ran proteins to perturb nuclear assembly and function, The addition of a bacterially expressed mutant form of Ran (T24N-Ran), which was predicted to be primarily in the GDP-bound state, profoundly disrupted nuclear assembly and DNA replication in extracts, We further examined the molecular mechanism by which T24N-Ran disrupts normal nuclear activity and found that T24N-Ran binds tightly to the RCC1 protein within the extract, resulting in its inactivation as a GEF. The capacity of T24N-Ran-blocked interphase extracts to assemble nuclei from de-membranated sperm chromatin and to replicate their DNA could be restored by supplementing the extract with excess RCC1 and thereby providing excess GEF activity, Conversely, nuclear assembly and DNA replication were both rescued in extracts lacking RCC1 by the addition of high levels of wild-type GTP-bound Ran protein, indicating that RCC1 does not have an essential function beyond its role as a GEF in interphase Xenopus extracts.