Ohba T, Seki T, Azuma Y, Nishimoto T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.
J Biol Chem. 1996 Jun 21;271(25):14665-7. doi: 10.1074/jbc.271.25.14665.
RCC1 is a guanine nucleotide exchanging factor acting on nuclear G protein Ran. Premature chromatin condensation occurs in the temperature-sensitive rcc1- mutant of the BHK21 cell line, tsBN2, at the restrictive temperature. This observation can be explained if the premature activation of MPF is normally inhibited by GTP-Ran. In the absence of RCC1, GDP-Ran predominates, resulting in MPF activation. However, experiments with Ran mutants to determine whether GTP- or GDP-Ran prevents activation of MPF have yielded conflicting results. In order to clarify this point, we have microinjected nucleotide-bound Ran, instead of mutated Ran, into the nuclei of tsBN2 cells treated to reduce RCC1-mediated guanine nucleotide exchange. GTP-Ran, GTPgammaS-Ran, and GDP-Ran all inhibited chromatin condensation. However, the inhibition of chromatin condensation by GDP-Ran could be completely abolished by co-injection with GDP, but not GTP. Thus, we conclude that GTP-Ran blocks the activation of MPF and that hydrolysis of GTP is not required to prevent MPF activation.
RCC1是一种作用于细胞核G蛋白Ran的鸟嘌呤核苷酸交换因子。在限制温度下，BHK21细胞系tsBN2的温度敏感型rcc1-突变体中会发生染色质过早凝聚。如果MPF的过早激活通常受到GTP-Ran的抑制，那么这一观察结果就能得到解释。在缺乏RCC1的情况下，GDP-Ran占主导地位，导致MPF激活。然而，用Ran突变体进行的实验，以确定是GTP-Ran还是GDP-Ran能阻止MPF激活，却得出了相互矛盾的结果。为了阐明这一点，我们将结合了核苷酸的Ran而非突变的Ran显微注射到经处理以减少RCC1介导的鸟嘌呤核苷酸交换的tsBN2细胞的细胞核中。GTP-Ran、GTPγS-Ran和GDP-Ran均抑制染色质凝聚。然而，GDP-Ran对染色质凝聚的抑制作用可通过与GDP共同注射而完全消除，但与GTP共同注射则不能。因此，我们得出结论，GTP-Ran可阻断MPF的激活，并且阻止MPF激活并不需要GTP水解。
