Furuta Maiko, Hori Tetsuya, Fukagawa Tatsuo
Department of Molecular Genetics, National Institute of Genetics, and Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka 411-8540, Japan.
Department of Molecular Genetics, National Institute of Genetics, and Graduate University for Advanced Studies (SOKENDAI), Mishima, Shizuoka 411-8540, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.
Mol Biol Cell. 2016 Jan 15;27(2):371-81. doi: 10.1091/mbc.E15-07-0497. Epub 2015 Nov 12.
RCC1, a guanine nucleotide exchange factor of the small GTPase Ran, plays various roles throughout the cell cycle. However, the functions of RCC1 in biological processes in vivo are still unclear. In particular, although RCC1 has multifunctional domains, the biological significance of each domain is unclear. To examine each domain of RCC1, we established an RCC1 conditional knockout chicken DT40 cell line and introduced various RCC1 mutants into the knockout cells. We found that nuclear reformation did not occur properly in RCC1-deficient cells and examined whether specific RCC1 mutants could rescue this phenotype. Surprisingly, we found that neither the nuclear localization signal nor the chromatin-binding domain of RCC1 is essential for its function. However, codisruption of these domains resulted in defective nuclear reformation, which was rescued by artificial nuclear localization of RCC1. Our data indicate that chromatin association of RCC1 during mitosis is crucial for its proper nuclear localization in the next interphase. Moreover, proper nuclear localization of RCC1 in interphase is essential for its function through its nucleotide exchange activity.
RCC1是小GTP酶Ran的鸟嘌呤核苷酸交换因子，在整个细胞周期中发挥着多种作用。然而，RCC1在体内生物学过程中的功能仍不清楚。特别是，尽管RCC1具有多功能结构域，但每个结构域的生物学意义尚不清楚。为了研究RCC1的每个结构域，我们建立了一个RCC1条件性敲除鸡DT40细胞系，并将各种RCC1突变体导入敲除细胞中。我们发现RCC1缺陷细胞中核重建不能正常发生，并研究了特定的RCC1突变体是否能挽救这种表型。令人惊讶的是，我们发现RCC1的核定位信号和染色质结合结构域对其功能都不是必需的。然而，这些结构域的共同破坏导致核重建缺陷，通过RCC1的人工核定位可以挽救这种缺陷。我们的数据表明，有丝分裂期间RCC1与染色质的结合对于其在下一个间期正确的核定位至关重要。此外，RCC1在间期的正确核定位通过其核苷酸交换活性对其功能至关重要。
