Quimby B B, Lamitina T, L'Hernault S W, Corbett A H
Department of Biochemistry, Emory University School of Medicine and the Department of Biology, Emory University, Atlanta, Georgia 30322, USA.
J Biol Chem. 2000 Sep 15;275(37):28575-82. doi: 10.1074/jbc.M005055200.
The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-beta. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis in Caenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-beta-mediated protein import.
小GTP酶Ran对于几乎所有的核质运输事件都是必不可少的。据推测，Ran通过在细胞质和核质之间建立Ran梯度来驱动大分子进出细胞核的定向运输。尽管Ran在细胞核和细胞质之间穿梭，但在稳态时它集中在细胞核中。我们发现，在出芽酵母酿酒酵母中，核运输因子2（NTF2）是将Ran集中在细胞核中所必需的。为了分析NTF2将Ran导入细胞核的机制，我们使用了多种核运输因子的突变体，并对NTF2复合物进行了生化分析。我们发现，当输入蛋白介导的蛋白质输入被破坏时，Ran仍集中在细胞核中，并证明NTF2不会与运输受体输入蛋白-β形成稳定的复合物。与NTF2在建立和维持Ran梯度中的关键作用一致，我们表明NTF2是秀丽隐杆线虫早期胚胎发育所必需的。我们的数据区分了NTF2导入Ran的两种可能机制，并证明Ran的导入独立于输入蛋白-β介导的蛋白质输入。
