State Key Laboratory of Bio-membrane and Membrane Biotechnology and Key Laboratory of Cell Proliferation and Differentiation of Ministry of Education, College of Life Sciences, Peking University, Beijing 100871, China.
Cell Res. 2012 Nov;22(11):1562-75. doi: 10.1038/cr.2012.113. Epub 2012 Jul 31.
The mechanism for nuclear envelope (NE) assembly is not fully understood. Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts. We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-α binds the chromatin NLS proteins rapidly. Meanwhile, importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-α on the chromatin NLS proteins, importin-β targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran-GTP on the chromatin generated by RCC1, importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.
核膜（NE）组装的机制尚不完全清楚。Importin-β 和小分子 GTPase Ran 已被牵连到 NE 组装过程的空间调节中。在这里，我们报告说，染色质结合的 NLS（核定位序列）蛋白通过 Importin-α 和 -β 为 NE 前体膜泡和核孔蛋白提供停靠位点，从而在 Xenopus 卵提取物中的 NE 组装过程中提供停靠位点。我们表明，随着丰富的 NLS 蛋白（如核质蛋白和组蛋白）快速募集到提取物中去膜精子染色质，Importin-α 快速结合染色质 NLS 蛋白。同时，Importin-β 结合细胞质 NE 前体膜泡和核孔蛋白。通过与染色质 NLS 蛋白上的 Importin-α 相互作用，Importin-β 将膜泡和核孔蛋白靶向到染色质表面。一旦在由 RCC1 产生的染色质上遇到 Ran-GTP，Importin-β 优先结合 Ran-GTP 并释放膜泡和核孔蛋白进行 NE 组装。通过用过量的可溶性 NLS 蛋白阻断 Importin-α 和 NLS 蛋白之间的相互作用，或通过从提取物中耗尽 Importin-β，可破坏 NE 组装。我们的发现揭示了 Xenopus 卵提取物中 NE 组装的一种新的分子机制。
