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首页> 外文期刊>Nucleus >Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin
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Analysis of the initiation of nuclear pore assembly by ectopically targeting nucleoporins to chromatin

机译：通过将核孔蛋白异位靶向染色质来分析核孔装配的启动

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Nuclear pore complexes (NPCs) form the gateway to the nucleus, mediating virtually all nucleocytoplasmic trafficking. Assembly of a nuclear pore complex requires the organization of many soluble sub-complexes into a final massive structure embedded in the nuclear envelope. By use of a LacI/LacO reporter system, we were able to assess nucleoporin (Nup) interactions, show that they occur with a high level of specificity, and identify nucleoporins sufficient for initiation of the complex process of NPC assembly in vivo . Eleven nucleoporins from different sub-complexes were fused to LacI-CFP and transfected separately into a human cell line containing a stably integrated LacO DNA array. The LacI-Nup fusion proteins, which bound to the array, were examined for their ability to recruit endogenous nucleoporins to the intranuclear LacO site. Many could recruit nucleoporins of the same sub-complex and a number could also recruit other sub-complexes. Strikingly, Nup133 and Nup107 of the Nup107/160 subcomplex and Nup153 and Nup50 of the nuclear pore basket recruited a near full complement of nucleoporins to the LacO array. Furthermore, Nup133 and Nup153 efficiently targeted the LacO array to the nuclear periphery. Our data support a hierarchical, seeded assembly pathway and identify Nup133 and Nup153 as effective “seeds” for NPC assembly. In addition, we show that this system can be applied to functional studies of individual nucleoporin domains as well as to specific nucleoporin disease mutations. We find that the R391H cardiac arrhythmia/sudden death mutation of Nup155 prevents both its subcomplex assembly and nuclear rim targeting of the LacO array.
机译：核孔复合物（NPC）形成了通往核的通道，几乎介导了所有核质运输。核孔复合物的组装需要将许多可溶性亚复合物组织成嵌入核膜中的最终块状结构。通过使用LacI / LacO报告系统，我们能够评估核孔蛋白（Nup）相互作用，显示它们以高水平的特异性发生，并鉴定出足以在体内启动NPC组装复杂过程的核孔蛋白。 。将来自不同亚复合物的11种核孔蛋白与LacI-CFP融合，并分别转染到包含稳定整合的LacO DNA阵列的人细胞系中。检查绑定到阵列的LacI-Nup融合蛋白的能力，以将其内源性核孔蛋白募集到核内LacO位点。许多人可以募集相同亚复合物的核孔蛋白，许多人也可以募集其他亚复合物。令人惊讶的是，Nup107 / 160亚复合物的Nup133和Nup107以及核孔篮的Nup153和Nup50向LacO阵列募集了几乎全部的核孔蛋白。此外，Nup133和Nup153有效地将LacO阵列靶向核外围。我们的数据支持分层的种子组装路径，并确定Nup133和Nup153是NPC组装的有效“种子”。此外，我们证明了该系统可以用于单个核孔蛋白结构域的功能研究以及特定的核孔蛋白疾病突变。我们发现Nup155的R391H心律不齐/猝死突变可阻止其亚复合体装配和LacO阵列的核边缘靶向。

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