Probing nuclear pore complex architecture with proximity-dependent biotinylation
Probing nuclear pore complex architecture with proximity-dependent biotinylation
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DOI：
10.1073/pnas.1406459111
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发表时间：
2014-06-17
影响因子：
11.1
通讯作者：
Roux, Kyle J.
中科院分区：
文献类型：
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作者：
Kim, Dae In;Birendra, K. C.;Roux, Kyle J.
邻近依赖性生物素鉴定（BioID）是用于鉴定体内发生的蛋白质缔合的方法。通过将混杂的生物素连接酶与活细胞中表达的感兴趣的蛋白质融合，BioID允许在限定的标记期间标记邻近的蛋白质。在这项研究中，我们使用BioID来研究人类核孔复合物（NPC），这是真核生物中最大的大分子组装体之一。NPC锚定在核膜内，介导许多细胞组分的核质运输。我们将BioID应用于Nup 107 -160复合物和Nup 93复合物（两种保守的NPC亚复合物）的组分。根据这些BioID融合蛋白在NPC中的位置，检测到一组明显不同的NPC成分。通过将BioID应用于位于非常稳定的Nup 107 -160亚复合物中的几种成分，我们改进了对这种高度保守的亚复合物的理解，部分原因是证明了Nup 43与Nup 85的直接相互作用。此外，通过使用非常稳定的Nup 107 -160结构作为分子标尺，我们定义了BioID的实际标记半径。这些研究进一步加深了我们对人类NPC组织的理解，并表明BioID是探索活细胞中大型蛋白质组装体的组成和组织的有价值的工具。
Proximity-dependent biotin identification (BioID) is a method for identifying protein associations that occur in vivo. By fusing a promiscuous biotin ligase to a protein of interest expressed in living cells, BioID permits the labeling of proximate proteins during a defined labeling period. In this study we used BioID to study the human nuclear pore complex (NPC), one of the largest macromolecular assemblies in eukaryotes. Anchored within the nuclear envelope, NPCs mediate the nucleocytoplasmic trafficking of numerous cellular components. We applied BioID to constituents of the Nup107-160 complex and the Nup93 complex, two conserved NPC subcomplexes. A strikingly different set of NPC constituents was detected depending on the position of these BioID-fusion proteins within the NPC. By applying BioID to several constituents located throughout the extremely stable Nup107-160 subcomplex, we refined our understanding of this highly conserved subcomplex, in part by demonstrating a direct interaction of Nup43 with Nup85. Furthermore, by using the extremely stable Nup107-160 structure as a molecular ruler, we defined the practical labeling radius of BioID. These studies further our understanding of human NPC organization and demonstrate that BioID is a valuable tool for exploring the constituency and organization of large protein assemblies in living cells.