In situ structural analysis of the human nuclear pore complex.
In situ structural analysis of the human nuclear pore complex.
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DOI：
10.1038/nature15381
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发表时间：
2015-10-01
期刊：
影响因子：
64.8
通讯作者：
Beck M
中科院分区：
文献类型：
--
作者：
von Appen A;Kosinski J;Sparks L;Ori A;DiGuilio AL;Vollmer B;Mackmull MT;Banterle N;Parca L;Kastritis P;Buczak K;Mosalaganti S;Hagen W;Andres-Pons A;Lemke EA;Bork P;Antonin W;Glavy JS;Bui KH;Beck M
核孔复合物是真核细胞的重要组成部分，它介导核质交换。阐明其110 MDa结构提出了一个艰巨的挑战，需要在原位结构生物学方法。大约30个核孔蛋白中有15个是结构化的，并形成Y-和内环复合物。这两个主要的支架模块以多个拷贝组装成八重旋转对称结构，该结构融合内核膜和外核膜以形成直径约60 nm的中央通道。支架用转运通道Nups装饰，其通常含有苯丙氨酸（FG）重复序列并介导与货物复合物的相互作用。虽然Y-复合物的部分结构排列已被阐明，但尚不清楚它究竟是如何原位寡聚的。在这里，我们结合低温电子断层扫描与质谱，生化分析，微扰实验和结构建模，以生成迄今为止最全面的NPC结构模型。我们的数据表明，以前未知的蛋白质接口跨Y-复合物和内环复合物的成员。我们证明，更高的真核细胞转运通道Nup 358（RanBP 2）在Y-复合物寡聚化中具有以前未预料到的作用。我们的研究结果模糊了支架和运输通道Nups之间的界限。我们的结论是，与包被囊泡类似，相同结构构建块的多个副本--尽管组成相同--参与不同的局部相互作用和构象。
Nuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.