Reconstitution of the nuclear pore complex

核孔复合物的重建核孔复合物（Nuclear pore complex，NPC）嵌入核被膜中，介导细胞核和细胞质之间的转运。NPC是一种巨大的大分子集合体，由多个拷贝的Ca。30种不同的核孔蛋白（或NUPS）。一个关键的问题是，大约30个Nups如何在物理上相互作用形成ca。60 MDa结构。我们最近建立了一个新的模式生物Chaetomium thermophilum（一种真核嗜热菌），为大Nups的结构分析铺平了道路，这是不可能与嗜温生物。最终的目标是从原子的角度来看待NPC支架，并解开结构原理，这些原理允许各种NPC模块组织成~ 60 MDa的八角形NPC框架。具体来说，我们希望进一步发展我们正在进行的NPC项目，所有30个Nups和衍生的NPC模块如何连接并最终锚定到核孔膜上。在这个项目中，我们的目标是系统地纯化和表征Nups和Nup亚复合物直接从真核嗜热菌，重组之间的相互作用的一个完整的孔膜蛋白Ndc 1和成员的Y形Nup 84复合物进入脂质体系统，建立一个NPC的原型单位的假设八倍对称支架，并最终重建mRNA输出机制在核篮Mlp 1蛋白。

Reconstitution of the nuclear pore complex Nuclear pore complexes (NPCs) embedded in the nuclear envelope mediate the transport between the nucleus and cytoplasm. NPCs are huge macromolecular assemblies, which are built up by multiple copies of ca. 30 different nucleoporins (or Nups). A key question is how the ~30 Nups physically interact to form the ca. 60 MDa structure. We have recently established a new model organism Chaetomium thermophilum (a eukaryotic thermophile) that paved the way for the structural analysis of large Nups which was not possible with mesophilic organisms. The ultimate goal is to come to an atomic view of the NPC scaffold and unravel the structural principles, which allow the various NPC modules to become organized into the ~60MDa octagonal NPC framework. Specifically, we wish to further develop our ongoing NPC projects, how all the 30 Nups and derived NPC modules become connected and eventually anchored to the nuclear pore membrane. In this project, we aim to systematically purify and characterize Nups and Nup subcomplexes directly from a eukaryotic thermophile, reconstitute interactions between an integral pore membrane protein Ndc1 and members of the Y-shaped Nup84 complex into a liposomal system, build up an NPC protomer unit of the assumed eightfold symmetrical scaffold, and finally reconstitute the mRNA export machinery at the nuclear basket Mlp1 protein.