Reconstitution of the nuclear pore complex

核孔复合体的重建嵌入核膜中的核孔复合体(NPC)介导了核质之间的运输。核孔蛋白是由大约30种不同的核孔蛋白(或核蛋白)的多个拷贝组成的巨大的大分子集合体。一个关键问题是~30个核子是如何相互作用形成约60个丙二醛结构的。我们最近建立了一个新的模式生物-嗜热毛壳菌(一种真核嗜热细菌)，为分析大型核仁的结构铺平了道路，这是中温生物所不可能做到的。最终目标是获得NPC支架的原子视图，并解开结构原则，这些原则允许各种NPC模块组织成~60MDa八角形NPC框架。具体地说，我们希望进一步发展我们正在进行的NPC项目，即如何将所有30个NUP和派生的NPC模块连接起来，并最终固定在核孔膜上。在这个项目中，我们的目标是直接从真核嗜热细胞中系统地纯化和鉴定NUPS和NUP亚复合体，将完整的孔膜蛋白NDc1与Y型Nup84复合体成员之间的相互作用重建到脂质体系统中，建立一个假设的八重对称支架的NPC原型单元，并最终重建核篮子Mlp1蛋白的mRNA输出机制。

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.