Reconstitution of the nuclear pore complex

点击翻译按钮获取中文摘要

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.