Periodic Reporting for period 4 - NPC-BUILD (The Nuclear Pore Basket – Functional Architecture of a Membrane Remodeling Machine)
Okres sprawozdawczy: 2022-08-01 do 2024-01-31
of cellular integrity. One of the most elaborate proteinaceous assemblies facilitating macromolecular exchange within a cell is the nuclear pore complex (NPC). The NPC is the sole gateway facilitating bidirectional transport of proteins, nucleic
acids and viruses between the nucleoplasm and the cytoplasm. It is embedded in the nuclear envelope (NE) at sites where the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) are fused. In S. cerevisiae, each NPC is composed of more than 500 subunits, collectively known as nucleoporins (Nups). Due to the highly modular architecture of the NPC and its eightfold rotational symmetry along the axis perpendicular to the NE, all Nups are present in multiples of 8 copies with only about 30 different Nups constituting an assembly of over 50 MDa. Most nucleoporins constituting the symmetric core of the NPC are present in biochemically stable subcomplexes. A key open question in the field of NPC research is how this fascinating structure is assembled. Given that the NPC is embedded in a membrane it is imperative to understand how the NPC interacts with lipids and how this interaction could you drive the overall could you drive the overall assembly process. We therefore set out to reconstitute key interactions between nucleoporins and a membrane in a defined in vitro system in order to get mechanistic insights into the process of NPC assembly.
While exploring these biochemical avenues we have developed protocols for nucleoporin-lipid reconstitution, which will be of great utility for the entire NPC field. The expected results at the end of this project are that we will be able to create ring-like NPC subassemblies, which could represent a minimal NPC scaffold on a membrane.