The Nuclear Pore Basket – Functional Architecture of a Membrane Remodeling Machine

Informazioni relative al progetto

NPC-BUILD

ID dell’accordo di sovvenzione: 772032

DOI 10.3030/772032

Data della firma CE 26 Gennaio 2018

Data di avvio 1 Febbraio 2018

Data di completamento 31 Gennaio 2025

Finanziato da

• EXCELLENT SCIENCE - European Research Council (ERC)

Costo totale € 2 178 488,00

Contributo UE € 2 178 488,00

Coordinato da

MEDIZINISCHE UNIVERSITAET WIEN
Austria

Periodic Reporting for period 3 - NPC-BUILD (The Nuclear Pore Basket – Functional Architecture of a Membrane Remodeling Machine)

Periodo di rendicontazione: 2021-02-01 al 2022-07-31

Sintesi del contesto e degli obiettivi generali del progetto

The compartmentalization of intracellular milieu into membrane-bound organelles is the defining feature of a eukaryotic cell. Various molecular mechanisms of macromolecular transport between different compartments ensure preservation
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.

Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti

We have made major progress towards reconstituting subassemblies of the NPC on a synthetic model membrane. First, we were able to identify key lipid-interacting domains in several nucleoporins. Second, we were able to define conditions under which they bind to a model membrane in vitro. Third, we were able to observe higher-order oligomerization of NPC subassemblies. Fourth, we could observe membrane remodeling events such as high curvature induction, which likely reflect how the membrane pore underlying the NPC is formed.

Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora)

To the best of our knowledge, we have succeeded for the first time in reconstituting NPC subassemblies in a membrane environment, which is the natural "habitat" of the NPC. In doing so we have paved the way for a mechanistic dissection of how NPC biogenesis is accomplished.
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.