Rebuilding Nuclear Pore Complex In Vitro

Barbato, Suncica. Rebuilding Nuclear Pore Complex In Vitro. 2020, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/79113/

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The hallmark of a eukaryotic cell is the segregation of genetic material within the cell’s nucleus. The nucleus is separated from the cytoplasm by a double lipid bilayer termed nuclear envelope (NE) that contains a number of proteinaceous channels known as nuclear pore complexes (NPCs).
NPCs enable molecular transport between the nucleus and cytoplasm, termed nucleocytoplasmic transport (NCT), which is bidirectional, fast and selective. Three major groups of proteins are continuously trafficking across the NPC’s central channel to orchestrate NCT. These are transport receptors, known as karyopherins (Kaps), signal- specific cargoes and a small GTPase (i.e., Ran) that sustains the process.
Interestingly, NPCs restrict or promote cargo translocation via biochemical selectivity and not size exclusion per se. Recent findings suggest that the NPC transport selective barrier is regulated by Kaps. This so-called Kap-centric regulation considers Kaps as integral constituents of the NPC that reinforce its selective barrier against large nonspecific macromolecules while simultaneously promoting the transport of specific cargoes. Nonetheless, an understanding of how Kaps contribute to NPC function remains incomplete.
The aim of this study was to rebuild NPC function in vitro.
First, we investigated how Kaps might reinforce the NPC transport barrier to establish a gradient of Ran guanosine triphosphate (RanGTP) and Ran guanosine diphosphate (RanGDP) in the nucleus and cytoplasm, respectively. Here, we show that the binding of RanGTP to Kaps at the NPC prevents its leakage into the cytosol.
Next, we show that two NPC pore membrane proteins are able to self-assemble into 20 nm- diameter nanopores following their reconstitution into lipid bilayers. This work represents a key step toward using nanopores as a de novo platform to construct additional NPC mimics with the aim of promoting NCT-like selective transport.
Advisors:Lim, Roderick Y.H. and Herrmann-Lerdon, Harald
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Nanobiology Argovia (Lim)
UniBasel Contributors:Lim, Roderick Y.H.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:13970
Thesis status:Complete
Number of Pages:116
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss139704
edoc DOI:
Last Modified:11 Mar 2021 09:28
Deposited On:01 Mar 2021 15:05

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