Markova, Evgenia A. (2021) Reconstituting the human COPII secretion system. PhD thesis, Birkbeck, University of London.
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Abstract
The human coat protein II (COPII) secretion system is an essential membrane trafficking system that exports cargo from the endoplasmic reticulum. While significant insight into the COPII secretion system has been gained using yeast as a model organism, many questions about the COPII secretion system in higher eukaryotes remain unanswered: do human COPII proteins form carriers for cargo export? What membrane morphologies do human COPII proteins generate, and how are those morphologies regulated to accommodate the wide range of cargoes exported from the endoplasmic reticulum? To understand the functioning of human COPII, I established a minimal in vitro reconstitution system using purified proteins and model membranes. I purified human COPII proteins as expressed in E. coli and insect cells and established that they were capable of membrane binding using liposome flotation assays. Furthermore, I used GTP hydrolysis assays to characterize the enzymatic activity of the Sar1 GTPase, the initiating factor of COPII assembly. I observed that Sar1 has an intrinsic GTP hydrolysis activity, which is stimulated by other COPII components, indicating that the purified components have retained their biologically relevant functionality. Finally, I analysed human COPII assemblies on membranes using electron microscopy. I established that purified human COPII components are sufficient for the deformation of model membranes and the generation of a range of diverse morphologies. Using cryo-electron microscopy, I observed that human COPII components form two discernible layers on membranes. The establishment of this in vitro reconstitution platform allows us to address outstanding questions about the functioning of this complex system, such as the importance of different COPII component paralogues in humans and the role of GTP hydrolysis in COPII-mediated membrane deformation. Furthermore, it is the first step to the structural analysis of human COPII assembled on membranes, which can provide insight into global COPII arrangement.
Metadata
| Item Type: | Thesis |
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| Copyright Holders: | The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted. |
| Depositing User: | Acquisitions And Metadata |
| Date Deposited: | 18 Nov 2021 14:34 |
| Last Modified: | 01 Nov 2023 15:03 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/46760 |
| DOI: | https://doi.org/10.18743/PUB.00046760 |
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