Coat assembly and membrane remodelling: understanding regulation of protein secretion

Project Information

CRYTOCOP

Grant agreement ID: 852915

DOI

10.3030/852915

Project closed

EC signature date 2 October 2019

Start date 1 November 2019

End date 30 April 2025

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 175,00

EU contribution

€ 1 499 175,00

Coordinated by

BIRKBECK COLLEGE - UNIVERSITY OF LONDON
United Kingdom

Periodic Reporting for period 4 - CRYTOCOP (Coat assembly and membrane remodelling: understanding regulation of protein secretion)

Reporting period: 2024-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

This project is focused on understanding the mechanisms of protein trafficking between intracellular compartments.
In particular we study the transport of newly synthesised proteins from the Endoplasmic Reticulum to the Golgi apparatus. This is a fundamental step in eukaryotic cell biology, and it is mediated by a protein complex name COPII, which forms a coat around the transport vesicles while concentrating cargo inside them.
A third of all proteins are transported into COPII-coated vesicles, and disruption of this for certain cargo proteins is linked to a number of genetic diseases.
Despite this, there are many unanswered questions with regards to the molecular mechanisms of COPII: how does it assemble to bend membranes? How is the process regulated? What is the morphological variability of COPII-coated vesicles?
The objectives of this proposal are to answer these questions using a multiscale approach, ranging from high resolution details of in vitro reconstituted reactions, to FIB/SEM studies of the COPII coat in cells.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have used cryo-electron tomography and subtomogram averaging to obtain high-resolution insights into COPII coat assembled on membranes in vitro. This revealed a complex and partially redundant network of interactions participate to coat assembly and drives membrane deformation, leading us to propose a model for regulation of membrane morphology by the coat (Hutchings et al 2021).
We have reconstituted COPII-coated membrane carriers using purified S. cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide novel insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated. (Pyle et al Nature Structural and Molecular Biology DOI: 10.1038/s41594-024-01413-4 (2024) and Pyle, E., et al. Faraday Discuss. DOI: 10.1039/D2FD00022A. (2022))
Upon move to the Crick institute, we have had direct access to cryo-FIB/SEM and have optimised a cryo-correlative approach to target COPII ER exit sites in cells. We obtained an RPE-1 cell line from Jon Audhya’s lab in Madison Wisconsin, We performed a complete morphological analysis of the coat, which will settle current controversies on the mechanism of COPII transport in animal cells. (Downes et al, under review, and on Biorxiv BiorXiv DOI:10.1101/2025.07.29.667472 (2025)

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Applying cryo-ET across scales, from high resolution (3.8A) structures from in vitro reconstituted sameples to in situ quantitative analysis in cells, we have revealed the molecular mechanisms mediating membrane remodelling and vesicular transport in the early secretory pathway.
This contributed novel discoveries on the mechanisms of COPII coat assembly, resolved disputes regarding ER exit modes in mammalian systems, and allowed us to contribute to novel data analysis methodologies (e.g. Zivanov, J., et al. Elife. DOI: 10.7554/eLife.83724 (2022)).

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