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)
