Cryo-electron tomography and subtomogram averaging studies of the COPII coat assembled on membranes

Eukaryotic cells are internally organised by membrane compartments—transport between which is essential for cell survival. Highly conserved coat proteins orchestrate this mem-brane trafficking by coupling membrane remodelling into coated vesicles and incorporation of transport cargo. Proteins are transported from the endoplasmic reticulum to the Golgi in coat protein complex II (COPII)-coated vesicles. The coat comprises an inner and outer layer, which polymerise on the ER membrane to recruit diverse cargo and form carriers of variable size. A third of the yeast and human proteome are believed to undergo this transport step, including large structures like procollagen and pre-chylomicrons. Despite the wealth of genetic and cell biological data, mechanisms for COPII action and regulation are speculative. Electron cryo-tomography (ECT) enables 3D visualisation of pleiomorphic structures with atomic resolution. Here, COPII activity is reconstituted with giant unilamellar vesicles (GUVs) and visualised using ECT. Using recent developments in data collection and processing, the membrane-assembled coat layers are analysed using subtomogram averaging (StA) at subnanometre resolution. As seen previously, COPII yields a prominent network of coated tubules. Disruption of the outer coat assembly still leads to tubulation, raising questions over the role of the coat layers. A 4.9 Å inner coat StA reconstruction reveals interactions with the disrupted outer coat essential to lattice formation and tubulation. Comparison of the inner coat from assemblies where the outer coat is either able or unable to polymerise suggests the outer coat—aside from previously proposed scaffolding properties—has a role in inner coat organisation and assembly. New features of the outer coat subunits are also revealed. In collaboration with Liz Miller (MRC-LMB), genetic and biochemical evidence for the importance of these interactions is provided. This thesis provides insight not just into COPII assembly, but also on possible regulatory mechanisms in dictating carrier morphology