Mechanisms of dynamic COPII coat assembly to drive vesicle formation from the endoplasmic reticulum.

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Protein secretion is an essential process that is conserved across all eukaryotes and is initiated by COPII-coated vesicles that bud from the endoplasmic reticulum (ER) carrying nascent secretory and membrane proteins. COPII vesicles are formed by five conserved "coat" proteins that self-assemble to simultaneously select cargo proteins and sculpt the membrane into a vesicle carrier. To achieve this, the coat must fulfil several seemingly contradictory principles: it must be (1) sufficiently robust to exert significant force to bend the membrane; (2) intrinsically unstable to permit uncoating, which drives fusion with the Golgi membrane and protein/lipid delivery; and (3) be architecturally adaptable, such that the morphology of transport carriers can encompass small vesicles (60-80 nm) as well as larger structures that carry cargos like pro-collagen (300nm rods) or lipoprotein particles (300nm spheres). Understanding how the COPII coat assembly pathway is initiated, regulated, and modified to generate transport intermediates with diverse morphologies is the focus of my project. I propose to investigate how the COPII assembly pathway proceeds in the context of dynamic protein-protein and protein-lipid interactions, and aim to bring new methodologies to solve the problem of how coat assembly is modulated to adapt to specific physiological needs. I will develop a real-time imaging assay that visualizes coat assembly on supported bilayers. Such reconstitution will allow analysis of the dynamics of the coat in the context of physiologically relevant states, including nucleotide (GTP), cargo, and accessory proteins. Moreover, capitalizing on the library of specific coat mutants available in the Miller lab will further reveal mechanisms that govern coat assembly and adaptivity.