Investigation of the assembly mechanisms of COPI-coated vesicles using cryo-electron microscopy

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In eukaryotic cells, proteins are mainly synthesized in the endoplasmic reticulum to be transported to the correct cellular compartment or secreted through the secretory pathway. Additionally, eukaryotic cells receive important clues from their environment, clues that are transmitted via the endocytosis pathway. The transport of proteins (cargo) within the secretory and endocytic pathways relies on the assembly and disassembly of protein coats onto cellular membranes. The coat components recruit cargo, generate highly curved membrane areas, facilitate vesicle scission and recruit uncoating and other regulatory factors. To date, three major types of vesicles have been described moving cargo between different cellular compartments: clathrin-, COPI (coat protein complex I)- and COPII (coat protein complex II)-coated vesicles. Despite the fact that these vesicles traffic between different cellular compartments and that their coats are composed of different proteins, it has been hypothesized that the basic design principles of assembly of the coat and cargo recruitment are similar but with distinct differences in ordered to allow specificity of transport. This hypothesis was based on similarities between COPII- and clathrin-coated vesicles. However, little is known regarding the actual structure of COPI components and the structure of the fully-assembled COPI vesicles. The research proposed here seeks to further our understanding of the underlying principles of COPI vesicle assembly using high-resolution molecular cryo-electron microscopy (single particle and electron tomography image analysis) combined with molecular modeling using Molecular Dynamics. To this end, we will determine the 3D structure of purified isolated entire COPI complex (CM7) and the sub-complexes CM3 (coat forming complex) and CM4 (adaptor-like complex involved). This will allow understanding of the subunits arrangement with the coatamer complex and provide critical information to understand how the complex may function and be regulated. We will also study the conditions in vitro that promote coat assembly and coatamer polymerization. Influence of factors, shown to have an impact in vivo on the efficiency of vesicles formation will be tested. These include lipids, lipid composition of the membrane, dilysine containing peptides, Arf1 and various ArfGAPs. Once oligomers are obtained in vitro, their 3D structures will be determined using single particle and/or electron tomography and sub-volumes averaging in order to understand how different coatamer assemble into a protein coat and to determine the different subunit interactions involved in this process. Taken together, these studies will provide the structural basis for understanding the molecular mechanisms involved in the assembly of COPI-coated vesicles as well as highlight the similarities and differences between COPI-, COPII- and clathrin-coated vesicles assembly mechanisms. In particular, these studies will reveal how the different COPI subunits interact with each other to assemble into large oligomeric complexes, detect any conformation changes that occur during assembly and define the intermolecular contacts responsible of assembly. The interdisciplinary nature of these projects will provide trainees the opportunity to learn biochemical techniques, cryo-electron microscopy and 3DEM reconstruction using state-of-the-art instrumentation available at the McGill Facility for Electron Microscopy Research (FEMR) and Molecular Dynamics. The current proposal fits within the long-term objectives of my research program that are to understand the molecular mechanisms and structural principles involved in the interaction of protein and membrane leading to membrane penetration, scission and fusion.