Molecular Mechanisms underlying selection of cargo, scission, and coat release of intracellular vesicular carriers

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In eukaryotes, protein-coated vesicular carriers mediate intracellular transport of membranes and proteins. We are interested in the structure and the molecular mechanisms that underlie the formation and function of such vesicles, their selective incorporation of protein and lipid cargo, and their un-coating. We have previously defined the minimal protein machinery that is necessary and sufficient to form COPI vesicles through reconstitution studies with recombinant coat proteins and defined liposomes. Whereas basic structural elements resemble each other in a variety of coat protein subunits that form COPI-, COPII-, and Clathrin-coated vesicles (CCVs), the overall structure of the COPI coat is different from the other established coats. This structural divergence might reflect different mechanisms for the uptake of cargo molecules into these carriers. Moreover, unlike the COPII and Clathrin coats that form stepwise, the COPI coat is recruited to the donor membrane in a single step. Yet, the mechanisms of coat recruitment, and possibly of membrane scission, share certain similarities among all classes of vesicular carriers. Driven by structural insight into the COPI coat, in this application we propose to investigate i) the structural basis for the COPI scission reaction, and whether a general mechanism exists that applies for all membrane scission events catalyzed by small GTP-binding proteins, ii) the selectivity of different COPI and COPII coat protein isoforms for the uptake of protein and membrane lipid cargo, and iii) how GTPase-activating proteins catalyze the removal of the COPI coat and possibly aid coatomer to be recycled to its soluble form.