The long-term goal this research program is the elucidation of the role of clathrin and the clathrin coat structure in the membrane dynamics of cells, i.e. the processes of receptor-mediated endocytosis, secretion, Golgi processes, transcellular shuttling and membrane recycling. Considerable evidence suggests that the coat structure in vivo is a highly dynamic element and a prerequisite to understanding coated membrane true function is likely to e an elucidation of the physiological pathways of coat assembly and disassembly. We have partially purified a coated vesicle protein (assembly protein; AP) that specifically promotes, and is stoichiometrically incorporated into, reassembled clathrin coat structures under physiologically g relevent conditions. Using biochemical and morphological techniques, including light scattering, ultracentrifugation and electron microscopy, the in vitro nature of the AP-mediated reaction and its in vivo involvement in coated membrane dynamics will be studied. The kinetic parameters of coat formation will be determined. AP will be purified by co-cycling with clathrin and characterized. Polyclonal and monoclonal antibodies to AP, particularly those blocking AP-mediated reassembly, will be prepared and used to determine the intracellular localization of AP. Fluorescently-labelled AP will be microinjected into cultured cells and its kinetics of incorporation and distribution relative to clathrin within the cell will be studied. The effect of microinjection of anti-AP antibodies on coated membrane-mediated processes will be evaluated. These studies will help to elucidate the role of AP in clathrin coated membrane dynamics, and thus, will have broad implications for our understanding of many critical intracellular membrane processes.
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