Our goal is to understand the mechanisms governing the formation, targeting, and fusion of transport vesicles. We have reconstituted these processes in cell-free extracts containing Golgi membrane fractions, and have accumulated evidence supporting the working hypothesis that protein transport between membrane compartments in this cell-free system is due to the budding of transport vesicles from one cisterna of a Golgi stack followed by fusion of the vesicle with the next cisterna. We now hope to purify two key cytoplasmic components (C and B) that have been identified as necessary for vesicle budding and fusion respectively. Furthermore, we hope to purify a recently discovered NEM-sensitive factor (NSF) that is bound to the membranes in an ATP-dependent fashion. NSF seems to be needed for membrane fusion, and the activity of NSF is greatly stimulated by long chain acyl Coenzyme A, acting as a cofactor. We hope to purify both NSF and its presumed """"""""receptor"""""""" that binds it to Golgi membranes, as well as to elucidate the basis of the Coenzyme A requirement, which may be for an acylation-decylation cycle in which NSF participates that regulates a step leading to fusion of transport vesicles. The role of transport vesicles as intermediates will be studied by electron microscope immunocytochemistry, an important technique which will also be exploited to determine the nature of transport intermediates which accumulate when individual transport components (like NSF) are eliminated, offering clues as to the functional role of the eliminated component.
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