IN VITRO ANALYSIS OF SECRETORY VESICLE DOCKING

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This project is concerned with the understanding of how docking and fusion processes involved in the interactions between post-Golgi derived vesicles and target plasma membrane lead to either constitutive or regulated secretion. These processes appear to share molecular components, as suggested by the isolation of synaptic vesicle and presynaptic plasma membrane proteins by means of components involved in constitutive intra-Golgi transport (NEM-sensitive factor, NSF). We assume, as a working hypothesis, that the basic molecular machinery responsible for constitutive docking and fusion processes acquires additional elements that turn it off; in resting conditions and "on" in conditions triggered by external stimuli, depending on the kind of cell. The specific goal of this study is the analysis of the molecular requirements of regulated exocytosis using homologous and heterologous in vitro reconstitution systems. To explore this hypothesis an experimental strategy is proposed involving: a)in vitro reconstitution of the docking and undocking of labeled synaptic vesicles in immuno- isolated presynaptic nerve terminals, b) the analysis of the specificity in the binding of different neuronal post-Golgi derived vesicles to active zones and c) the search for inhibitors of the post Golgi constitutive secretion using reconstituting perforated Saccharomyces cerevisiae cells with synaptic proteins.