The overall Iong-term goal is the reconstitution of regulated exocytosis from purified components. In particular, the molecular mechanisms and components controlling the stepwise assembly of the v-/t SNARE fusion machinery at the neuronal synapse will be analyzed. A completely reconstituted SNARE-mediated fusion assay and a semi-reconstituted assay, measuring the fusion of synaptic vesicles with t-SNARE liposomes will provide the necessary functional readout systems. The molecular roles of Rab3, Rim 1 in vesicle tethering and subsequent t-SNARE assembly will be determined. It will be analyzed whether and how Rab3, Rim 1, Munc 13-1 can relieve the inhibitory block of Munc18-1 and which function they have in the subsequent SNAREpin assembly. To understand the mechanisms underlying calcium triggered exocytosis, the calcium sensor synaptotagmin I and some of its isoforms will be reconstituted into liposomes in the presence of additional regulatory components, such as Snapin and complexins, and their effects on membrane fusion will be analyzed in detail. Factors required for v-SNARE activation on synaptic vesicles will be identified and characterized. Knowledge of the basic mechanisms mediating neurotransmitter release will help us understand the molecular process of synaptic modulation, and medically relevant alterations, such as neuro-degenerative diseases and epilepsy. In general, an understanding of relevant mechanisms and gene products mediating neurotransmission will become important for preventive medicine, disease diagnosis, and treatment.
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