Rab3a and rab5 are members of the rab-family of small GTP-binding proteins. Recent work by us and by others has shown that both proteins are highly concentrated in nerve terminals where they reside on membranes of the synaptic vesicle cycle. Rab3A is specific for exocytotic synaptic vesicles and is found selectively in neurons and neuroendocrine cells. Rab5 is ubiquitous and appears to be associated with early endosomes in neurons as well as in other cells. while the function of the small GTP- binding proteins is not yet understood, they appear to cycle between GTP- and GDP-forms which parallels a membrane association-dissociation cycle. Previous work by our laboratory has provided direct evidence for membrane dissociation of rab3A upon activation of exocytosis. It is supposed that small GTP-binding proteins act as cyclic molecular switches controlling membrane traffic since disruption of their cycle disrupts membrane traffic in a highly selective and specific manner. In this application it is proposed to study the cycles of rab3A and rab5, and to a lesser extent of rab4 and rab7 that are residents of early and late endosomes, respectively, in nerve terminals and to investigate their connections with exocytosis and membrane recycling of synaptic vesicles. In particular, the steps of the rab3A cycle shall be investigated. this involves the characterization of GTP-cleavage in the context of exocytosis, the characterization of membrane dissociation and association including that of the protein partners involved such as the GTPase activating protein and the putative membrane receptor of rab3A on the synaptic vesicle surface. Rab5 will be used as a paradigm for an endocytic small GTP-binding protein within the nerve terminal. In addition, rab4 and rab7 will be used as tools to characterize endosomal compartments in nerve terminals. It is planned to examine whether synaptic vesicle recycling requires passage through compartments containing rab5, rab4 and rab7, whether this passage involves GTP- hydrolysis of rab5 and whether rab5 undergoes an association-dissociation cycle similar to that of rab3. In the last part of the proposal, plans are outlined to use cell-free assays for exocytosis as well as for vesicle fusion and budding at the endosomal stage to address the potential involvement of rab3A and rab5 in exocytosis and endosome function within nerve terminals.
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