GTP-binding proteins of the Sec4/Ypt1/rab subfamily of the Ras superfamily are thought to play an important role in controlling vesicular traffic along the secretory and endocytic pathways of all cells. The precise mechanism of action of these proteins remains to be elucidated. We propose here to study the role of rab proteins in the exo-endocytotic recycling of neuronal synaptic vesicles (SVs) and endocrine synaptic-like microvesicles (SLMVs). The large body of information already available on the biochemical composition of these organelles makes them optimal model systems to investigate the molecular mechanisms involved in membrane budding and membrane fusion. This project will be organized around four specific aims. First, the precise recycling pathway of SVs/SLMVs will be further investigated using a neuroendocrine cell line as a model system. Primary goal of this work is to test the hypothesis that SV/SLMV recycling involves transit through endosomes and sorting from endosomal membranes. Second, assays to study specific steps of the recycling of SLMVs will be developed. These will include assays to quantify vesicle exocytosis independent of neurotransmitter release. Third, these assays will be used to study the role of rab3a, rab4 and rab5 in specific steps of SLMV recycling. Fourth, the function in neurons and endocrine cells of the mammalian protein Mss4 which can suppress the secretory defect of the sec4-8 mutant of Saccaromyces cerevisiae will be investigated. The sec4-8 yeast strain harbors a point mutation in the GTP-binding protein Sec4 which is associated with secretory vesicles. Mss4 is a guanine nucleotide exchange protein homologous the yeast protein DSS4.
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