Unveiling the molecular mechanisms of vesicular trafficking, membrane fusion, and intravesicular acidification

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In order for a complex system of organs and tissues that compose an organism to maintain homeostasis and co-ordinate the intricate set of activities that are vital for its survival and well-being, the cells that comprise these organs and tissues must be able to communicate with and regulate each other. It is the secretory vesicles within these cells that house the vast array of contents that cells use to achieve this cell-to-cell communication. Thus, the regulation of these processes is an important area of inquiry in the field of fundamental cellular biology. Interestingly, these processes have been shown to be strongly influenced by the acidification of the intravesicular compartment. Keeping this in mind, our long-term goal is to determine how vesicular trafficking, membrane fusion, and intravesicular acidification are regulated in neurons, neuroendocrine cells, and immune cells. As described in a Nobel prize-winning discovery, SNAREs are critical for vesicular trafficking and membrane fusion. These SNAREs are classified as either v-SNAREs (e.g., synaptobrevin), found on the vesicular membrane, or t-SNAREs (e.g., syntaxin-1, SNAP-25), found on the membrane of target organelles. It is the association of these v- and t-SNAREs that is believed to regulate vesicular trafficking and to underlie the initiation of membrane fusion. In being important components of cellular function, it is no surprise that the interaction of v-SNAREs and t-SNAREs seems to be regulated by myriad proteins; however, the entire nature of this regulation remains a mystery. Intending to solve this mystery, our short-term goal is to elucidate the roles of two distinct classes of potential SNARE-regulating proteins: the Voa subunit of vacuolar ATPases (V-ATPases) and CAPS1 (Calcium-dependent activator protein for secretion 1). Based on preliminary work of us and others, we hypothesize that: 1) The Voa subunit has critical roles in dense-core vesicle trafficking in neuroendocrine cells and in rapid exocytosis of synaptic vesicles in neurons. 2) CAPS1 binds to syntaxin-1, a key t-SNARE protein, to facilitate SNARE complex assembly and membrane fusion. The Specific Aims of this research proposal are: 1) To determine the isoform-specific function of Voa isoforms in intracellular organelle targeting, intravesiclular acidification, vesicular trafficking, and exocytosis in neuroendocrince cells and neurons. 2) To elucidate the biochemical factors that are involved in the physical interactions between CAPS1 and syntaxin-1 and to elucidate the physiological significance of the CAPS1-syntaxin-1 interaction in vesicular fusion in neuroendocrine cells and in the organism. Taken together, our proposed work will provide fundamental, novel, and exciting insight into how vesicular trafficking, membrane fusion, and intravesicular acidification are regulated by the Voa subunit of V-ATPase and CAPS1.