CHAPTER 2 Vti 1 a functions in dense-core vesicle biogenesis

The SNARE-protein Vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated Vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal CCs, but localizes to a compartment near the Trans-Golgi-Network, partially overlapping with Syntaxin6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca-channels at the PM, partly due to fewer vesicles of reduced size and Synaptobrevin-2 content. In contrast, release kinetics and Ca-sensitivity remains unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, Vti1b, does not exacerbate the Vti1a phenotype and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term reexpression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short-term expression (hours) was ineffective, consistent with Vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that Vti1a functions in vesicle generation and Ca-channel trafficking, but is dispensable for transmitter release.