Molecular Mechanisms of V-SNARE Function in Secretory Granule Exocytosis

Abstract

Insulin is released by regulated exocytosis and docking of insulin granules at the plasma membrane, followed by the assembly of the secretory machinery is required for insulin release. Newly formed granules require a complex maturation process to become release-ready and the formation of these fusion competent mature secretory granules involves a series of discrete and unique events comprised of granule sorting, maturation, docking, priming, and finally Ca2+-dependent fusion. Soluble NSF attachment receptor (SNARE) proteins are the conserved core machinery required for all intracellular membrane fusion events. While SNAREs specifically control fusion, vesicular SNAREs (v-SNAREs) perform additional roles that are still elusive. In secretory cell types, v-SNAREs are pivotal for targeting vesicles to different locations, mediating both sorting and membrane protein distribution and reductions in v-SNAREs result in defective regulated exocytosis. Using total internal reflection fluorescence (TIRF) microscopy, we studied the role of VAMP8 in secretory granule exocytosis in pancreatic beta cells. We found that VAMP8 associates with early, late and recycling endosome compartment, rather than insulin containing granules. However, VAMP8 traffics and fuses at the plasma membrane specifically with the recycling endosomal compartment. VAMP8 expression compromised secretory granule fusion, whereas VAMP2 expression facilitated granule fusion, delineating a VAMP8-dependent fusion step between recycling endosomes and the plasma membrane. Truncation of the VAMP8 C-terminal region resulted in attenuated exocytosis and a change in secretion kinetics. Moreover, secreting insulin measurements are negatively correlated with increased VAMP8 copy number in human islets. We conclude that VAMP8 vesicles play an important role in granule genesis and sorting of essential membrane proteins regulating insulin secretion. Additionally, the TMD of VAMP8 may be necessary for initiating fusion upon stimulation and appears to have some direct interaction on insulin secretion. This work should fill critical gaps in our understanding of regulated secretion and the treatment of type-2 diabetes.