Localization and Functional Role of Synaptotagmin III in Insulin Secretory Vesicles in Pancreatic β-Cells

Pancreatic β-cells secrete insulin by Ca²⁺-triggered exocytosis of insulin-containing large dense-core vesicles. Synaptotagmin is a Ca²⁺/phospholipid-binding protein and is a good candidate for the Ca²⁺ sensor for exocytosis of synaptic vesicles in neurons. In the present study, we generated a polyclonal antibody against synaptotagmin III, and found that synaptotagmin III immunoreactivity was present at high levels in insulincontaining pancreatic islet cells and insulin-secreting clonal MIN6 cells. In subcellular fractionations of MIN6 cells, synaptotagmin III was recovered in the vesicular fractions containing both insulin and vesicleassociated membrane protein-2 (VAMP-2), but not in synaptophysin-positive fractions. The secretory vesicles immunoprecipitated by anti-VAMP-2 antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MIN6 cells with anti-synaptotagmin III antibody significantly inhibited Ca²⁺-triggered insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic β-cells, and further strongly suggest that synaptotagmin III is the Ca²⁺ sensor in the exocytosis of insulin secretory vesicles.