LOCALIZATION AND FUNCTIONAL-ROLE OF SYNAPTOTAGMIN-III IN INSULIN SECRETORY VESICLES IN PANCREATIC BETA-CELLS

Pancreatic beta-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+ sens or for exocytosis of synaptic vesicles in neurons. In the present stud y, we generated a polyclonal antibody against synaptotagmin III, and f ound that synaptotagmin III immunoreactivity was present at high level s in insulin-containing pancreatic islet cells and insulin-secreting c lonal MIN6 cells. In subcellular fractionations of MIN6 cells, synapto tagmin III was recovered in the vesicular fractions containing both in sulin and vesicle-associated membrane protein-2 (VAMP-2), but not in s ynaptophysin-positive fractions. The secretory vesicles immunoprecipit ated by anti-VAMP-a antibody contained synaptotagmin III and insulin. In addition, treatment of streptolysin-O-permeabilized MING cells with anti-synaptotagmin III antibody significantly inhibited Ca2+-triggere d insulin secretion. These results indicate that synaptotagmin III is localized in insulin-containing dense-core vesicles in pancreatic beta -cells, and further strongly suggest that synaptotagmin III is the Ca2 + sensor in the exocytosis of insulin secretory vesicles.