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+ 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 insulin-containing 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 vesicle-associated 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 beta-cells, and further strongly suggest that synaptotagmin III is the Ca2+ sensor in the exocytosis of insulin secretory vesicles.
Mizuta
M
M
Third Department of Internal Medicine, Miyazaki Medical College, Japan.
Kurose
T
T
Miki
T
T
Shoji-Kasai
Y
Y
Takahashi
M
M
Seino
S
S
Matsukura
S
S
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000502:physiology; D007150:Immunohistochemistry; D007163:Immunosorbent Techniques; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000648:ultrastructure*; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000502:physiology*; D008565:Membrane Proteins / Q000032:analysis; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000502:physiology*; D050683:R-SNARE Proteins; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins; D050857:Synaptotagmins
D000906:Antibodies; D002135:Calcium-Binding Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; C494291:Syt3 protein, rat; D050857:Synaptotagmins