SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE-FACTOR ATTACHMENT PROTEIN AND N-ETHYLMALEIMIDE-INSENSITIVE FACTORS ARE REQUIRED FOR CA2-STIMULATED EXOCYTOSIS OF INSULIN()

Ca2+ stimulates exocytosis in permeabilized insulin-secreting cells. T o investigate the putative cytosolic components involved in the Ca2+ r esponse, HIT-T15 cells (a pancreatic B-cell line) were permeabilized w ith streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell prepar ation the secretory response to Ca2+ but not to guanosine 5'-[gamma-th io]triphosphate was lost as a function of time and could be restored b y rat brain cytosol in a concentration-dependent manner. Reconstitutiv e activity of rat brain cytosol was found in a high-molecular-mass hea t-labile partially N-ethylmaleimide (NEM)-sensitive fraction. The NEM- sensitive factor (NSF) and the soluble NSF attachment protein (alpha-S NAP) were found to be expressed in HIT-T15 cells and largely lost (abo ut 30% remaining) from porated cells, Recombinant alpha-SNAP partially reconstituted the Ca2+ response when added to the permeabilized cells , Moreover, alpha-SNAP restored the effect of NEM-treated cytosol to t he level observed for untreated cytosol, In contrast, NSF was ineffect ive when preincubated alone or with NEM-treated cytosol. Our results i ndicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.