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. To investigate the putative cytosolic components involved in the Ca2+ response, HIT-T15 cells (a pancreatic B-cell line) were permeabilized with streptolysin-O, a procedure that allows rapid exchange of soluble components including macromolecules. We found that in this cell preparation the secretory response to Ca2+ but not to guanosine 5'-[gamma-thio]triphosphate was lost as a function of time and could be restored by rat brain cytosol in a concentration-dependent manner. Reconstitutive activity of rat brain cytosol was found in a high-molecular-mass heat-labile partially N-ethylmaleimide(NEM)-sensitive fraction. The NEM-sensitive factor (NSF) and the soluble NSF attachment protein (alpha-SNAP) were found to be expressed in HIT-T15 cells and largely lost (about 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 the level observed for untreated cytosol. In contrast, NSF was ineffective when preincubated alone or with NEM-treated cytosol. Our results indicate that both alpha-SNAP and NEM-insensitive cytosolic factors are involved in Ca2+-mediated exocytosis from endocrine HIT-T15 cells.
Kiraly-Borri
C E
CE
Division de Biochimie Clinique, Département de Médecine, Centre Médical Universitaire, Geneva, Switzerland.
Morgan
A
A
Burgoyne
R D
RD
Weller
U
U
Wollheim
C B
CB
Lang
J
J
D000818:Animals; D001426:Bacterial Proteins; D015153:Blotting, Western; D001923:Brain Chemistry; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D002352:Carrier Proteins / Q000494:pharmacology / Q000502:physiology*; D002460:Cell Line; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D008565:Membrane Proteins / Q000494:pharmacology / Q000502:physiology*; D052066:N-Ethylmaleimide-Sensitive Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013301:Streptolysins / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D001426:Bacterial Proteins; D002352:Carrier Proteins; D007328:Insulin; D008565:Membrane Proteins; D011994:Recombinant Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013301:Streptolysins; D033921:Vesicular Transport Proteins; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide; D002118:Calcium