Soluble N-ethylmaleimide-sensitive-factor attachment protein and N-ethylmaleimide-insensitive factors are required for Ca2+-stimulated exocytosis of insulin

被引:61
作者
KiralyBorri, CE
Morgan, A
Burgoyne, RD
Weller, U
Wollheim, CB
Lang, JC
机构
[1] CTR MED UNIV GENEVA,DEPT MED,DIV BIOCHIM CLIN,CH-1211 GENEVA 4,SWITZERLAND
[2] UNIV LIVERPOOL,PHYSIOL LAB,LIVERPOOL L69 3BX,MERSEYSIDE,ENGLAND
[3] UNIV MAINZ,INST MED MIKROBIOL,HOCHHAUS AUGUSTUSPLATZ,D-55101 MAINZ,GERMANY
关键词
D O I
10.1042/bj3140199
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
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页码:199 / 203
页数:5
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