Interaction of SNARE proteins in rat parotid acinar cells

The protein–protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.