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.
Takuma
T
T
Department of Oral Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido, Japan. takuma@hoku-iryo-u.ac.jp
Arakawa
T
T
Tajima
Y
Y
D000251:Adenosine Triphosphatases / Q000032:analysis; D000818:Animals; D001923:Brain Chemistry; D002352:Carrier Proteins / Q000032:analysis; D003594:Cytoplasmic Granules / Q000737:chemistry; D005033:Ethylmaleimide; D015151:Immunoblotting; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000032:analysis*; D051938:Munc18 Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000032:analysis*; D010306:Parotid Gland / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D012471:Salivary Proteins and Peptides / Q000032:analysis*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013439:Sulfhydryl Reagents; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C494212:STX1A protein, human; D012471:Salivary Proteins and Peptides; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C494213:Stx1a protein, rat; C497944:Stxbp3 protein, rat; D013439:Sulfhydryl Reagents; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D005033:Ethylmaleimide