nSec-1 (munc-18) interacts with both primed and unprimed syntaxin 1A and associates in a dimeric complex on adrenal chromaffin granules

The target-SNARE syntaxin 1A is an essential component of the core machiner y required for regulated exocytosis (where SNARE is the soluble N-ethylmale imide-sensitive fusion protein-attachment protein receptor). Syntaxin 1A in teracts with a variety of other proteins, two of which, N-ethylmaleimide-se nsitive fusion protein (NSF) and alpha-soluble NSF attachment protein (alph a-SNAP) have been suggested to impart a conformational rearrangement on thi s protein during a reaction referred to as priming. We have studied the eff ect of the primed state on the binding properties of syntaxin 1A and we hav e confirmed that primed syntaxin 1A no longer associated with alpha-SNAP or its cognate vesicle-SNARE, vesicle-associated membrane protein (VAMP). Und er such conditions, however, it retained the ability to bind to nSec-1. It has been demonstrated that nSec-1, a regulatory protein also involved in ne uronal exocytosis, binds syntaxin 1A with high affinity in vitro, although evidence for this physical interaction occurring in vivo has proven elusive . We analysed the subcellular distribution of these two proteins in fractio ns from bovine adrenal medulla and detected syntaxin 1A and nSec-1 in both plasma membrane and chromaffin-granule fractions. Using a cross-linking app roach with chromaffin-granule membranes we detected a putative dimeric comp lex composed of approx. 54% total granule membrane nSec-1 and approx. 30% t otal syntaxin 1A. The results of this study therefore suggest the possibili ty of nSec-1 interactions with primed syntaxin 1A and demonstrate a potenti ally significant interaction of syntaxin 1A and nSec-1 on the membranes of chromaffin granules.