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 machinery required for regulated exocytosis (where SNARE is the soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor). Syntaxin 1A interacts with a variety of other proteins, two of which, N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-soluble NSF attachment protein (alpha-SNAP) have been suggested to impart a conformational rearrangement on this protein during a reaction referred to as priming. We have studied the effect of the primed state on the binding properties of syntaxin 1A and we have confirmed that primed syntaxin 1A no longer associated with alpha-SNAP or its cognate vesicle-SNARE, vesicle-associated membrane protein (VAMP). Under 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 neuronal 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 fractions from bovine adrenal medulla and detected syntaxin 1A and nSec-1 in both plasma membrane and chromaffin-granule fractions. Using a cross-linking approach with chromaffin-granule membranes we detected a putative dimeric complex composed of approx. 54% total granule membrane nSec-1 and approx. 30% total syntaxin 1A. The results of this study therefore suggest the possibility of nSec-1 interactions with primed syntaxin 1A and demonstrate a potentially significant interaction of syntaxin 1A and nSec-1 on the membranes of chromaffin granules.
Haynes
L P
LP
The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool L69 3BX, U.K.
Morgan
A
A
Burgoyne
R D
RD
D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002459:Cell Fusion; D002462:Cell Membrane / Q000378:metabolism; D002837:Chromaffin Granules / Q000378:metabolism*; D019281:Dimerization; D008565:Membrane Proteins / Q000378:metabolism; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D011485:Protein Binding; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D013572:Synaptic Vesicles / Q000378:metabolism; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins