Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 27, Issue 4, Pages 669-685Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E15-09-0638
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Funding
- Natural Sciences and Engineering Research Council of Canada [298461]
- Heart and Stroke Foundation of Ontario [0171]
- Canadian Institute of Health Research [MOP-93665, 130573]
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Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states (closed vs. open) of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of closed syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the open syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between closed syntaxin-1A and Munc18-1, whereas the same mutation in the open syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.
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