title: Conformational states of syntaxin-1 govern the necessity of N-peptide binding in exocytosis of PC12 cells and Caenorhabditis elegans.
abstract: 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.
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Seungmee
authors.initials: S
authors.lastname: Park
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Na-Ryum
authors.initials: NR
authors.lastname: Bin
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada.
authors.forename: Maaran
authors.initials: M
authors.lastname: Rajah
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada.
authors.forename: Byungjin
authors.initials: B
authors.lastname: Kim
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Ting-Chieh
authors.initials: TC
authors.lastname: Chou
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada.
authors.forename: Soo-Young Ann
authors.initials: SY
authors.lastname: Kang
authors.affiliation: Division of Genetics and Development, Krembil Discovery Tower, University Health Network, Toronto, ON M5T 2S8, Canada.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Sugita
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Leon
authors.initials: L
authors.lastname: Parsaud
authors.affiliation: Division of Signaling Biology, MaRS Toronto Medical Discovery Tower, Ontario Cancer Institute, University Health Network, Toronto, ON M5G 1L7, Canada Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada.
authors.forename: Matthew
authors.initials: M
authors.lastname: Smith
authors.affiliation: Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada Division of Genetics and Development, Krembil Discovery Tower, University Health Network, Toronto, ON M5T 2S8, Canada.
authors.forename: Philippe P
authors.initials: PP
authors.lastname: Monnier
authors.affiliation: Division of Genetics and Development, Krembil Discovery Tower, University Health Network, Toronto, ON M5T 2S8, Canada Division of Signaling Biology, MaRS Toronto Medical Discovery Tower, Ontario Cancer Institute, University Health Network, Toronto, ON M5G 1L7, Canada.
authors.forename: Mitsuhiko
authors.initials: M
authors.lastname: Ikura
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada.
authors.forename: Mei
authors.initials: M
authors.lastname: Zhen
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, ON M5T 2S8, Canada Department of Physiology, University of Toronto, Toronto, ON M5S 1A8, Canada ssugita@uhnres.utoronto.ca.
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005089:Exocytosis; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism / Q000502:physiology*; D016716:PC12 Cells; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D050827:Syntaxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029742:Caenorhabditis elegans Proteins; D051938:Munc18 Proteins; D010455:Peptides; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1; C494215:unc-64 protein, C elegans