title: A pivotal role for pro-335 in balancing the dual functions of Munc18-1 domain-3a in regulated exocytosis.
abstract: Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Gayoung Anna
authors.initials: GA
authors.lastname: Han
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Seungmee
authors.initials: S
authors.lastname: Park
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Na-Ryum
authors.initials: NR
authors.lastname: Bin
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Chang Hun
authors.initials: CH
authors.lastname: Jung
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and.
authors.forename: Byungjin
authors.initials: B
authors.lastname: Kim
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and.
authors.forename: Prashanth
authors.initials: P
authors.lastname: Chandrasegaram
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and.
authors.forename: Maiko
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and.
authors.forename: Indira
authors.initials: I
authors.lastname: Riadi
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Liping
authors.initials: L
authors.lastname: Han
authors.affiliation: From the Division of Fundamental Neurobiology, Toronto Western Research Institute, University Health Network, Toronto, Ontario M5T 2S8 and the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada ssugita@uhnres.utoronto.ca.
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D019943:Amino Acid Substitution; D005089:Exocytosis / Q000502:physiology*; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011392:Proline / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism
chemical_list: D018832:Molecular Chaperones; D051938:Munc18 Proteins; D050827:Syntaxin 1; D011392:Proline
keywords: Dense Core Vesicles; Exocytosis; Membrane Fusion; Munc18; Secretion; Soluble NSF Attachment Protein Receptor (SNARE); Vesicles