title: Domain 3a of Munc18-1 plays a crucial role at the priming stage of exocytosis.
abstract: Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, 399 Bathurst Street, Toronto, Ontario, M5T 2S8, Canada.
authors.forename: Gayoung Anna
authors.initials: GA
authors.lastname: Han
authors.forename: Na-Ryum
authors.initials: NR
authors.lastname: Bin
authors.forename: Soo-Young Ann
authors.initials: SY
authors.lastname: Kang
authors.forename: Liping
authors.initials: L
authors.lastname: Han
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D000818:Animals; D005089:Exocytosis / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology*; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; C497939:Stxbp1 protein, rat; C497942:Stxbp2 protein, rat
keywords: Exocytosis; Munc18; SNARE; Secretory vesicles; Syntaxin