title: The Munc18-1 domain 3a loop is essential for neuroexocytosis but not for syntaxin-1A transport to the plasma membrane.
abstract: Munc18-1 plays a dual role in transporting syntaxin-1A (Sx1a) to the plasma membrane and regulating SNARE-mediated membrane fusion. As impairment of either function leads to a common exocytic defect, assigning specific roles for various Munc18-1 domains has proved difficult. Structural analyses predict that a loop region in Munc18-1 domain 3a could catalyse the conversion of Sx1a from a 'closed', fusion-incompetent to an 'open', fusion-competent conformation. As this conversion occurs at the plasma membrane, mutations in this loop could potentially separate the chaperone and exocytic functions of Munc18-1. Expression of a Munc18-1 deletion mutant lacking 17 residues of the domain 3a loop (Munc18-1(Δ317-333)) in PC12 cells deficient in endogenous Munc18 (DKD-PC12 cells) fully rescued transport of Sx1a to the plasma membrane, but not exocytic secretory granule fusion. In vitro binding of Munc18-1(Δ317-333) to Sx1a was indistinguishable from that of full-length Munc18-1, consistent with the critical role of the closed conformation in Sx1a transport. However, in DKD-PC12 cells, Munc18-1(Δ317-333) binding to Sx1a was greatly reduced compared to that of full-length Munc18-1, suggesting that closed conformation binding contributes little to the overall interaction at the cell surface. Furthermore, we found that Munc18-1(Δ317-333) could bind SNARE complexes in vitro, suggesting that additional regulatory factors underpin the exocytic function of Munc18-1 in vivo. Together, these results point to a defined role for Munc18-1 in facilitating exocytosis linked to the loop region of domain 3a that is clearly distinct from its function in Sx1a transport.
authors.affiliation: Queensland Brain Institute, The University of Queensland, Brisbane QLD 4072, Australia.
authors.forename: Sally
authors.initials: S
authors.lastname: Martin
authors.forename: Vanesa M
authors.initials: VM
authors.lastname: Tomatis
authors.forename: Andreas
authors.initials: A
authors.lastname: Papadopulos
authors.forename: Michelle P
authors.initials: MP
authors.lastname: Christie
authors.forename: Nancy T
authors.initials: NT
authors.lastname: Malintan
authors.forename: Rachel S
authors.initials: RS
authors.lastname: Gormal
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Martin
authors.forename: Brett M
authors.initials: BM
authors.lastname: Collins
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Meunier
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D051938:Munc18 Proteins; D050600:SNARE Proteins; C494213:Stx1a protein, rat; C497939:Stxbp1 protein, rat; D050827:Syntaxin 1
keywords: Munc18-1; Neuroexocytosis; Syntaxin-1