title: Rescue of Munc18-1 and -2 double knockdown reveals the essential functions of interaction between Munc18 and closed syntaxin in PC12 cells.
abstract: Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.
authors.affiliation: Division of Fundamental Neurobiology, University Health Network, Toronto, Ontario, M5T 2S8, Canada.
authors.forename: Liping
authors.initials: L
authors.lastname: Han
authors.forename: Tiandan
authors.initials: T
authors.lastname: Jiang
authors.forename: Gayoung A
authors.initials: GA
authors.lastname: Han
authors.forename: Nancy T
authors.initials: NT
authors.lastname: Malintan
authors.forename: Li
authors.initials: L
authors.lastname: Xie
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Tse
authors.forename: Herbert Y
authors.initials: HY
authors.lastname: Gaisano
authors.forename: Brett M
authors.initials: BM
authors.lastname: Collins
authors.forename: Frederic A
authors.initials: FA
authors.lastname: Meunier
authors.forename: Shuzo
authors.initials: S
authors.lastname: Sugita
mesh_terms: D000818:Animals; D001665:Binding Sites; D055785:Gene Knockdown Techniques; D006801:Humans; D008958:Models, Molecular; D051938:Munc18 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D016716:PC12 Cells; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D013816:Thermodynamics; D020798:Two-Hybrid System Techniques
chemical_list: D051938:Munc18 Proteins; D011506:Proteins; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C497939:Stxbp1 protein, rat; C497942:Stxbp2 protein, rat; C055324:phospholipase A2-activating protein