title: SNARE-catalyzed fusion events are regulated by Syntaxin1A-lipid interactions.
abstract: Membrane fusion is a process that intimately involves both proteins and lipids. Although the SNARE proteins, which ultimately overcome the energy barrier for fusion, have been extensively studied, regulation of the energy barrier itself, determined by specific membrane lipids, has been largely overlooked. Our findings reveal a novel function for SNARE proteins in reducing the energy barrier for fusion, by directly binding and sequestering fusogenic lipids to sites of fusion. We demonstrate a specific interaction between Syntaxin1A and the fusogenic lipid phosphatidic acid, in addition to multiple polyphosphoinositide lipids, and define a polybasic juxtamembrane region within Syntaxin1A as its lipid-binding domain. In PC-12 cells, Syntaxin1A mutations that progressively reduced lipid binding resulted in a progressive reduction in evoked secretion. Moreover, amperometric analysis of fusion events driven by a lipid-binding-deficient Syntaxin1A mutant (5RK/A) demonstrated alterations in fusion pore dynamics, suggestive of an energetic defect in secretion. Overexpression of the phosphatidic acid-generating enzyme, phospholipase D1, completely rescued the secretory defect seen with the 5RK/A mutant. Moreover, knockdown of phospholipase D1 activity drastically reduced control secretion, while leaving 5RK/A-mediated secretion relatively unaffected. Altogether, these data suggest that Syntaxin1A-lipid interactions are a critical determinant of the energetics of SNARE-catalyzed fusion events.
authors.affiliation: Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA. adlam@umich.edu
authors.forename: Alice D
authors.initials: AD
authors.lastname: Lam
authors.forename: Petra
authors.initials: P
authors.lastname: Tryoen-Toth
authors.forename: Bill
authors.initials: B
authors.lastname: Tsai
authors.forename: Nicolas
authors.initials: N
authors.lastname: Vitale
authors.forename: Edward L
authors.initials: EL
authors.lastname: Stuenkel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001905:Botulinum Toxins / Q000378:metabolism; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D050356:Lipid Metabolism; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D016716:PC12 Cells; D010641:Phenotype; D010712:Phosphatidic Acids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D051938:Munc18 Proteins; D010712:Phosphatidic Acids; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D001905:Botulinum Toxins; C096324:botulinum toxin type C