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Vol. 19, Issue 2, 485-497, February 2008

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SNARE-catalyzed Fusion Events Are Regulated by Syntaxin1A–Lipid Interactions

Alice D. Lam^*,, Petra Tryoen-Toth, Bill Tsai, Nicolas Vitale, and Edward L. Stuenkel^*,

*Department of Molecular and Integrative Physiology, Neuroscience Program, and Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI 48109; and Département Neurotransmission et Sécrétion Neuroendocrine, Unité Mixte de Recherche 7168/LC2, Centre National de la Recherche Scientifique/Université Louis Pasteur, 67084 Strasbourg, France

Submitted February 21, 2007; Revised October 29, 2007; Accepted November 5, 2007
Monitoring Editor: Patrick Brennwald

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.

Address correspondence to: Alice D. Lam (adlam{at}umich.edu)

Abbreviations used: BoNT-C, botulinum neurotoxin type C; ECL, enhanced chemiluminescence; FRET, fluorescence resonance energy transfer; hGH, human growth hormone; LPA, lysophosphatidic acid; PA, phosphatidic acid; PC, phosphatidylcholine; PC-12, pheochromocytoma-12; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; PLD1, phospholipase D1; PSF, prespike foot; PSS, physiological saline solution; SNAP25, synaptosomal-associated protein of 25 kDa; Syn1A, Syntaxin1A; VAMP2, vesicle-associated membrane protein-2.