Barszczewski Marcin, Chua John J, Stein Alexander, Winter Ulrike, Heintzmann Rainer, Zilly Felipe E, Fasshauer Dirk, Lang Thorsten, Jahn Reinhard
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Mol Biol Cell. 2008 Mar;19(3):776-84. doi: 10.1091/mbc.e07-05-0498. Epub 2007 Dec 19.
Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.
神经元和神经内分泌细胞中的调节性胞吐作用需要形成一种稳定的可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物，该复合物由突触小泡蛋白-2/囊泡相关膜蛋白2、25 kDa的突触体相关蛋白（SNAP-25）和 syntaxin 1组成。随后，该复合物在α-SNAP和与不同细胞活动相关的ATP酶——ATP酶N-乙基马来酰亚胺敏感因子（NSF）的协同作用下被分解。我们报告称，NSF抑制会导致α-SNAP在质膜上聚集成簇。这种聚集是由α-SNAP与未复合的syntaxin结合介导的，因为用肉毒杆菌神经毒素C1切割syntaxin或使用针对syntaxin SNARE基序的抗体进行竞争会消除聚集。α-SNAP的结合有力地抑制了分泌颗粒的Ca(2+)依赖性胞吐作用和SNARE介导的脂质体融合。膜聚集以及胞吐作用和脂质体融合的抑制可被NSF抵消，但使用在NSF激活方面存在缺陷的α-SNAP突变体时则不会。我们得出结论，α-SNAP通过与syntaxin SNARE基序结合来抑制胞吐作用，进而阻止SNARE组装，揭示了α-SNAP在驱动胞吐性膜融合的SNARE循环中的一个意外作用位点。
