Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis.
Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis.
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DOI：
10.1074/jbc.m803355200
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发表时间：
2008-11-14
期刊：
影响因子：
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通讯作者：
Chapman ER
中科院分区：
文献类型：
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作者：
Gaffaney JD;Dunning FM;Wang Z;Hui E;Chapman ER
Synaptotagmin (syt) 1定位于突触囊泡，结合Ca2+，调节神经元胞吐。syt1包含两个Ca2+结合基序，称为C2A和C2B。在这项研究中，我们使用重组的SNARE（可溶性n -乙基马来酰亚胺敏感因子附着受体）介导的融合实验来检测syt1分离的C2结构域的功能。我们报道，将磷脂酰乙醇胺纳入重构的SNARE囊泡中，可以使分离的C2B而不是C2A调节Ca2+触发的融合。在Ca2+ 50激活的融合中，分离的C2B结构域的EC比完整的syt1胞质结构域（C2AB）低6倍。磷脂酰乙醇胺增加了C2AB-和c2b调节的融合的速率和效率，而不影响它们结合膜嵌入syntaxin-SNAP-25 （t-SNARE）复合物的能力。在等摩尔浓度下，分离的C2A结构域是C2B-的有效抑制剂，而不是c2ab调节的融合；因此，C2A在融合实验中有明显不同的作用，这取决于它是否与C2B相连。最后，扫描C2AB的丙氨酸突变揭示了C2B结构域内四组不同的突变，这些突变在snre介导的融合调控中发挥作用。令人惊讶的是，用位于Ca2+/膜结合环C2B的另一端的丙氨酸取代Arg-398，在体外减少C2AB t-SNARE结合和Ca2+触发的融合，而不影响Ca2+触发的与磷脂酰丝氨酸的相互作用或囊泡聚集。此外，一些突变解除了syt1的夹紧和刺激功能，表明这两种活动是由C2B中不同的结构决定因素介导的。
Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC for Ca2+ 50-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.