Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, 210023 Nanjing, China.
Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309.
Proc Natl Acad Sci U S A. 2018 Sep 4;115(36):E8421-E8429. doi: 10.1073/pnas.1802645115. Epub 2018 Aug 20.
Soluble -ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze membrane fusion by forming coiled-coil bundles between membrane bilayers. The SNARE bundle zippers progressively toward the membranes, pulling the lipid bilayers into close proximity to fuse. In this work, we found that the +1 and +2 layers in the C-terminal domains (CTDs) of SNAREs are dispensable for reconstituted SNARE-mediated fusion reactions. By contrast, all CTD layers are required for fusion reactions activated by the cognate Sec1/Munc18 (SM) protein or a synthetic Vc peptide derived from the vesicular (v-) SNARE, correlating with strong acceleration of fusion kinetics. These results suggest a similar mechanism underlying the stimulatory functions of SM proteins and Vc peptide in SNARE-dependent membrane fusion. Unexpectedly, we identified a conserved SNARE-like peptide (SLP) in SM proteins that structurally and functionally resembles Vc peptide. Like Vc peptide, SLP binds and activates target (t-) SNAREs, accelerating the fusion reaction. Disruption of the t-SNARE-SLP interaction inhibits exocytosis in vivo. Our findings demonstrated that a t-SNARE-SLP intermediate must form before SNAREs can drive efficient vesicle fusion.
可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体（SNAREs）通过在双层膜之间形成卷曲螺旋束来催化膜融合。SNARE 束逐渐向膜靠拢，将脂质双层拉近融合。在这项工作中，我们发现 SNAREs 的 C 端结构域（CTDs）中的+1 和+2 层对于重建的 SNARE 介导的融合反应是可有可无的。相比之下，所有的 CTD 层对于由同源 Sec1/Munc18（SM）蛋白或源自囊泡（v-）SNARE 的合成 Vc 肽激活的融合反应都是必需的，这与融合动力学的强烈加速相关。这些结果表明，SM 蛋白和 Vc 肽在 SNARE 依赖性膜融合中的刺激功能具有类似的机制。出乎意料的是，我们在 SM 蛋白中鉴定出一种保守的 SNARE 样肽（SLP），它在结构和功能上类似于 Vc 肽。与 Vc 肽一样，SLP 结合并激活靶（t-）SNARE，加速融合反应。破坏 t-SNARE-SLP 相互作用会抑制体内的胞吐作用。我们的发现表明，在 SNARE 能够驱动有效的囊泡融合之前，必须形成 t-SNARE-SLP 中间体。
