Determinants of liposome fusion mediated by synaptic SNARE proteins
Determinants of liposome fusion mediated by synaptic SNARE proteins
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DOI：
10.1073/pnas.0400044101
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发表时间：
2004-03-02
影响因子：
11.1
通讯作者：
Jahn, R
中科院分区：
文献类型：
--
作者：
Schuette, CG;Hatsuzawa, K;Jahn, R
突触胞吐作用需要可溶性 N-乙基马来酰亚胺敏感因子附着蛋白受体 (SNARE) 蛋白突触蛋白 1、SNAP-25 和突触糖素 (VAMP)。将 SNARE 组装成稳定的核心复合物应该会催化膜融合，并且用突触 SNARE 蛋白重构的脂蛋白体会自发地彼此融合。我们现在表明，突触 SNARE 介导的脂质体融合受到肉毒杆菌神经毒素 E (BoNT/E) 的抑制，但可以通过补充 SNAP-25 的 C 端部分来挽救。此外，已知可阻断嗜铬细胞胞吐作用的 SNAP-25 特异性抗体会阻止融合，并且 R-SNARE synaptobrevin 2、endobrevin/VAMP-8 和 tomosyn 的可溶片段会竞争融合。反应过程中没有观察到簇状囊泡的积累。含有 SNARE 的蛋白脂质体的快速人工聚类可提高低浓度但不饱和脂质体浓度下的融合率。我们得出的结论是，脂质体融合速率由 SNARE 的内在特性决定，而不是由之前的对接步骤决定。
Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobirevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT/E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin/VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step.