Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia.
Biophys J. 2010 Nov 3;99(9):2936-46. doi: 10.1016/j.bpj.2010.09.011.
The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in ∼20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., ~10 and ~20 ms, respectively [corrected].
脂质环境对突触融合蛋白 2 (Syb2) 囊泡与靶 SNARE 复合膜对接和融合的影响在具有高时间分辨率的平面支撑膜融合测定中进行了研究。之前，我们表明，在大约 20 毫秒内，大约需要八个 SNARE 复合物来融合磷脂酰胆碱 (PC) 和胆固醇模型膜。在这里，我们展示了实验，其中在 Syb2 囊泡膜中仅或在支撑双层膜和 Syb2 囊泡中以不同比例添加磷脂酰丝氨酸 (PS) 和磷脂酰乙醇胺 (PE) 到 PC/胆固醇混合物中。我们发现 PS 和 PE 都降低了融合的概率，并且这种降低完全由囊泡膜中的脂质组成来解释。然而，当囊泡（和靶膜）中的 PE 含量从 0 增加到 30%时，对接效率增加。快速激活 SNARE 复合物的分数随着 PE 含量的增加而减少。当至少 5%的 PS 和 10%的 PE 存在于两个膜中或仅 5%和 30%的 PE 存在于囊泡膜中时，只需 3 个 SNARE 复合物就足以支持膜融合。尽管所需 SNARE 的数量较少，但 SNARE 激活和融合的速度几乎与之前在重组 PC/胆固醇双层中报道的一样快，即分别为10 和20 毫秒[更正]。
