Eidelman O, Schlegel R, Tralka T S, Blumenthal R
J Biol Chem. 1984 Apr 10;259(7):4622-8.
Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.
通过对辛基葡糖苷进行去污剂透析，将来自水疱性口炎病毒的纯化G蛋白重组成卵磷脂囊泡。如果蛋白质与脂质的比例较高（约0.3摩尔%蛋白质）且去污剂去除缓慢，则可获得均匀的重组囊泡群体。使用电子显微镜和荧光能量转移对重组囊泡的融合活性进行检测。病毒包膜蛋白介导的融合活性取决于pH值、温度和靶膜脂质组成。如使用负染电子显微镜所示，将重组囊泡在低pH值下与含有带负电荷脂质的小单层囊泡一起孵育，会导致出现大的螺旋状结构。此过程不会导致囊泡包裹的水性标记物泄漏。融合速率依赖于pH值，pK约为4，融合的表观活化能为16±1千卡/摩尔。G蛋白介导的融合对含有磷脂酰丝氨酸或磷脂酸的靶膜表现出很大的偏好。在任何脂质组成中加入36%的胆固醇对融合速率没有影响。这些重组囊泡提供了一个系统，用于研究病毒刺突蛋白诱导的pH依赖性融合机制。
