Studies on the mechanism of membrane fusion: role of phosphate in promoting calcium ion induced fusion of phospholipid vesicles.

The role of phosphate in enhancing the calcium-induced fusion of phosphatidylserine (PS) vesicles has been examined by using the new fluorescent (terbium/dipicolinic acid) assay described by Wilschut et al. (1980) [Wilschut, J., Düzgüneş, N., Fraley, R., & Papahajopoulos, D. (1980) Biochemistry (preceding paper in this issue)]. In the presence of physiological levels of phosphate, the calcium concentration required for fusion of PS vesicles was lowered significantly (3-4-fold), and the rate of vesicle fusion was increased dramatically (up to 1000-fold). The fusion of PS vesicles by calcium and phosphate is shown to be specific and critically dependent on temperature, pH, ion concentrations, and the composition of the calcium phosphate crystalline phase present during the incubation. The results indicate that a significant enhancement in vesicle fusion occurs only when calcium phosphate precipitation is initiated in the presence of PS vesicles, suggesting that crystal nucleation on the vesicle surface is a prerequisite for fusion. Calcium and phosphate were shown to promote phospholipid phase separations and vesicle fusion under conditions (e.g., mixtures of PS and phosphatidylcholine) in which calcium alone is ineffective, indicating that formation of PS calcium phosphate complexes may facilitate the molecular segregation of PS into distinct domains. These experiments underline the important role that phosphate may play in calcium-mediated fusion phenomena in biological membranes.