Fusion of Phospholipid Vesicles Induced by Clathrin: Modulation of Fused Liposome Size

Abstract

The effects of lipid composition, temperature, and ionic strength on critical events in the membrane fusion reaction induced by clathrin, such as membrane binding and selfassociation of clathrin, membrane aggregation, and actual fusion, and the size of fused liposomes, were examined using large unilamellar vesicles (LUV) containing acidic phospholipids. When membrane fusion and aggregation of LUV with different lipid compositions were initiated at 25°C in 0.1 M NaCl by lowering the pH of the medium from 7.40 to 4.75 in the presence of clathrin, two types of reaction processes were observed: fusion and aggregation of LUV containing phosphatidylserine and phosphatidic acid occurred slowly with a long fusion lag-phase and reached a high level, whereas the two reactions of LUV containing phosphatidylglycerol and phosphatidylinositol were induced faster with a much shorter fusion lag-time and leveled off in a shorter time. Similar differences in the fusion reactions were observed in media at different temperatures and ionic strengths with any type of LUV: slow and extensive aggregation and fusion occurred at low temperature and/or high ionic strength, but faster, less extensive reactions occurred at high temperature and/or low ionic strength, indicating that the two types of reaction pattern are due to the dependencies of the aggregation and fusion reactions on the temperature and ionic strength, but not on the lipid composition. The sizes of fused liposomes were large under the former conditions but smaller under the latter, and the ratio of the rates of self-association to membrane binding of clathrin was found to be correlated with the rates and levels of these critical events and the sizes of fused liposomes. These results suggest that the fusion reaction and the size of fused liposomes are modulated by the balance between the self-association and membrane binding reactions of the protein. A novel fusion mechanism is proposed to explain the modulation of the size of fused liposomes.