Extending Techniques to Prepare Asymmetric Vesicles to Additional Lipid Compositions: Lipid Structure Affects the Ability to Maintain Lipid Asymmetry

Abstract

We recently developed a method in which methyl-beta-cyclodextrin (MβCD)-induced lipid exchange was used to prepare model membrane vesicles with a stable asymmetry and which, like plasma membranes, have an outer leaflet rich in sphingomyelin (SM) and have an inner leaflet rich in ordinary glycerophospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylserine. Experiments were carried out to determine how the phospholipid head group and acyl chain structure affected the ability to form stable asymmetric vesicles with SM-rich outer leaflets. In addition to the examples given above, stable asymmetric vesicles could be formed with inner leaflets rich in phosphatidylglycerol or cardiolipin. When PC with different acyl chain structures were compared, asymmetric vesicles with inner leaflets rich in PC could be formed when PC had one saturated acyl chain and one mono or polyunsaturated acyl chain, two monounsaturated acyl chains of various lengths, or when the PC had phytanoyl acyl chains, but not when the PC had two polyunsaturated acyl chains, although SM could be exchanged into vesicles in every case. The lack of asymmetry when vesicles had lipids with two polyunsaturated acyl chains was due to fast flip-flop. Based on these studies, and because lipids with two polyunsaturated acyl chains are of very low abundance in nature, we conclude that, at least in the absence of proteins, natural membranes of various compositions can maintain stable lipid asymmetry. These studies show that asymmetric vesicles with a wide variety of inner leaflet lipids can be prepared.