Asymmetric Role of Negative Curvature Lipids on the SNARE Mediated Vesicle Docking and Fusion

Abstract

N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are fundamental machinery in the cellular membrane fusion process. Much of focuses of studies have been placed on the formation of core ternary SNARE complex and key accessory proteins, but the role of membrane environment in SNARE complex assembly is still unclear. We have used neuronal SNARE proteins (syntaxin-1HT, soluble SNAP-25 and VAMP-2) reconstituted proteoliposomes to study the effect of the cholesterol on the membrane fusion. Cholesterol is one of the most abundant components of eukaryotic plasma membrane. Bulk lipid mixing assay revealed that the addition of cholesterol to both acceptor and donor vesicles promote lipid mixing. However, when cholesterol was added to only one of the vesicle, VAMP-2 containing vesicle was found to contribute more towards the overall effect. Surprisingly, a similar result was observed using another negative curvature lipid POPE. Single vesicle fusion assay further revealed that this asymmetry comes from the fact that the vesicle docking and fusion of syntaxin-1HT/SNAP-25 and VAMP-2 containing vesicles are promoted differently in the presence of negative curvature lipids. A molecular mechanism behind this effect is discussed in the context of protein organization in different membrane environment. This study demonstrates the importance of membrane context in which SNARE proteins are being studied in in vitro system.