Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p

Homotypic fusion of the endoplasmic reticulum (ER) is required for generating and maintaining the characteristic reticular ER membrane structures. This organelle membrane fusion process depends on the ER-bound dynamin-related GTPases, such as atlastins in animals and Sey1p in yeast. Here, to investigate whether specific lipid molecules facilitate GTPase-dependent ER membrane fusion directly, we comprehensively evaluated membrane docking and lipid mixing of reconstituted proteoliposomes bearing purified Sey1p and a set of ER-mimicking lipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and ergosterol. Remarkably, we revealed that each specific lipid species contributed little to membrane docking mediated by Sey1p. Nevertheless, Sey1p-dependent lipid mixing was strongly reduced by omitting three major acidic lipids from the ER-mimicking set and, moreover, was entirely abolished by omitting either phosphatidylethanolamine or ergosterol. Our reconstitution studies thus established that physiological lipid composition is vital for lipid bilayer rearrangements in GTPase-mediated homotypic ER membrane fusion.