Abstract
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.
Highlights
Sey1p-bearing proteoliposomes were reconstituted with the untagged form of Sey1p, which had been digested by human rhinovirus 3C protease to cleave the N-terminal polyhistidine tag, and with the preformed protein-free liposomes bearing the endoplasmic reticulum (ER)-mimicking lipids (Table 1), using a detergent-assisted insertion method[6,18]
After incubating the reaction mixtures containing the biotin-labeled and rhodamine (Rh)-labeled liposomes bearing Sey1p with streptavidin-coated beads (Fig. 2c), Rh fluorescence of the co-isolated Rh/Sey1p and biotin/Sey1p liposomes was measured to quantify the physical interactions between these Sey1p liposomes (Fig. 2d)
Using current newly developed assays to analyze Sey1p-dependent proteoliposomal membrane docking (Fig. 2c,d) and lipid mixing (Fig. 2e–h), we explored the essential role of lipids in homotypic ER membrane fusion mediated by the Sey1p GTPase and GTP (Fig. 3, Table 1)
Summary
This Sey1p-dependent lipid mixing strictly requires GTP binding/hydrolysis and the presence of Sey1p proteins on both opposing membranes destined for fusion (Fig. 2f,g), as expected from the results from our current membrane docking assays (Fig. 2d).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
