Organizing Large GTPase Atlastin‐Mediated Membrane Fusion by Programmable DNA Origami Platforms

ABSTRACT In eukaryotic cells, the endoplasmic reticulum (ER) utilizes membrane fusion to form interconnected networks. GTP hydrolysis‐triggered conformational changes in ER‐anchored atlastins (ATLs) have been shown to be responsible for this process, which is facilitated by the amphipathic helix within its C‐terminal tail (AH ATL‐C ). However, how the multiple ATL molecules cooperate to complete membrane tethering and subsequent fusion is still not known. Here, we used a programmable DNA origami ring to generate uniformly sized proteoliposomes containing a controlled maximum number of Drosophila ATL (dmATL) molecules. Using this system, we demonstrated at the single‐event level that one pair of dmATL molecules is capable of mediating membrane tethering but not lipid mixing. To investigate the subsequent fusion event, we developed programmable DNA soccer‐ball frameworks as a template for the generation of tethered liposomes. With a combination of molecular dynamics simulations and DNA‐ring‐templated single‐vesicle fusion assays, we found that the AH ATL‐C of dmATL is sufficient to promote the merging of closely apposed membranes by enhancing bilayer fluctuations under specific lipid composition condition. Our results provide mechanistic insights into dmATL‐mediated homotypic membrane fusion, and our adaptable DNA nanostructures offer powerful tools for studying other biological processes requiring predetermined numbers of molecules or involving tethered membranes.