Figure 3.

Atg3-catalyzed GL1 lipidation reaction studied using uniform-size liposomes. (a) Schematic illustrations of GL1-DOPE conjugate (left) and the expected lipidation outcomes on liposomes with differential membrane curvatures (right, radii of curvature = 70 nm, 43 nm, and 18 nm). (b) GL1-lipidation efficiencies on extruded, sonicated and sorted liposomes (59.2% DOPC, 30% DOPE, 10% DOPS, and 0.8% rhodamine-DOPE) characterized by gel electrophoresis (top row, stained by Coomassie Blue) and immunoblot against GL1 with an antibody that preferentially recognizes the GL1-PE conformation (bottom row). The numbers (in nm) above lanes represent the nominal pore size of the filters (extruded liposomes) or measured mean diameters (sorted liposomes). Reaction mixtures from the same experiment were loaded in two gels that were run and processed in parallel. This experiment was repeated 5 times with similar results (see Supplementary Fig. 28).