Interaction of Faa1 with membranes. (A and B) Microscopy-based membrane recruitment assays analyzing Faa1 binding to different liposome compositions. Liposomes contained POPC and additionally, the amount of POPS, PI3P, or PI4P indicated in the figures and 0.5% lissamine rhodamine-DHPE. Quantifications show mean ± SD (n = 3). LP = liposome. Two-sided t test: * < 0.05, ** < 0.01. (C) Cosedimentation assay directly comparing Faa1 binding to differently sized liposomes to test for curvature dependency including quantification. Liposome composition resembles Atg9 vesicles with 44% POPC, 6% POPS, 44% liver PI, 6% POPE, and 0.5% lissamine rhodamine-DHPE, whereas liver PI was substituted with POPC in “Atg9-like LPs + 25% PI” and for “POPC only” liposomes contained 99.5% POPC and 0.5% lissamine rhodamine-DHPE. Data are means ± SD (n = 3). One-way ANOVA: ns = not significant. LP = liposome, SN = supernatant, P = pellet. (D) Dynamic light scattering evaluation of the average radius of liposomes from C. When labeled “0.4 µm” the liposomes were extruded only through a 0.4 µm filter while those labeled “0.05 µm” were additionally extruded through a 0.05 µm filter. Two-sided t test: **** < 0.0001. Source data are available for this figure: SourceData FS1.