Faa1 binds to negatively charged membranes with preference for PI3P/PI4P. (A) Microscopy-based membrane binding assay showing that Faa1 is recruited to liposomes dependent on the net charge of the membranes. Faa1-EGFP was immobilized on GFP-trap beads and incubated with different lipid compositions. For visualization, they were supplemented with lissamine rhodamine-1,2-dihexadecanoylphosphatidylethanolamine (DHPE). Data are means ± SD (n = 3). LP = liposome. (B) Cosedimentation assay confirming the results from Fig. 2 A. Data are means ± SD (n = 3). LP = liposome, SN = supernatant, P = pellet. Liposome compositions in 2A and 2B resemble Atg9 vesicles with 44% POPC, 6% POPS, 44% liver PI, 5.5% POPE, and 0.5% lissamine rhodamine-DHPE. For liposomes containing PI3P, the percentage of PI was reduced while a decreased percentage of PI was substituted with POPC. POPC-only liposomes contained 99.5% POPC and 0.5% lissamine rhodamine-DHPE. (C) Cosedimentation assay shows that Faa1 is preferentially recruited to liposomes containing phosphatidylinositides when compared to liposomes with the same net charge. All liposomes are composed of POPC with additionally the lipids indicated in the figures and 0.5% lissamine rhodamine-DHPE. Data are means ± SD (n = 4). One-way ANOVA: * < 0.05, ** < 0.01. SN = supernatant, P = pellet. (D) Microscopy-based GUV binding assay testing Faa1-mCherry recruitment to flat membranes. GUVs were composed of POPC with additionally the lipids indicated in the figures and 1.5% NBD-DPPE. Source data are available for this figure: SourceData F2.