Membrane binding of Faa1 is important for its activity. (A) Scheme of the coupled enzymatic assay for testing the activity of Faa1. (B) Coupled enzymatic assay following the activity of Faa1 in the presence of different liposomes by measuring the absorbance of NADH at 340 nm. Liposome composition resembles Atg9 vesicles with 44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, and 2.5% PI3P, whereas PI3P was substituted for liver PI in “Atg9-like LPs w/o PI3P” and for “POPC only” liposomes contained 100% POPC. Data are means ± SD (n = 3). The dotted line serves as a reference for 0. LP = liposome. (C) Enzymatic assay comparing Faa1-WT to Faa1-4D and Faa1-6D. Data are means ± SD (n = 3). Liposome composition resembles Atg9 vesicles + PI3P (44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, 2.5% PI3P). The dotted line serves as a reference for 0. (D) Survival of indicated strains during starvation + cerulenin after 0, 1, 3, and 6 h of treatment. Data are means ± SD (n = 3; 100 cells/strain per replicate). One-way ANOVA: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (E) Fluorescent microscopy assessment of autophagosome biogenesis in indicated strains after 1 h nitrogen starvation + cerulenin. Left: Representative images of the analyzed strains. Dashed lines indicate cell boundaries, arrowheads autophagosomes (AP). Scale bar, 1 µm. Right: Respective quantification of autophagosomes. Data are means ± SEM (n = 3; 50 cells/strain per replicate). One-way ANOVA: **** < 0.0001. (F) Autophagic flux of indicated strains expressing 2xGFP-ATG8 after 0, 3, and 6 h of starvation + cerulenin. Data are means ± SD (n = 3). One-way ANOVA: * < 0.05, ** < 0.01, **** < 0.0001. Source data are available for this figure: SourceData F4.