Figure S3.
Activity, membrane recruitment, and expression of Faa1. (A) Coupled enzymatic assay comparing the activities of FadD in the absence of liposomes and Faa1 in the presence of Atg9 vesicle-like liposomes (44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, 2.5% PI3P). Data are means ± SD (n = 3). (B) The autophagic flux of indicated strains expressing 2xGFP-Atg8 after 0, 3, and 6 h of starvation. Upon fusion of the autophagosome with the vacuole to form an autolysosome, Atg8 is rapidly degraded while GFP remains intact for a longer time and can be detected as free GFP by Western blot analysis. Data are means ± SD (n = 4). (C) Fluorescent microscopy assessment of autophagosome biogenesis in indicated strains after 1 and 1 h 15 min of nitrogen starvation and glucose depletion (0.01% wt/vol). Top, representative images of the analyzed strains at indicated time points. Dashed lines indicate cell boundaries. Scale bar, 1 µm. Bottom, respective quantification of AP. Data are means ± SEM (n = 3; 50 cells/strain per replicate). One-way ANOVA: * < 0.05, ** < 0.01. AP = autophagosome. (D) Autophagic flux of indicated strains expressing 2xGFP-Atg8 after 0, 3, and 6 h of starvation and glucose depletion (0.01% wt/vol). Data are means ± SD (n = 5). (E) Cosedimentation assay of Faa-WT-10xHis and Faa1-4D-10xHis with Atg9 vesicle-like LPs or POPC LPs with or without 5% DGS-NTA lipids including quantification. Liposome composition resembled Atg9 vesicles (“Atg9-like LPs”) with 44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, and 2.5% PI3P or only contained 100% POPC (“POPC LPs”). For liposomes containing 5% DGS-NTA, the percentage of POPC was reduced. Data are means ± SD (n = 3). LP = liposome, SN = supernatant, P = pellet. (F) Normalized protein levels of genomically tagged Faa1-WT, Faa1-4D, Faa1-WT-FYVE, and Faa1-4D-FYVE by Western blot quantification. Data are means ± SD (n = 4). Source data are available for this figure: SourceData FS3.

Activity, membrane recruitment, and expression of Faa1. (A) Coupled enzymatic assay comparing the activities of FadD in the absence of liposomes and Faa1 in the presence of Atg9 vesicle-like liposomes (44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, 2.5% PI3P). Data are means ± SD (n = 3). (B) The autophagic flux of indicated strains expressing 2xGFP-Atg8 after 0, 3, and 6 h of starvation. Upon fusion of the autophagosome with the vacuole to form an autolysosome, Atg8 is rapidly degraded while GFP remains intact for a longer time and can be detected as free GFP by Western blot analysis. Data are means ± SD (n = 4). (C) Fluorescent microscopy assessment of autophagosome biogenesis in indicated strains after 1 and 1 h 15 min of nitrogen starvation and glucose depletion (0.01% wt/vol). Top, representative images of the analyzed strains at indicated time points. Dashed lines indicate cell boundaries. Scale bar, 1 µm. Bottom, respective quantification of AP. Data are means ± SEM (n = 3; 50 cells/strain per replicate). One-way ANOVA: * < 0.05, ** < 0.01. AP = autophagosome. (D) Autophagic flux of indicated strains expressing 2xGFP-Atg8 after 0, 3, and 6 h of starvation and glucose depletion (0.01% wt/vol). Data are means ± SD (n = 5). (E) Cosedimentation assay of Faa-WT-10xHis and Faa1-4D-10xHis with Atg9 vesicle-like LPs or POPC LPs with or without 5% DGS-NTA lipids including quantification. Liposome composition resembled Atg9 vesicles (“Atg9-like LPs”) with 44% POPC, 6% POPS, 41.5% liver PI, 6% POPE, and 2.5% PI3P or only contained 100% POPC (“POPC LPs”). For liposomes containing 5% DGS-NTA, the percentage of POPC was reduced. Data are means ± SD (n = 3). LP = liposome, SN = supernatant, P = pellet. (F) Normalized protein levels of genomically tagged Faa1-WT, Faa1-4D, Faa1-WT-FYVE, and Faa1-4D-FYVE by Western blot quantification. Data are means ± SD (n = 4). Source data are available for this figure: SourceData FS3.

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