Figure 5.
Physiological Faa1 membrane localization is required for efficient lipid incorporation and maintenance of autophagic flux. (A) Quantification of a FRET assay measuring the incorporation of acyl-CoA into the membrane. The normalized difference in fluorescence intensity between activated (+CoA) and not activated (−CoA) Faa1-WT and Faa1-4D after 30 min is depicted. Liposome composition resembled Atg9 vesicles with 44% POPC, 6% POPS, 2% POPE, 44% liver PI, 2% NBD-DPPE, and 2% lissamine rhodamine-DHPE. Data are means ± SD (n = 3). Two-sided t test: ** < 0.01. (B) The maximum reaction rate of Faa1-WT-10xHis and Faa1-4D-10xHis when incubated with Atg9-like liposomes or POPC liposomes with or without 5% DGS-NTA was measured with a coupled enzymatic assay. 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. (C) Left: Localization of Faa1-WT, Faa1-4D, Faa1-WT-FYVE, and Faa1-4D-FYVE in cells coexpressing mCherry-ATG8. Indicated strains were imaged via fluorescence microscopy after starvation + cerulenin treatment (3 h), scale bars, 1 µm. Right: Quantification of survival of indicated strains during starvation + cerulenin after 0, 1, 3, and 6 h of treatment. Data are means ± SD (n = 4; 100 cells/strain per replicate). One-way ANOVA: * < 0.05, **** < 0.0001. (D) Representative images of the vacuolar signal of indicated strains after 6 h of starvation + cerulenin. (E) Autophagic flux of indicated strains expressing mCherry-ATG8 after 6 h of starvation + cerulenin. Data are means ± SD (n = 3). (F) Model for the role of Faa1 during autophagosome formation. Source data are available for this figure: SourceData F5.

Physiological Faa1 membrane localization is required for efficient lipid incorporation and maintenance of autophagic flux. (A) Quantification of a FRET assay measuring the incorporation of acyl-CoA into the membrane. The normalized difference in fluorescence intensity between activated (+CoA) and not activated (−CoA) Faa1-WT and Faa1-4D after 30 min is depicted. Liposome composition resembled Atg9 vesicles with 44% POPC, 6% POPS, 2% POPE, 44% liver PI, 2% NBD-DPPE, and 2% lissamine rhodamine-DHPE. Data are means ± SD (n = 3). Two-sided t test: ** < 0.01. (B) The maximum reaction rate of Faa1-WT-10xHis and Faa1-4D-10xHis when incubated with Atg9-like liposomes or POPC liposomes with or without 5% DGS-NTA was measured with a coupled enzymatic assay. 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. (C) Left: Localization of Faa1-WT, Faa1-4D, Faa1-WT-FYVE, and Faa1-4D-FYVE in cells coexpressing mCherry-ATG8. Indicated strains were imaged via fluorescence microscopy after starvation + cerulenin treatment (3 h), scale bars, 1 µm. Right: Quantification of survival of indicated strains during starvation + cerulenin after 0, 1, 3, and 6 h of treatment. Data are means ± SD (n = 4; 100 cells/strain per replicate). One-way ANOVA: * < 0.05, **** < 0.0001. (D) Representative images of the vacuolar signal of indicated strains after 6 h of starvation + cerulenin. (E) Autophagic flux of indicated strains expressing mCherry-ATG8 after 6 h of starvation + cerulenin. Data are means ± SD (n = 3). (F) Model for the role of Faa1 during autophagosome formation. Source data are available for this figure: SourceData F5.

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