Fig. 5

Gcn5 acetylates Atg8 at K13 and represses autophagy. a Mass spectrometry of the peptides containing K13 or K38 acetylation. b Acetylation level of GFP-tagged Atg8 or mutated Atg8 proteins. The double mutated Atg8^K13R-K38R was abbreviated to Atg8^2KR. GFP tagged proteins were immunoprecipitated with GFP-agarose and immunoblotted by α-AcK. c In vitro acetylation assay using immunoprecipitated Atg8^K13R and Gcn5-Flag. d Translocation of GFP-Atg8 during autophagy process. Mycelia expressing GFP-Atg8 were grown in CM and then transferred into MM-N. The localization of GFP-Atg8 was observed at indicated time points by confocal microscopy. e Localization of the wild type GFP-Atg8 and Atg8^K13R. Histone 1 (H1)-mCherry and CMAC staining served as nuclear and vacuolar markers respectively. Bar = 10 μm. f Protein abundance of GFP-Atg8 and GFP-Atg8^K13R in cytoplasm and nucleus. Cytoplasmic and nuclear GFP-Atg8 or GFP-Atg8^K13R were fractionated and analyzed by immunoblot assay using the anti-GFP antibody. GAPDH and Histone 3 (H3) were detected as internal controls for the cytoplasmic and nuclear fractions, respectively. g Autophagy flux of GFP-Atg8 and GFP-Atg8^K13R strain upon nitrogen starvation. h Relocalization of GFP-Atg8 or GFP-Atg8^K13R during autophagy induction. Bar = 10 μm. i TEM images of autophagic structures (blue arrows) in mycelia of GFP-Atg8 and GFP-Atg8^K13R