Fig. 4

Atg8 is acetylated by Gcn5. a GFP-Atg8 and Gcn5-mCherry co-localized in nuclei. The dual-labeled strain was grown in CM and observed with a confocal microscope. Bar = 10 μm. b Co-IP analysis of the interaction between Atg8 and Gcn5. GFP-Atg8 and Gcn5-mCherry were detected using an anti-GFP or anti-mCherry antibody, respectively. Proteins samples were also detected with anti-GAPDH antibody as an internal control. c Pull-down analysis of Atg8-6×His and Gcn5-GFP. 6×His-tagged Atg8 purified from E. coli was incubated with the lysate of the fungal strain expressing Gcn5-GFP. Gcn5-GFP was immunoprecipitated using an anti-GFP antibody, and the precipitated complex was analyzed by immunoblotting using anti-His or anti-GFP antibody. d Acetylation level of Atg8 in WT or Δgcn5. The GFP-Atg8 was immunoprecipitated with GFP-agarose from the whole mycelial lysate of the WT or Δgcn5 grown in CM or MM-N, the acetylation level of Atg8 was detected with an anti-pan-acetyl-lysine monoclonal antibody (α-AcK). e, f Acetylation of Atg8 catalyzed by Gcn5 in vitro. Purified Atg8-6×His from E. coli was incubated with increased amounts of Gcn5-GFP purified from F. graminearum (e) or Gcn5-6×His purified from E. coli (f) and then detected with α-AcK antibody. The relative intensities of acetylated Atg8 bands were quantified with ImageJ. The fold change is relative to the control reaction without Gcn5-GFP or Gcn5-6×His supplementation. The bands in the control reaction were set as 1.00