Extended Data Fig. 2: Lysine acetylation effects on ADA2 function.
(a) Protein interaction between ADA2 full-length, truncated fragments, or the K263R point mutation version and GCN5 in yeast two hybrid assays. (b) Effects of the point mutations (K263R and K263Q) for ADA2 nucleus localization in rice protoplasts. At least two repetitions were performed. (c) Tests of ADA2 function to promote the acetyltransferase activity of GCN5. In vitro HAT assay was performed using recombinant GCN5-His as acetyltransferases and recombinant H3.3-His as substrates with or without ADA2-His recombinant protein in 50 µM acetyl-CoA. GCN5E233AD274A-His is GCN5 catalytically dead mutant, GCN5Y485A-His is a bromodomain mutation that disrupts acetyl-lysine binding. (d) Tests of effect of the ADA2 7KR point mutations on acetyltransferase activity of GCN5. In vitro HAT assay was performed using recombinant GST-GCN5 as acetyltransferases and recombinant H3.3-His as substrates with or without ADA2-His or ADA27KR-His recombinant protein in 50 µM Acetyl-CoA. The assays in c and d were analyzed by immunoblotting with anti-H3, anti-pan-H3 acetylation, anti-H3K9 acetylation, anti-H3K14 acetylation, anti-acetyl-lysine and anti-ADA2 antibody. Data in c and d are the two repetitions (except the controls used in c and d are different).
