Fig. 4: Tpk2-catalyzed phosphorylation of Rpd3 inhibits its deacetylase activity.
a Analysis of Ada3 acetylation in WT and Rpd3-2SA mutant when treated with YP + 2% glucose (Glu) and YP + 2% sucrose (Suc) for 0.5 h. b Analysis of Ada3 acetylation in WT, Rpd3-2SA, and tpk2∆ mutants when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h by immunofluorescence. Scale bars, 5 μm. c Analysis of Ada3 acetylation in WT and Rpd3-2SD mutant when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. d Analysis of the effect of Tpk2-mediated Rpd3 phosphorylation on its activity to deacetylate Ada3. Rpd3-FLAG was immunoprecipitated from WT and Rpd3-2SA mutant by anti-FLAG beads. Wt Rpd3-FLAG and Rpd3-2SA-FLAG were phosphorylated by purified Tpk2-CBP for 1 h. Tpk2-CBP was then washed away and purified SAGA was added for in vitro deacetylase assay. e Analysis of the effect of Tpk2-catalyzed Rpd3 phosphorylation on His-Rpd3-mediated Ada3 deacetylation. The purified recombinant His-Rpd3 and His-Rpd3-2SA were phosphorylated by Tpk2-CBP as in Fig. 4d and then used for in vitro deacetylase assay with purified SAGA. f Analysis of the effect of Tpk2-catalyzed Rpd3 phosphorylation on Rpd3-catalyzed histone deacetylation. His-Rpd3 and His-Rpd3-2SA were phosphorylated by Tpk2-CBP and then used for in vitro histone deacetylation assay using nucleosomes as the substrate. For Fig. (a–f), data represent means ± SE; n = 3 biological independent experiments; two-way ANOVA with Tukey’s multiple comparisons tests (a–c) and Å Ãdák’s multiple comparisons tests (d–f) were used for statistical analysis.
