Fig. 6: Tpk2 phosphorylates Ash1 to reduce the interaction between Rpd3L and SAGA.

a Tpk2-catalyzed Ash1 phosphorylation reduced the interaction between purified His-Ash1 and purified SAGA as determined by in vitro Co-IP. His-Ash1 was pre-phosphorylated by purified Tpk2-CBP and then incubated with purified SAGA for immunoprecipitation with nickel beads. b Loss of Tpk2 increased the interaction between Ubp8 and Ash1. c Ash1 was phosphorylated at S149 and S388. Ash1 was immunoprecipitated from WT, Ash1-S149A, Ash1-S388A and Ash1-2SA (Ash1-S149A S388A) mutants and its phosphorylation level was determined by immunoblots with phospho-PKA antibody. d The purified recombinant His-Ash1 was phosphorylated at S149 and S388 by purified Tpk2-CBP in vitro. e Analysis of Ash1 phosphorylation in WT, Ash1-2SA and Ash1-2SD mutants when cells were treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. f Analysis of the interaction between Ubp8 and Ash1 in WT, Ash1-2SA and Ash1-2SD mutants when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. g Mutation of Ash1-2SA enhanced the interaction between Rpd3L (Rxt2-CBP) and SAGA as determined by in vitro Co-IP. h Tpk2-catalyzed Ash1 phosphorylation reduced the interaction between Ubp8 and Ash1 as determined by in vitro Co-IP. i Immunoblot analysis of Ada3 acetylation in WT, Ash1-2SA and Ash1-2SD mutants when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. j Analysis of Ada3 acetylation in WT and Ash1-2SA mutant when cells were treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h by immunofluorescence. Scale bars, 5 μm. For (b, c, f–i), data represent means ± SE; n = 3 biological independent experiments; two tailed unpaired t-tests (b, g), one-way ANOVA with Tukey’s multiple comparisons tests (c), and two-way ANOVA with Tukey’s multiple comparisons tests (f, h, i) were used for statistical analysis. For (a, d, e, j), shown is the typical example of at least two biological independent experiments.