Fig. 5: Ash1 and Ubp8 mediate the direct interaction between Rpd3L and SAGA.

a Sucrose treatment reduced the interaction between Rpd3-FLAG and SAGA as determined by in vivo Co-IP. Rpd3-FLAG cells were treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. Rpd3-FLAG was immunoprecipitated with anti-FLAG beads and the co-IPed SAGA was detected with indicated antibodies. b Tpk2-catalyzed Rpd3L phosphorylation reduced its interaction with SAGA as determined by in vitro Co-IP. c The interaction between Rpd3-FLAG and Ada3 was reduced in sgf73∆, ubp8∆, cti6∆, rxt2∆, dep1∆, and ash1∆ mutants. d Summary for Co-IP results (c, Supplementary Fig. 5e–g) that determined the proteins required for Ada3-Rpd3 interaction. “+”: the subunit required for Ada3-Rpd3 interaction. “-”: the subunit not required for Ada3-Rpd3 interaction. “N/A”: as loss of Gcn5 reduced Ada3 level and loss of Spt7 compromised SAGA structure integrity, their contribution to the interaction between Ada3 and Rpd3 was unknown. e The purified recombinant Ubp8-FLAG directly interacted with purified recombinant His-Ash1 as determined by in vitro Co-IP. f The purified recombinant Ubp8-FLAG directly interacted with purified Rpd3L (Rxt2-CBP) as determined by in vitro Co-IP. g Ash1 and Ubp8 were required for sucrose to reduce the interaction between Rpd3 and Ada3. The endogenously expressed Rpd3-FLAG was immunoprecipitated from WT, ash1∆, and ubp8∆ mutants when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. h Ash1 and Ubp8 were required for sucrose to induce Ada3 acetylation as determined by immunofluorescence. Scale bars, 5 μm. i Analysis of Ada3 deacetylase activity of Rpd3L when Wt SAGA and SAGA ubp8∆ were used as the substrates. j Proposed model for Ubp8 and Ash1 act together to mediate the interaction between SAGA and Rpd3L and promote Rpd3-catalyzed Ada3 deacetylation. For (g, i), data represent means ± SE; n = 3 biological independent experiments; two-way ANOVA with Tukey’s multiple comparisons tests (g) and Šídák’s multiple comparisons tests (i) were used for statistical analysis. For (a–c, e, f, h) shown is the typical example of at least two biological independent experiments.