Fig. 2: The Gpr1-Gpa2-Cyr1-Tpk2 pathway is required for sucrose to repress Rpd3L-mediated Ada3 deacetylation. | Nature Communications

Fig. 2: The Gpr1-Gpa2-Cyr1-Tpk2 pathway is required for sucrose to repress Rpd3L-mediated Ada3 deacetylation.

From: PKA plays a conserved role in regulating gene expression and metabolic adaptation by phosphorylating Rpd3/HDAC1

Fig. 2: The Gpr1-Gpa2-Cyr1-Tpk2 pathway is required for sucrose to repress Rpd3L-mediated Ada3 deacetylation.The alternative text for this image may have been generated using AI.

a IP-mass spectrometry analysis of proteins co-purified with Rpd3 and Tpk2. The endogenously expressed Rpd3-FLAG and Tpk2-FLAG were individually immunoprecipitated from cells with anti-FLAG beads. The identified unique peptides and sequence coverage were listed. b Tpk2 interacted with Rpd3 as determined by Co-IP and reciprocal IP. c Rpd3L interacted with purified recombinant His-Tpk2 as determined by in vitro Co-IP. d Loss of Tpk2 enhanced the interaction between endogenously expressed Ada3 and Rpd3. e Analysis of Ada3 acetylation in WT and tpk2Δ mutant when treated with YP + 2% glucose (Glu) or YP + 2% sucrose (Suc) for 0.5 h. f Analysis of Ada3 deacetylase activity of Rpd3L purified from WT and tpk2Δ mutant. g The interaction between Tpk2 and Bcy1 was reduced when cells were shifted from YP + 2% glucose (Glu) to YP + 2% sucrose (Suc). h Diagram showing sucrose treatment activates Tpk2 via the Gpr1-Gpa2-cAMP pathway in budding yeast. i Analysis of intracellular cAMP levels in WT, gpr1∆ and gpa2∆ mutants when grown in YP, YP + 2% glucose (YP + Glu) or YP + 2% sucrose (YP + Suc) medium for 10 min. j, k Analysis of Ada3 acetylation in WT, gpr1∆ and gpa2∆ mutants when treated with YP + 2% glucose (YP + Glu) or YP + 2% sucrose (YP + Suc) for 0.5 h by immunoblots (j) and immunofluorescence (k). For Fig. k, cell nuclei was stained by DAPI (in blue). Ada3 acetylation was stained red with anti-Ada3-K14ac and anti-Ada3-K182ac antibodies. Anti-Ada3 antibody was used as a control. Shown are merged images. Scale bars, 5 μm. For (d–g, i, j), data represent means ± SE; n = 3 biological independent experiments; two tailed unpaired t-tests (d), one-way ANOVA with Tukey’s multiple comparison tests (g), two-way ANOVA with Tukey’s multiple comparisons tests (e, i, j) and Šídák’s multiple comparisons tests (f) were used for statistical analysis. For (b, c, k), shown is the typical example of at least two biological independent experiments.

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