Fig 2: Tpk2-catalyzed Jhd2 phosphorylation enhances Not4-mediated Jhd2 ubiquitination and its turnover by the proteasome pathway.a Western blot analysis of Jhd2 in WT, Jhd2-S321A, S340A, and tpk2Δ mutants. b Analysis of Jhd2 stability in WT, Jhd2-S321A, S340A, and tpk2Δ mutants. c, d Analysis of the effect of MG132 or inactivation of Cim3 on Jhd2 protein levels. e Analysis of the ubiquitination of Jhd2 in WT, Jhd2-S321A, S340A, not4Δ, tpk2Δ, not4Δ Jhd2-S321A, S340A, and not4Δ tpk2Δ mutants. f Analysis of Jhd2, Rpd3 and H3K4 methylation in WT, Jhd2-S321A, S340A, tpk2Δ, not4Δ, Jhd2-S321A, S340A not4Δ, and tpk2Δ not4Δ mutants. g Co-IP assay showing Jhd2-S321A, S340A mutation reduced the interaction between Jhd2 and Not4. h Tpk2-catalyzed Jhd2 phosphorylation increased the interaction between Not4 and Jhd2. i Proposed model showing that Tpk2-catalyzed Jhd2 phosphorylation promotes Not4-mediated ubiquitination and subsequent proteasome degradation of Jhd2. For a–d, f, data represent the mean ± SEM of at least three biological independent experiments. Two-sided t-tests were used for statistical analysis. Source data are provided as a Source data file.

Fig 3: Ino80 interacts with Rpd3 and regulates autophagy.(A) Left: Heatmap showing the occupancy of Rpd3 [log2(Rpd3 IP/input)] at 18 core autophagy machinery genes by ChIP-seq. Right: Heatmap showing the transcriptional changes [log2(rpd3Δ/WT)] of 18 core ATG genes in rpd3Δ mutant by RNA-seq. (B) IP–mass spectrometry analysis of proteins copurified with Rpd3 and Ino80. The endogenously expressed Rpd3-FLAG and Ino80-FLAG were individually immunoprecipitated from cells with anti-FLAG agarose beads. (C and D) Rpd3 interacts with Ino80 as determined by co-IP assay (C) and reciprocal IP (D). The endogenously expressed Rpd3-FLAG and Ino80-FLAG were immunoprecipitated with anti-FLAG agarose beads. The untagged strain was used as a negative control. Asterisk indicates the nonspecific band. (E) Analysis of the autophagy activity in WT and INO80 complex subunit gene-null mutants as determined by the GFP-Atg8 processing assay. (F) Quantification of the immunoblot data in Fig. 1E and fig. S1H. (G) Analysis of the autophagy activity in WT, ino80Δ, arp8Δ, and nhp10Δ mutants as determined by the fluorescence assay. The autophagic cells were defined as cells with clear vacuolar GFP fluorescence. (H) Analysis of the transcription of ATG genes in WT, ino80Δ, arp8Δ, and nhp10Δ mutants as determined by qRT-PCR. The constitutively expressed PYK1 serves as a negative control. (I) Analysis of the autophagy activity in WT, rpd3Δ, ino80Δ, and rpd3Δ ino80Δ mutants as determined by the fluorescence assay. For (E) to (I), data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 4: Rpd3 deacetylates Ino80 to regulate the occupancy of H2A.Z at autophagy-related genes.(A) Left: Heatmap showing the occupancy of H2A.Z [log2(H2A.Z IP/input)] at 18 core autophagy machinery genes by ChIP-seq. Right: Heatmap showing the transcriptional changes [log2(htz1Δ/WT)] of 18 core ATG genes in htz1Δ mutant by RNA-seq. (B) ChIP-qPCR analysis of H2A.Z occupancy at ATG gene promoters in WT, swr1Δ, and ino80Δ mutants when cells were grown in YPD medium. PYK1 serves as a negative control. (C) ChIP-qPCR analysis of H2A.Z occupancy at ATG gene promoters in WT, Ino80-K929R, rpd3Δ, and Ino80-K929R rpd3Δ mutants. (D) qRT-PCR analysis of the transcription of ATG genes in WT, htz1Δ, rpd3Δ, and rpd3Δ htz1Δ mutants when grown in YPD medium. (E and F) Effect of HTZ1 deletion on autophagy activity as determined by the GFP-Atg8 processing assay (E) and fluorescence assay (F). (G) Analysis of autophagy activity in WT, htz1Δ, ino80Δ, and ino80Δ htz1Δ mutants as determined by the GFP-Atg8 processing assay. (H) qRT-PCR analysis of the transcription of ATG genes in WT, Ino80-K929R, htz1Δ, and Ino80-K929R htz1Δ mutants. For (B) to (H), data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: Tpk2-catalyzed Jhd2 phosphorylation maintains normal chronological life span and promotes autophagy.a Venn diagram showing genes co-regulated by Jhd2-S321A, S340A and tpk2Δ mutants. b Left panel: GSEA analysis of longevity regulating genes in Jhd2-S321A, S340A and tpk2Δ mutants. Right panel: Heatmap showing the transcription of longevity regulating genes in Jhd2-S321A, S340A and tpk2Δ mutants. c Chronological life span analysis of WT, Jhd2-S321A, S340A and tpk2Δ mutants. d Venn diagram showing genes co-regulated by Jhd2-S321A, S340A and rpd3Δ mutants. e GSEA analysis of autophagy genes regulated by tpk2Δ, Jhd2-S321A, S340A, jhd2Δ and rpd3Δ mutants. f Heatmap showing the binding of Jhd2 and Rpd3 at autophagy genes in WT and Jhd2-S321A, S340A mutant. Heatmap for the transcription of autophagy genes in jhd2Δ, Jhd2-S321A, S340A, tpk2Δ and rpd3Δ mutants was also provided. g ChIP-qPCR analysis of Jhd2 (top panel) and Rpd3 (bottom panel) occupancy at representative autophagy genes in WT, Jhd2-S321A, S340A and tpk2Δ mutants. PYK1 was used as a negative control. h RT-qPCR analysis of representative autophagy genes in WT, Jhd2-S321A, S340A, tpk2Δ, rpd3Δ, rpd3Δ Jhd2-S321A, S340A, and rpd3Δ tpk2Δ mutants. i, j Analysis of autophagy activity in WT, Jhd2-S321A, S340A, tpk2Δ, rpd3Δ, rpd3Δ Jhd2-S321A, S340A, and rpd3Δ tpk2Δ mutants. k Proposed model for regulation of autophagy by Tpk2-catalyzed Jhd2 phosphorylation. b, e, The P value was calculated using a permutation test with 1,000 permutations. c, g–j, Data represent the mean ± SEM of three biological independent experiments. Two-sided t-tests were used for statistical analysis. Source data are provided as a Source data file.