Fig. 3: The Gcn2-Gcn4 pathway positively regulated V-ATPase-dependent autophagy.

a Volcano plot of transcriptional changes in SEY6210 VMA2-AID*-9MYC cells treated with YPD + 300 μM 3-IAA for 4 h compared to the one treated with YPD + 0.1% DMSO for 4 h. DESeq2 was used with the Wald test (two-sided) to assess differential expression. Adjusted p-values were calculated using the Benjamini–Hochberg procedure to control for the FDR. Differentially expressed genes, padj<0.05, log2 (fold change)>1.5 were colored in blue (downregulated in cells treated with YPD + 300 μM 3-IAA for 4 h) and red (upregulated in cells treated with YPD + 300 μM 3-IAA for 4 h). Differentially expressed Gcn4 target genes were labeled in blue with black circles. Differentially expressed RPGs were labeled in red with black circles. b SEY6210 VMA2-AID*-9MYC arg4∆ lys2∆ cells were grown in SILAC medium supplemented with non-labeled lysine and arginine (R0K0) or stable isotype-labeled lysine and arginine (R6K4) to early log phase (OD600 = 0.02 ~ 0.04), centrifuged and resuspended in SILAC medium (R0K0) + 0.1% DMSO or SILAC medium (R6K4) + 300 μM 3-IAA for 8 h. Cell pellets were harvested and analyzed by two-way SILAC. Selected upregulated and downregulated pathways adapted from G-profiler are presented. c, d SEY6210 VMA2-AID*-9MYC cells were grown to mid-log phase in YPD (0 h), centrifuged and resuspended in YPD + 0.1% DMSO, YPD + 300 μM 3-IAA, or SD-N medium for the indicated times. Three biological replicates were repeated with similar results. c WT and gcn4∆ cell lysates were prepared and analyzed by western blot. Gcn4 protein was detected with an anti-Gcn4 antibody. *, non-specific band. d WT, gcn2∆, and gcn4∆ cell lysates were prepared and analyzed by western blot. e Schematic of V-ATPase-dependent autophagy. V-ATPase dysfunction activates Gcn2 kinase activity and induces Gcn4 translation to promote autophagy.