Fig. 4: A genome-wide suppressor screen reveals that the Trp biosynthetic pathway inhibits V-ATPase-dependent autophagy via Gcn4 but not Gcn2.

a Schematic of suppressor screen design. For the first round of the suppressor screen, one biological replicate was performed for each transformant from the 1588 genome tiling library. A ratio of free GFP versus total GFP below 0.2 was considered as a suppressive effect on V-ATPase-dependent autophagy. For the second round of individual gene overexpression validation, 3 different colonies were tested, and candidates were considered suppressor genes if more than two colonies showed a suppressive effect. b Summary of 32 suppressor genes. c SEY6210 VMA2-AID*-9MYC cells (pRS307-CUP1p-GFP-ATG8) transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-TAP plasmid were grown to mid-log phase in YPD (0 h), centrifuged and resuspended in YPD + 0.1% DMSO or YPD + 300 μM 3-IAA for 8 h. Cell lysates were prepared and analyzed by western blot. Three biological replicates were repeated with similar results. d SEY6210 VMA2-AID*-9MYC cells (pRS307-CUP1p-GFP-ATG8) with overexpressed Trp permease Tat2 protein (pRS405-CUP1p-TAT2-3xHA) were grown to mid-log phase in YPD (0 h), centrifuged and resuspended in YPD + 0.1% DMSO or YPD + 300 μM 3-IAA with or without 1 mM Trp for 8 h. Cell lysates were processed and analyzed by western blot. Three biological replicates were repeated with similar results. e Volcano plot of transcriptional changes in cells transformed with pRS405-CUP1p-TRP1-3xHA plasmid compared to empty pRS405 plasmid after 4 h in YPD + 300 µM 3-IAA medium. 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 are colored in blue (downregulated in cells transformed with pRS405-CUP1p-TRP1-3xHA plasmid) and red (upregulated in cells transformed with pRS405-CUP1p-TRP1-3xHA plasmid). Gcn4 target genes and RPGs were highlighted with black circles. f SEY6210 VMA2-AID*-9MYC gcn4∆ cells and WT cells transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-3xHA plasmid were grown to mid-log phase in YPD medium (0 h), centrifuged and resuspended in YPD + 0.1% DMSO or YPD + 300 μM 3-IAA medium for 8 h. Cell lysates were prepared and analyzed by western blot. Gcn4 protein was detected with anti-Gcn4 antibody. *, non-specific band. Three biological replicates were repeated with similar results. g BY4742 WT, trp1∆, and gcn2∆ trp1∆ 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 amino acid starvation medium for the indicated times. Cell lysates were processed and analyzed by western blot. p-Ser51-Sui2/eIF2α was detected with a phospho-specific antibody. Three biological replicates were repeated with similar results. h Schematic of V-ATPase-dependent autophagy. Trp prototrophy inhibits Gcn4 induction and suppresses autophagy flux upon Vma2 degradation.