Fig. 6: Ribosome biogenesis inhibits Gcn4 induction in the context of V-ATPase-dependent autophagy.

a–c 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. a Total RNA was extracted from SEY6210 VMA2-AID*-9MYC cells transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-TAP plasmid or SEY6210 VMA2-AID*-9MYC cells treated with or without 1 mM niacin. The mRNA level of individual genes was normalized to the mRNA of the corresponding genes in cells transformed with an empty vector treated with YPD + 0.1% DMSO for 4 h, which was set to 1. The data represent the average of 3 independent biological replicates. Statistical analysis was carried out by a one-tailed unpaired t-test. The summary data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant (n = 3). b SEY6210 VMA2-AID*-9MYC WT and gcn4Δ cells transformed with empty pRS405 plasmid and pRS405-CUP1p-RPS6A-3xHA plasmid were harvested, processed and analyzed by western blot. Gcn4 protein was detected with an anti-Gcn4 antibody. *, non-specific band. Three biological replicates were repeated with similar results. c SEY6210 VMA2-AID*-9MYC cells transformed with empty pRS405 plasmid and pRS405-CUP1p-RPS6A-TAP plasmid were harvested, processed and analyzed by western blot. Three biological replicates were repeated with similar results. d Schematic of V-ATPase-dependent autophagy. Trp prototrophy prevents downregulation of ribosome biogenesis upon Vma2 temporal degradation and therefore inhibits Gcn4 induction to block autophagy flux. NAD⁺ homeostasis does not affect ribosome biogenesis and Gcn4 induction. Three biological replicates were repeated with similar results.