Fig. 1: Inducible degradation of V-ATPase subunits activates autophagy flux in nutrient-rich medium.

a Summary of previously published data about the effects of FL-associated ATP6V1B2/VMA2 mutants on autophagy flux in human cells and in yeast. In both systems, an FL-associated R to Q mutation on a conserved residue in ATP6V1B2/VMA2 causes deacidification of lysosomal/vacuolar compartments and an increase of basal autophagy flux in nutrient-rich medium. This autophagy flux triggered by dysfunctional V-ATPase under nutrient-rich conditions is less robust than the one induced by starvation. b Schematic of inducible degradation of the target protein (Vma2) via the auxin-inducible degron system. Addition of the auxin molecule 3-IAA recruited the adapter protein OsTIR1 and the Cul1 E3 ubiquitin ligase complex to Vma2-AID*−9MYC. After poly-ubiquitination, the majority of Vma2-AID*-9MYC was degraded by the 26S proteasome. c–e 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, YPD + 500 nM conc. A, or SD-N medium for the indicated times. c Cell lysates were prepared, subjected to 10% SDS-PAGE, and analyzed by western blot. The ratio of free GFP to total GFP (free GFP plus GFP-Atg8) was quantified to indicate autophagy flux. Pgk1 was used as a loading control. The MYC blot detected Vma2-AID*-9MYC and OsTIR-9MYC. Three biological replicates were repeated with similar results. d Pho8-SEP cells were harvested and analyzed by flow cytometry to monitor vacuolar deacidification. A.U., arbitrary units. e WT, atg1∆ and pep4∆ cell lysates were processed and analyzed using the GFP-Atg8 processing western blot assay. *, non-specific band. Three biological replicates were repeated with similar results. f Hypothetical model of V-ATPase-dependent autophagy. V-ATPase-dependent autophagy describes the autophagy-activating effect of V-ATPase impairment in nutrient-rich medium. Under nutrient-rich conditions, the basal autophagy flux is very low, and the activating effect of V-ATPase dysfunction outweighs the defects in vacuolar hydrolytic activity; thus, the net outcome is enhancement of autophagy flux. During nitrogen starvation, autophagy activation induced by the starvation signal is much stronger than the one caused by V-ATPase dysfunction; the net result of V-ATPase dysfunction (i.e., the decrease in vacuolar hydrolytic activity) on starvation-induced autophagy is inhibitory.