Fig. 2: V-ATPase-dependent autophagy is distinct from nitrogen starvation-induced autophagy.

a–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 + 200 nM rapamycin, or SD-N medium for the indicated times. a ATG13-TAP cell lysates were analyzed by western blot. A 0.5-h treatment with the TORC1 inhibitor rapamycin triggers robust dephosphorylation of Atg13-TAP. Three biological replicates were repeated with similar results. b WLY176 WT and atg13∆ cell lysates were collected for the Pho8∆60 assay. Pho8∆60 activity was normalized to WT cells cultured in SD-N for 4 h (set to 100%). Statistical analysis was carried out by a two-tailed unpaired t-test with different variance. The summary data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant (n = 3). c SEY6210 WT and atg11∆ cell lysates were collected and analyzed by western blot. Three biological replicates were repeated with similar results. d RPS6A-GFP WT, atg1∆, doa1∆, and ubp3∆ cell lysates were collected for western blot analysis. DMSO (0.1%) or 3-IAA (300 μM) were re-added every 12 h to maintain low levels of Vma2-AID*-9MYC. Statistical analysis was carried out by a one-tailed unpaired t-test with different variances. The summary data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant (n = 3). e Total RNA was extracted from SEY6210 WT cells. The mRNA level of individual genes was normalized to the mRNA of the corresponding genes in cells 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 with different variance. The summary data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, and NS, not significant (n = 3). f Hypothetical model of V-ATPase-dependent autophagy. The autophagy triggered by V-ATPase dysfunction features active TORC1, cargo selectivity, and less transcriptional activation of ATG genes.