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. 2025 Sep 26;16(1):8508.
doi: 10.1038/s41467-025-63472-5.

V-ATPase-dependent induction of selective autophagy

Yuxiang Huang  1 , Dimitra Dialynaki #  1 , Yuchen Lei #  1 , Zhihai Zhang #  1 , Charles R Evans  2 , Daniel J Klionsky  3
Affiliations

V-ATPase-dependent induction of selective autophagy

Yuxiang Huang et al. Nat Commun. .

Abstract

The general consensus is that the vacuolar-type H+-translocating ATPase (V-ATPase) is critical for macroautophagy/autophagy. However, there is a fundamental conundrum because follicular lymphoma-associated mutations in the V-ATPase result in lysosomal/vacuolar deacidification but elevated autophagy activity under nutrient-replete conditions and the underlying mechanisms remain unclear. Here, working in yeast, we show that V-ATPase dysfunction activates a selective autophagy flux termed "V-ATPase-dependent autophagy ". By combining transcriptomic and proteomic profiling, along with genome-wide suppressor screening approaches, we found that V-ATPase-dependent autophagy is regulated through a unique mechanism distinct from classical nitrogen starvation-induced autophagy. Tryptophan metabolism negatively regulates V-ATPase-dependent autophagy via two parallel effectors. On the one hand, it activates ribosome biogenesis, thus repressing the translation of the transcription factor Gcn4/ATF4. On the other hand, tryptophan fuels NAD+ de novo biosynthesis to inhibit autophagy. These results provide an explanation for the mutational activation of autophagy seen in follicular lymphoma patients.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
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.
Fig. 2
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.
Fig. 3
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.
Fig. 4
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.
Fig. 5
Fig. 5. NAD+ homeostasis acts downstream of Trp prototrophy and negatively regulates V-ATPase-dependent autophagy.
a–f BY4742 or SEY6210 VMA2-AID*-9MYC cells (pRS307-CUP1p-GFP-ATG8) 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 with or without 1 mM niacin addition for the indicated times. a BY4742 WT, trp1∆, and bna4∆ cell lysates were prepared and analyzed by western blot. Three biological replicates were repeated with similar results. b SEY6210 VMA2-AID*-9MYC whole cell lysates were collected, and intermediate metabolites involved in the kynurenine pathway were analyzed. Statistical analysis was carried out using 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). c SEY6210 WT cell lysates were processed and analyzed by western blot. Three biological replicates were repeated with similar results. d SEY6210 WT and gcn4∆ cell lysates were prepared 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. 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 without niacin addition 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). f SEY6210 WT and gcn2∆ 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. g Schematic of V-ATPase-dependent autophagy. NAD+ biosynthesis acts downstream of Trp metabolism but is not responsible for inhibition of Gcn4 induction.
Fig. 6
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.
Fig. 7
Fig. 7. Rpc53 phosphorylation regulates V-ATPase-dependent autophagy.
a Schematic showing the phosphosites in Rpc53. The zoomed-in sequence shown with a proposed Kns1 motif (RXXS/TP) underlined and the phosphosite boxed in gray. Two overlapping GSK3 motifs (S/TXXXS/T) direct Mck1 phosphorylation of primed substrates (blue arrows) at phosphosites boxed in gray. Adapted from Lee et al., (2012). During nitrogen starvation or rapamycin treatment, Rpc53 is hyperphosphorylated and inactivated. As a result, ribosome biogenesis is repressed. b–d SEY6210 VMA2-AID*-9MYC cells (pRS307-CUP1p-GFP-ATG8) 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. Three biological replicates were repeated with similar results. b RPC53-TAP cell lysates were prepared and analyzed by western blot. Rapamycin treatment was the positive control that was known to induce complete Rpc53 phosphorylation. Rpc53-TAP protein was detected with anti-PAP antibody. The upper band represents phosphorylated Rpc53 (p-Rpc53-TAP) with lower mobility on an SDS-PAGE gel. c SEY6210 RPC53-TAP and RPC53T228A-TAP cell lysates were processed and analyzed by western blot. Gcn4 protein was detected with an anti-Gcn4 antibody. *, non-specific band. d RPC53-TAP and RPC53T228A-TAP cells transformed with empty pRS405 plasmid and pRS405-CUP1p-TRP1-3xHA plasmid were harvested, processed, and analyzed by western blot. e Graphical model of V-ATPase-dependent autophagy. 1 A, FL-associated mutations of the V-ATPase subunits cause vacuolar deacidification. 1 B, unlike nitrogen starvation-induced autophagy, the TORC1 complex remains active in this context. 2 A, The Gcn2-Gcn4 pathway is activated by the vacuolar deacidification signal. 2B, Rpc53 is partially phosphorylated by Kns1 and Mck1 and thus inactivated. This induces a downregulation of ribosome biogenesis. 2 C, the downregulation of ribosome biogenesis releases the inhibitory effect on Gcn4 translational activation, thus further stimulating the Gcn2-Gcn4 pathway. 3 A, Trp prototrophy partially inhibits Rpc53 phosphorylation, and therefore maintains ribosome biogenesis in an active state. 3B, in addition, the Trp biosynthetic pathway fuels the intracellular NAD+ pool, which inhibits V-ATPase-dependent autophagy without affecting Gcn4 induction.
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