The V-ATPase/ATG16L1 axis is controlled by the VH subunit.

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the VH subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The VH/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking VH fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within VH that mediates ATG16L1 binding. A neuronal VH isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, VH controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.