Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation

ABSTRACT: Summary Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens. Graphical abstract Highlights • Subtractive CRISPR screen identifies genes involved in non-canonical LC3 lipidation• v-ATPase regulates LC3 lipidation at erroneously neutral compartments• RALGAP complex involved in M2 proton channel induced LC3 lipidation• ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation Ulferts et al. identify v-ATPase as the central regulator of ATG16L1 WD40 domain-dependent of LC3 lipidation. This lipidation can be prevented by Salmonella SopF and counteracted by ATG4D, the predominant ATG4 paralog responsible for LC3 delipidation. The RalGAP complex affects influenza virus M2-induced LC3 lipidation by affecting M2 trafficking.

SUBMITTER: Ulferts R 

PROVIDER: S-EPMC8567314 | biostudies-literature |

REPOSITORIES: biostudies-literature