PT  - JOURNAL ARTICLE
AU  - Rachel Ulferts
AU  - Elena Marcassa
AU  - Lewis Timimi
AU  - Liam C Lee
AU  - Andrew Daley
AU  - Beatriz Montaner
AU  - Suzanne D. Turner
AU  - Oliver Florey
AU  - J. Kenneth Baillie
AU  - Rupert Beale
TI  - Subtractive CRISPR screen identifies factors involved in non-canonical LC3 lipidation
AID  - 10.1101/2020.11.18.388306
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.11.18.388306
4099  - http://biorxiv.org/content/early/2020/11/18/2020.11.18.388306.short
4100  - http://biorxiv.org/content/early/2020/11/18/2020.11.18.388306.full
AB  - Although commonly associated with autophagosomes, LC3 can also be recruited to membranes in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. LC3 is attached to membranes by covalent lipidation that depends on recruitment of the ATG5-12-16L1 complex. Non-canonical LC3 lipidation requires the C-terminal WD40 domain of ATG16L1 that is dispensable for canonical autophagy. We devised a subtractive CRISPR knock-out screening strategy to investigate the requirements for non-canonical LC3-lipidation. This correctly identified the enzyme complexes directly responsible for LC3-lipidation. We additionally identified the RALGAP complex as important for M2-induced, but not ionophore drug induced LC3 lipidation. In contrast, we identified ATG4D as responsible for LC3 recycling in M2-induced and basal LC3-lipidation. Identification of a vacuolar ATPase subunit in the screen suggested a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug induced LC3-lipidation leads to association of the vacuolar ATPase and ATG16L1 and can be antagonised by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.Competing Interest StatementThe authors have declared no competing interest.