Abstract
SummaryAlthough 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.
Highlights
Autophagy is a catabolic process characterized by the delivery of cytoplasmic material to the lysosome for degradation (Mizushima and Komatsu, 2011)
The proton channel activity of M2 is required for M2induced light chain 3 (LC3) lipidation We previously showed that influenza A virus (IAV) induces LC3 lipidation and relocalization to the plasma membrane through a pathway that differs from canonical autophagy (Beale et al, 2014)
Other WD40 CTD-dependent LC3 lipidation processes target endo-lysosomal vesicles and components overlap with the pathway required for IAV entry into the cell (Florey et al, 2011; Sanjuan et al, 2007)
Summary
Autophagy is a catabolic process characterized by the delivery of cytoplasmic material to the lysosome for degradation (Mizushima and Komatsu, 2011). LC3 becomes covalently conjugated to phosphatidylethanolamine (PE) at sites forming double-membrane autophagosomes (Mizushima et al, 1998). Lipidation depends on the activity of two ubiquitin-like conjugation systems comprising ATG3, ATG5, ATG7, ATG10, and ATG12 (Kaufmann et al, 2014). ATG5 and ATG12 form a complex with ATG16L1 that catalyzes the transfer of activated LC3 to PE, in a manner analogous to an E3 ligase. Localization of this complex determines site specificity of LC3 lipidation (Fujioka et al, 2014). The soluble form of LC3 is referred to as LC3-I, and the PE-conjugated form as LC3-II
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