The WD 40 domain of ATG 16L1 is required for its non‐canonical role in lipidation of LC 3 at single membranes

Abstract

A hallmark of macroautophagy is the covalent lipidation of LC3 and insertion into the double‐membrane phagophore, which is driven by the ATG16L1/ATG5‐ATG12 complex. In contrast, non‐canonical autophagy is a pathway through which LC3 is lipidated and inserted into single membranes, particularly endolysosomal vacuoles during cell engulfment events such as LC3‐associated phagocytosis. Factors controlling the targeting of ATG16L1 to phagophores are dispensable for non‐canonical autophagy, for which the mechanism of ATG16L1 recruitment is unknown. Here we show that the WD repeat‐containing C‐terminal domain (WD40 CTD) of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non‐canonical autophagy, but dispensable for canonical autophagy. Using this strategy to inhibit non‐canonical autophagy specifically, we show a reduction of MHC class II antigen presentation in dendritic cells from mice lacking the WD40 CTD. Further, we demonstrate activation of non‐canonical autophagy dependent on the WD40 CTD during influenza A virus infection. This suggests dependence on WD40 CTD distinguishes between macroautophagy and non‐canonical use of autophagy machinery.

Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

“…Furthermore, we tested LC3 lipidation induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 lM) treatment, shown to induce the selective degradation of mitochondria (Narendra et al, 2008), and similarly observed defective LC3 lipidation in ATG16L1 LD -expressing cells (Fig 5B). Consistent with previous results, these treatments required the activity of ATG16L1 WT to support LC3 lipidation (Fig 5D and E;Fletcher et al, 2018), whereas LC3 lipidation was strongly diminished in ATG16L1 LD -expressing cells. Given that noncanonical LC3 lipidation can occur on single membranes and requires the ATG5 complex but not additional upstream autophagy machinery, such as WIPI2 or the ULK1 complex, we further examined whether ATG16L1 LD could support LC3 lipidation during treatment with monensin, ammonium chloride (NH 4 Cl) or CCCP (100 lM), known to act as ionophores and/or lysosomotropic agents (Jacquin et al, 2017).…”

“…Interestingly, a recent study shows that the recruitment of WIPI2-to PI3P-positive recycling endosomes requires both its lipid and protein interactions with PI3P and RAB11A, respectively (Puri et al, 2018). Furthermore, ATG16L1-dependent lipidation of LC3 on single membranes, for example during treatment with ionophores and lysosomotropic agents, has been shown to occur independently of FIP200, WIPI2 and PI3P (Florey et al, 2015;Fletcher et al, 2018). Furthermore, ATG16L1-dependent lipidation of LC3 on single membranes, for example during treatment with ionophores and lysosomotropic agents, has been shown to occur independently of FIP200, WIPI2 and PI3P (Florey et al, 2015;Fletcher et al, 2018).…”

“…Given the central role of the ATG5 complex during various forms of LC3 conjugation, including both canonical and non-canonical autophagy (Fletcher et al, 2018), we aimed to investigate how the ATG5 complex is recruited to autophagy-related membranes. In this study, we have identified highly conserved sequences within the coiled-coil domain (CCD) of ATG16L1 that mediate its direct interaction with lipids, thereby enhancing its PAS localisation and autophagic activity.…”

The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal respiratory infection by influenza A virus at epithelial surfaces

“…Recent work shows that autophagy and phagocytosis can be linked by non-canonical autophagy. This is best characterized in 10 phagocytic cells where LC3 associated phagocytosis (LAP) is activated by Toll-like receptor signaling resulting in recruitment of autophagy marker protein LC3 to the phagosome membrane to enhance phagosome maturation [1][2][3][4][5] . In non-phagocytes a similar non-canonical pathway recruits LC3 to endo-lysosome compartments during the uptake of particulate material such as apoptotic cells and aggregated β-amyloid and 15 following membrane damage during pathogen entry or osmotic imbalance induced by lysosomotropic drugs 6,7 8-10 .…”

“…This prompted us to generate a mouse lacking the WD domain of ATG16L1 to study the role 10 played by non-canonical autophagy in vivo 21 . The mouse model (WD [Atg16L1 wd/wd ]) carries a stop codon after the coiled coil domain which removes amino acid residues at 266 and 319 in the linker region and conserved phenylalanine residues at positions 467 and 490 in the WD domain required for lipid binding of ATG16L1 and subsequent recruitment of ATG5-ATG12 to endo-lysosome membranes 2,22 . As a consequence the 15 mice are unable to conjugate LC3 to endo-lysosome membrane compartments 21 , but the mutation preserves the N-terminal ATG5-binding domain and glutamates at positions 226 and 230 in the CCD of ATG16L1 that are required for WIPI2 binding and autophagy 23 .…”