Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Abstract

29 30 Autophagy is a fundamental catabolic process essential for development, 31 homeostasis and proper immune function 1 . During autophagy, a cascade of 32 ATG proteins target intracellular cargoes for lysosomal degradation and 33 recycling 2 . This pathway utilises a unique post-translational modification, the 34 conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at 35 autophagosomes, which modulates cargo selection and maturation. 36 ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway 37 involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal 38compartments, which plays a key role in phagocytosis and other processes 3 . It 39 has been widely assumed that SMAC involves the same lipidation of ATG8 to 40 PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes 41 alternative lipidation to phosphatidylserine (PS) during non-canonical 42 autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, 43 by pharmacological agents 4,5 , or during non-canonical autophagy processes such 44 as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8 , induces the 45 covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation 46 event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched 47 endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-48 PE adducts are differentially delipidated by isoforms of the ATG4 family, 49 indicating significant molecular distinctions and mechanisms between these two 50 species. 51Together, these results provide an important new insight into autophagy 52 signalling, revealing an alternative form of the hallmark ATG8-lipidation event, 53Main 60 61 A defining feature of autophagy is the lipidation of ATG8, a family of ubiquitin-like 62 proteins including mammalian LC3s (A/B/B2/C) and GABARAPs 63 (GABARAP/L1/L2) 9 . Nascent pro-ATG8 is first primed by a cysteine protease, 64 ATG4, to expose a conserved aromatic-Gly motif at its C-terminus 10 . A ubiquitin-65 like conjugation system, comprised of ATG7 (E1-like), ATG3 (E2-like) and 66 ATG16L1/ATG12/ATG5 (E3-like), then drives the covalent ligation of this glycine to 67 a lipid, phosphatidylethanolamine (PE), via an amide bond to its headgroup (Extended 68 Data Fig. 1a) 11,12 . This is a unique post-translational modification that recruits ATG8 69 to autophagosomal membranes, where it plays an important role in cargo loading and 70 autophagosome maturation 9,13 . The associated relocalisation of ATG8s, and the 71 characteristic protein bandshift between the unlipidated (ATG8-I) and lipidated 72 (ATG8-II) forms, are widely used to define and assay autophagy-related processes 73 14,15 . 74 75A second phospholipid, phosphatidylserine (PS), also bears an amino group in its 76 head moiety (Extended Data Fig. 1b), which can be conjugated to ATG8 in vitro 16 . 77However, in vivo, ATG8 lipidation is reported to occur exclusively to PE, in both 78 yeast 11 and mammalian cells 16 . The mechanism underlying cellular...

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“…We have shown that LC3 can also become conjugated to phosphatidylserine (PS)-enriched membranes during non-canonical autophagy. ATG4D, but not the other paralogues, appears to be uniquely capable for the de-conjugation of LC3-PS (Durgan et al, 2020).…”

“…Similar observations were made for GABAR-AP-L1 and GABARAP-L2. Elsewhere we describe a role for ATG4D in delipidation of phosphatidylserine conjugated LC3, which accumulates during WD40 CTD-dependent LC3-lipidation – including in response to IAV M2 – but not during canonical autophagy (Durgan et al, 2020). A missense mutation in ATG4D was found in Lagotto Romagnolo dogs with progressive neurological symptoms (Kyöstilä et al, 2015).…”

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“…Similar to LAP, LANDO requires Rubicon and ATG5 activity but not FIP200. Interestingly, a recent study has shown the ability of LC3 to be conjugated to another lipid, phosphatidylserine, expanding the possible roles of non-canonical autophagy pathways ( 41 ). Further studies are required to understand the role and the mechanism of these processes.…”

The regulatory factors and pathological roles of autophagy‐related protein 4 in diverse diseases: Recent research advances

“…The evolved virus can take advantage of the autophagy proteins to construct its skeleton in the early stages of infection. ATG4B can hydrolytically cleave an EV71 3C pro substrate in addition to priming ATG8‐PE, ATG8‐PS, 16 and LC3B‐ATG3 187 . Therefore, further studies should be conducted on the novel functions of ATG4s as a cysteine protease.…”

“…The C‐terminal residues of pro‐LC3 are immediately primed by a cysteine protease, ATG4B, producing the free cytoplasmic LC3‐I form and exposing a glycine at its COOH‐terminal. This renders the conjunction of LC3 with phosphatidylethanolamine (PE) on phagophore membranes mostly during canonical autophagy or alternatively to phosphatidylserine (PS) during noncanonical autophagy 13–16 . A succession of ubiquitin‐like proteins contributes to catalyzing the formation of an amide bond between the C‐terminal carboxyl group of LC3 and the hydrophilic head group amino of the lipid PE, producing covalent membrane‐bound LC3‐II 17,18 .…”