Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Abstract

Autophagy is a fundamental catabolic process essential for development, homeostasis and proper immune function 1 . During autophagy, a cascade of ATG proteins target intracellular cargoes for lysosomal degradation and recycling 2 . This pathway utilises a unique post-translational modification, the conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at autophagosomes, which modulates cargo selection and maturation. ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal compartments, which plays a key role in phagocytosis and other processes 3 . It has been widely assumed that SMAC involves the same lipidation of ATG8 to PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes alternative lipidation to phosphatidylserine (PS) during non-canonical autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, by pharmacological agents 4,5 , or during non-canonical autophagy processes such as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8 , induces the covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-PE adducts are differentially delipidated by isoforms of the ATG4 family, indicating significant molecular distinctions and mechanisms between these two species. Together, these results provide an important new insight into autophagy signalling, revealing an alternative form of the hallmark ATG8-lipidation event, so widely used to define and assay autophagy. Furthermore, ATG8-PS lipidation provides a specific ‘molecular signature’ for non-canonical autophagy, uncovering a novel means of detecting and monitoring this emerging pathway.

References 54

1. 1.

   Acheson J.F., Derewenda Z.S., Zimmer J. Architecture of the cellulose synthase outer membrane channel and its association with the periplasmic TPR domain. Structure. 2019;27:1855-1861.e3.

   Not in our library10.1016/j.str.2019.09.008

2. 2.
   Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells · 2019

   No notice
3. 3.

   Betin V.M., Lane J.D. Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J. Cell Sci. 2009;122:2554-2566.

   Not in our library10.1242/jcs.046250

4. 4.

   Choy A., Dancourt J., Mugo B., O’Connor T.J., Isberg R.R., Melia T.J., Roy C.R. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science. 2012;338:1072-1076.

   Not in our library10.1126/science.1227026

5. 5.

   Cunha L.D., Yang M., Carter R., Guy C., Harris L., Crawford J.C., Quarato G., Boada-Romero E., Kalkavan H., Johnson M.D.L. LC3-associated phagocytosis in myeloid cells promotes tumor immune tolerance. Cell. 2018;175:429-441.e16.

   Not in our library10.1016/j.cell.2018.08.061

6. 6.

   de Wit E., Spronken M.I., Bestebroer T.M., Rimmelzwaan G.F., Osterhaus A.D., Fouchier R.A. Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res. 2004;103:155-161.

   Not in our library10.1016/j.virusres.2004.02.028

7. 7.
   WIPI2 Links LC3 Conjugation with PI3P, Autophagosome Formation, and Pathogen Clearance by Recruiting Atg12–5-16L1 · 2014

   No notice
8. 8.

   Durgan J., Tseng Y.Y., Hamann J.C., Domart M.C., Collinson L., Hall A., Overholtzer M., Florey O. Mitosis can drive cell cannibalism through entosis. eLife. 2017;6:e27134.

   Not in our library10.7554/elife.27134

9. 9.

   Eck F., Phuyal S., Smith M.D., Kaulich M., Wilkinson S., Farhan H., Behrends C. ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis. J. Cell Sci. 2020;133:jcs243477.

   Not in our library10.1242/jcs.243477

10. 10.
    STING induces LC3B lipidation onto single-membrane vesicles via the V-ATPase and ATG16L1-WD40 domain · 2020

    No notice
11. 11.
    The WD40 domain of ATG16L1 is required for its non‐canonical role in lipidation of LC3 at single membranes · 2018

    No notice
12. 12.

    Florey O., Kim S.E., Sandoval C.P., Haynes C.M., Overholtzer M. Autophagy machinery mediates macroendocytic processing and entotic cell death by targeting single membranes. Nat. Cell Biol. 2011;13:1335-1343.

    Not in our library10.1038/ncb2363

13. 13.

    Florey O., Gammoh N., Kim S.E., Jiang X., Overholtzer M. V-ATPase and osmotic imbalances activate endolysosomal LC3 lipidation. Autophagy. 2015;11:88-99.

    Not in our library10.4161/15548627.2014.984277

14. 14.

    Fujita N., Itoh T., Omori H., Fukuda M., Noda T., Yoshimori T. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell. 2008;19:2092-2100.

    Not in our library10.1091/mbc.e07-12-1257

15. 15.

    Galluzzi L., Green D.R. Autophagy-independent functions of the autophagy machinery. Cell. 2019;177:1682-1699.

    Not in our library10.1016/j.cell.2019.05.026

16. 16.

    Gammoh N., Florey O., Overholtzer M., Jiang X. Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy. Nat. Struct. Mol. Biol. 2013;20:144-149.

    Not in our library10.1038/nsmb.2475

17. 17.

    Gao Y., Liu Y., Hong L., Yang Z., Cai X., Chen X., Fu Y., Lin Y., Wen W., Li S. Golgi-associated LC3 lipidation requires V-ATPase in noncanonical autophagy. Cell Death Dis. 2016;7:e2330.

    Not in our library10.1038/cddis.2016.236

18. 18.

    Hanada T., Noda N.N., Satomi Y., Ichimura Y., Fujioka Y., Takao T., Inagaki F., Ohsumi Y. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J. Biol. Chem. 2007;282:37298-37302.

    Not in our library10.1074/jbc.c700195200

19. 19.

    Hartler J., Triebl A., Ziegl A., Trötzmüller M., Rechberger G.N., Zeleznik O.A., Zierler K.A., Torta F., Cazenave-Gassiot A., Wenk M.R. Deciphering lipid structures based on platform-independent decision rules. Nat. Methods. 2017;14:1171-1174.

    Not in our library10.1038/nmeth.4470

20. 20.

    Heckmann B.L., Teubner B.J.W., Tummers B., Boada-Romero E., Harris L., Yang M., Guy C.S., Zakharenko S.S., Green D.R. LC3-associated endocytosis facilitates β-amyloid clearance and mitigates neurodegeneration in murine Alzheimer’s disease. Cell. 2019;178:536-551.e14.

    Not in our library10.1016/j.cell.2019.05.056

21. 21.

    Henault J., Martinez J., Riggs J.M., Tian J., Mehta P., Clarke L., Sasai M., Latz E., Brinkmann M.M., Iwasaki A. Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes. Immunity. 2012;37:986-997.

    Not in our library10.1016/j.immuni.2012.09.014

22. 22.

    Ichimura Y., Kirisako T., Takao T., Satomi Y., Shimonishi Y., Ishihara N., Mizushima N., Tanida I., Kominami E., Ohsumi M. A ubiquitin-like system mediates protein lipidation. Nature. 2000;408:488-492.

    Not in our library10.1038/35044114

23. 23.

    Jacquin E., Leclerc-Mercier S., Judon C., Blanchard E., Fraitag S., Florey O. Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation. Autophagy. 2017;13:854-867.

    Not in our library10.1080/15548627.2017.1287653

24. 24.

    Jacquin E., Fletcher K., Florey O. Imaging noncanonical autophagy and LC3-associated phagocytosis in cultured cells. Methods Mol. Biol. 2019;1880:295-303.

    Not in our library10.1007/978-1-4939-8873-0_19

25. 25.

    Johansen T., Lamark T. Selective autophagy: ATG8 family proteins, LIR motifs and cargo receptors. J. Mol. Biol. 2020;432:80-103.

    Not in our library10.1016/j.jmb.2019.07.016

26. 26.

    Kauffman K.J., Yu S., Jin J., Mugo B., Nguyen N., O’Brien A., Nag S., Lystad A.H., Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases. Autophagy. 2018;14:992-1010.

    Not in our library10.1080/15548627.2018.1437341

27. 27.

    Kim J.Y., Zhao H., Martinez J., Doggett T.A., Kolesnikov A.V., Tang P.H., Ablonczy Z., Chan C.C., Zhou Z., Green D.R., Ferguson T.A. Noncanonical autophagy promotes the visual cycle. Cell. 2013;154:365-376.

    Not in our library10.1016/j.cell.2013.06.012

28. 28.

    Kirisako T., Ichimura Y., Okada H., Kabeya Y., Mizushima N., Yoshimori T., Ohsumi M., Takao T., Noda T., Ohsumi Y. The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J. Cell Biol. 2000;151:263-276.

    Not in our library10.1083/jcb.151.2.263

29. 29.

    Klionsky D.J., Abdelmohsen K., Abe A., Abedin M.J., Abeliovich H., Acevedo Arozena A., Adachi H., Adams C.M., Adams P.D., Adeli K. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 2016;12:1-222.

    Not in our library10.1080/15548627.2015.1100356

30. 30.

    Kyöstilä K., Syrjä P., Jagannathan V., Chandrasekar G., Jokinen T.S., Seppälä E.H., Becker D., Drögemüller M., Dietschi E., Drögemüller C. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease. PLoS Genet. 2015;11:e1005169.

    Not in our library10.1371/journal.pgen.1005169

31. 31.

    Lystad A.H., Carlsson S.R., de la Ballina L.R., Kauffman K.J., Nag S., Yoshimori T., Melia T.J., Simonsen A. Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes. Nat. Cell Biol. 2019;21:372-383.

    Not in our library10.1038/s41556-019-0274-9

32. 32.

    Ma J., Becker C., Lowell C.A., Underhill D.M. Dectin-1-triggered recruitment of light chain 3 protein to phagosomes facilitates major histocompatibility complex class II presentation of fungal-derived antigens. J. Biol. Chem. 2012;287:34149-34156.

    Not in our library10.1074/jbc.m112.382812

33. 33.

    Martinez J., Malireddi R.K., Lu Q., Cunha L.D., Pelletier S., Gingras S., Orchard R., Guan J.L., Tan H., Peng J. Molecular characterization of LC3-associated phagocytosis reveals distinct roles for Rubicon, NOX2 and autophagy proteins. Nat. Cell Biol. 2015;17:893-906.

    Not in our library10.1038/ncb3192

34. 34.

    Martinez J., Cunha L.D., Park S., Yang M., Lu Q., Orchard R., Li Q.Z., Yan M., Janke L., Guy C. Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature. 2016;533:115-119.

    Not in our library10.1038/nature17950

35. 35.

    Mizushima N. The ATG conjugation systems in autophagy. Curr. Opin. Cell Biol. 2020;63:1-10.

    Not in our library10.1016/j.ceb.2019.12.001

36. 36.

    Mizushima N., Yoshimori T. How to interpret LC3 immunoblotting. Autophagy. 2007;3:542-545.

    Not in our library10.4161/auto.4600

37. 37.

    Nakatogawa H., Oh-oka K., Ohsumi Y. Lipidation of Atg8: how is substrate specificity determined without a canonical E3 enzyme? Autophagy. 2008;4:911-913.

    Not in our library10.4161/auto.6646

38. 38.

    Nguyen T.N., Padman B.S., Usher J., Oorschot V., Ramm G., Lazarou M. Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation. J. Cell Biol. 2016;215:857-874.

    Not in our library10.1083/jcb.201607039

39. 39.

    Oh-oka K., Nakatogawa H., Ohsumi Y. Physiological pH and acidic phospholipids contribute to substrate specificity in lipidation of Atg8. J. Biol. Chem. 2008;283:21847-21852.

    Not in our library10.1074/jbc.m801836200

40. 40.
    A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B · 2017

    No notice
41. 41.

    Rai S., Arasteh M., Jefferson M., Pearson T., Wang Y., Zhang W., Bicsak B., Divekar D., Powell P.P., Naumann R. The ATG5-binding and coiled coil domains of ATG16L1 maintain autophagy and tissue homeostasis in mice independently of the WD domain required for LC3-associated phagocytosis. Autophagy. 2019;15:599-612.

    Not in our library10.1080/15548627.2018.1534507

42. 42.

    Sanjuan M.A., Dillon C.P., Tait S.W., Moshiach S., Dorsey F., Connell S., Komatsu M., Tanaka K., Cleveland J.L., Withoff S., Green D.R. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007;450:1253-1257.

    Not in our library10.1038/nature06421

43. 43.

    Santiago C., Ballesteros A., Martínez-Muñoz L., Mellado M., Kaplan G.G., Freeman G.J., Casasnovas J.M. Structures of T cell immunoglobulin mucin protein 4 show a metal-Ion-dependent ligand binding site where phosphatidylserine binds. Immunity. 2007;27:941-951.

    Not in our library10.1016/j.immuni.2007.11.008

44. 44.

    Satoo K., Noda N.N., Kumeta H., Fujioka Y., Mizushima N., Ohsumi Y., Inagaki F. The structure of Atg4B-LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy. EMBO J. 2009;28:1341-1350.

    Not in our library10.1038/emboj.2009.80

45. 45.

    Sou Y.S., Tanida I., Komatsu M., Ueno T., Kominami E. Phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian Atg8 modifiers, LC3, GABARAP, and GATE-16. J. Biol. Chem. 2006;281:3017-3024.

    Not in our library10.1074/jbc.m505888200

46. 46.

    Sugawara K., Suzuki N.N., Fujioka Y., Mizushima N., Ohsumi Y., Inagaki F. Structural basis for the specificity and catalysis of human Atg4B responsible for mammalian autophagy. J. Biol. Chem. 2005;280:40058-40065.

    Not in our library10.1074/jbc.m509158200

47. 47.

    Syrjä P., Anwar T., Jokinen T., Kyöstilä K., Jäderlund K.H., Cozzi F., Rohdin C., Hahn K., Wohlsein P., Baumgärtner W. Basal autophagy is altered in Lagotto Romagnolo dogs with an ATG4D mutation. Vet. Pathol. 2017;54:953-963.

    Not in our library10.1177/0300985817712793

48. 48.

    Tanida I., Ueno T., Kominami E. Human light chain 3/MAP1LC3B is cleaved at its carboxyl-terminal Met121 to expose Gly120 for lipidation and targeting to autophagosomal membranes. J. Biol. Chem. 2004;279:47704-47710.

    Not in our library10.1074/jbc.m407016200

49. 49.

    Ulferts R., Marcassa E., Timimi L., Lee L.C., Daley A., Montaner B., Turner S.D., Florey O., Baillie J.K., Beale R. Subtractive CRISPR screen identifies factors involved in non-canonical LC3 lipidation. bioRxiv. 2020.

    Not in our library10.1016/j.celrep.2021.109899

50. 50.

    Wang Y., Zhang W., Jefferson M., Sharma P., Bone B., Kipar A., Coombes J.L., Pearson T., Man A., Zhekova A. The WD and linker domains of ATG16L1 required for non-canonical autophagy limit lethal respiratory infection by influenza A virus at epithelial surfaces. bioRxiv. 2020.

    Not in our library10.15252/embj.2020105543

51. 51.

    Yang A., Pantoom S., Wu Y.W. Elucidation of the anti-autophagy mechanism of the Legionella effector RavZ using semisynthetic LC3 proteins. eLife. 2017;6:e23905.

    Not in our library10.7554/elife.23905

52. 52.

    Yeung T., Gilbert G.E., Shi J., Silvius J., Kapus A., Grinstein S. Membrane phosphatidylserine regulates surface charge and protein localization. Science. 2008;319:210-213.

    Not in our library10.1126/science.1152066

53. 53.

    Yeung T., Heit B., Dubuisson J.F., Fairn G.D., Chiu B., Inman R., Kapus A., Swanson M., Grinstein S. Contribution of phosphatidylserine to membrane surface charge and protein targeting during phagosome maturation. J. Cell Biol. 2009;185:917-928.

    Not in our library10.1083/jcb.200903020

54. 54.

    Zhuang X., Magri A., Hill M., Lai A.G., Kumar A., Rambhatla S.B., Donald C.L., Lopez-Clavijo A.F., Rudge S., Pinnick K. The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication. Nat. Commun. 2019;10:377.

    Not in our library10.1038/s41467-019-08299-7

Cited by papers we hold 5

• The role of autophagy in viral infections · 2023
  No notice
• Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment · 2023
  Not checked
• ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling · 2023
  No notice
• V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy · 2022
  No notice
• Beyond autophagy: LC3-associated phagocytosis and endocytosis · 2022
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.