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Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes

Abstract

Covalent modification of LC3 and GABARAP proteins to phosphatidylethanolamine in the double-membrane phagophore is a key event in the early phase of macroautophagy, but can also occur on single-membrane structures. In both cases this involves transfer of LC3/GABARAP from ATG3 to phosphatidylethanolamine at the target membrane. Here we have purified the full-length human ATG12-5–ATG16L1 complex and show its essential role in LC3B/GABARAP lipidation in vitro. We have identified two functionally distinct membrane-binding regions in ATG16L1. An N-terminal membrane-binding amphipathic helix is required for LC3B lipidation under all conditions tested. By contrast, the C-terminal membrane-binding region is dispensable for canonical autophagy but essential for VPS34-independent LC3B lipidation at perturbed endosomes. We further show that the ATG16L1 C-terminus can compensate for WIPI2 depletion to sustain lipidation during starvation. This C-terminal membrane-binding region is present only in the β-isoform of ATG16L1, showing that ATG16L1 isoforms mechanistically distinguish between different LC3B lipidation mechanisms under different cellular conditions.

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Code availability

Code used to perform image analysis is available from the corresponding authors upon request.

Data availability

Source data for Figs. 1e,f, 2d–h, 3c–f, 4b,c,f–h, 5b–g, 6c–e and 7b,c,e–h and Supplementary Figs. 1b,f and 2e have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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References

  1. 1.

    Kabeya, Y. et al. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation. J. Cell. Sci. 117, 2805–2812 (2004).

  2. 2.

    Florey, O. & Overholtzer, M. Autophagy proteins in macroendocytic engulfment. Trends Cell Biol. 22, 374–380 (2012).

  3. 3.

    Li, M. et al. Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates. J. Biol. Chem. 286, 7327–7338 (2011).

  4. 4.

    Tanida, I., Tanida-Miyake, E., Ueno, T. & Kominami, E. The human homolog of Saccharomyces cerevisiae Apg7p is a protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3. J. Biol. Chem. 276, 1701–1706 (2001).

  5. 5.

    Tanida, I. et al. HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates. J. Biol. Chem. 279, 36268–36276 (2004).

  6. 6.

    Tanida, I., Tanida-Miyake, E., Komatsu, M., Ueno, T. & Kominami, E. Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. J. Biol. Chem. 277, 13739–13744 (2002).

  7. 7.

    Fujita, N. et al. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol. Biol. Cell. 19, 2092–2100 (2008).

  8. 8.

    Metlagel, Z., Otomo, C., Takaesu, G. & Otomo, T. Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12. Proc. Natl Acad. Sci. USA 110, 18844–18849 (2013).

  9. 9.

    Dooley, H. C. et al. WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5–16L1. Mol. Cell 55, 238–252 (2014).

  10. 10.

    Itakura, E., Kishi, C., Inoue, K. & Mizushima, N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Mol. Biol. Cell. 19, 5360–5372 (2008).

  11. 11.

    Vicinanza, M. et al. PI(5)P regulates autophagosome biogenesis. Mol. Cell 57, 219–234 (2015).

  12. 12.

    Gao, Y. et al. Golgi-associated LC3 lipidation requires V-ATPase in noncanonical autophagy. Cell Death Dis. 7, e2330 (2016).

  13. 13.

    Fletcher, K. et al. The WD40 domain of ATG16L1 is required for its non‐canonical role in lipidation of LC3 at single membranes. EMBO J. 37, e97840 (2018).

  14. 14.

    Martinez-Martin, N. et al. A switch from canonical to noncanonical autophagy shapes B cell responses. Science 355, 641–647 (2017).

  15. 15.

    Kim, J. H. et al. Insights into autophagosome maturatiothe figure legends. Experimentsn revealed by the structures of ATG5 with its interacting partners. Autophagy. 11, 75–87 (2015).

  16. 16.

    Otomo, C., Metlagel, Z., Takaesu, G. & Otomo, T. Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy. Nat. Struct. Mol. Biol. 20, 59–66 (2013).

  17. 17.

    Fujita, N. et al. Differential involvement of Atg16L1 in Crohn disease and canonical autophagy analysis of the organization of the Atg16L1 complex in fibroblasts. J. Biol. Chem. 284, 32602–32609 (2009).

  18. 18.

    Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415–424 (2014).

  19. 19.

    Sanjuan, M. A. et al. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450, 1253–1257 (2007).

  20. 20.

    Sanjuan, M. A., Milasta, S. & Green, D. R. Toll-like receptor signaling in the lysosomal pathways. Immunol. Rev. 227, 203–220 (2009).

  21. 21.

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

  22. 22.

    Jarsch, I. K. & Daste, F. & Gallop, J.L. Membrane curvature in cell biology: An integration of molecular mechanisms. J. Cell. Biol. 214, 375–387 (2016).

  23. 23.

    Kaufmann, A., Beier, V., Franquelim, H. G. & Wollert, T. Molecular mechanism of autophagic membrane-scaffold assembly and disassembly. Cell 156, 469–481 (2014).

  24. 24.

    Romanov, J. et al. Mechanism and functions of membrane binding by the Atg5–Atg12/Atg16 complex during autophagosome formation. EMBO J. 31, 4304–4317 (2012).

  25. 25.

    Mizushima, N., Noda, T. & Ohsumi, Y. Apg16p is required for the function of the Apg12p–Apg5p conjugate in the yeast autophagy pathway. EMBO J. 18, 3888–3896 (1999).

  26. 26.

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

  27. 27.

    Nishimura, T. et al. FIP200 regulates targeting of Atg16L1 to the isolation membrane. EMBO Rep. 14, 284–291 (2013).

  28. 28.

    Boada-Romero, E. et al. TMEM59 defines a novel ATG16L1-binding motif that promotes local activation of LC3. EMBO J. 32, 566–582 (2013).

  29. 29.

    Hu, J. et al. TMEM166/EVA1A interacts with ATG16L1 and induces autophagosome formation and cell death. Cell Death Dis. 7, e2323 (2016).

  30. 30.

    Fujita, N. et al. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin. J. Cell. Biol. 203, 115–128 (2013).

  31. 31.

    Mizushima, N. et al. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12–Apg5 conjugate. J. Cell. Sci. 116, 1679–1688 (2003).

  32. 32.

    Murthy, A. et al. A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature 506, 456–462 (2014).

  33. 33.

    Lundmark, R. & Carlsson, S. R. Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components. J. Biol. Chem. 278, 46772–46781 (2003).

  34. 34.

    Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome. Biol. 7, R100 (2006).

  35. 35.

    Berthold, M. R. KNIME: The Konstanz information miner. Stud. Class Data Anal. 2008, 319–326 (2008).

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Acknowledgements

We thank T. Fujita for help with the generation of ATG16L1 KO HEK293 cells and G. T. Bjørndal and N. T. Asp for help with cell culture. This work was partly supported by the Research Council of Norway (project number 221831) and through its Centres of Excellence funding scheme (project number 262652), as well as the Norwegian Cancer Society (project number 171318). Support for J.T.M. was provided by NIH grant (GM1000930), and support for K.J.K. was provided by Cellular and Molecular Biology NIH Training grant T32-GM007223. The research leading to these results has also received funding from the European Union Seventh Framework Programme (FP7-PEOPLE-2013-COFUND) under grant agreement no. 609020 - Scientia Fellows.

Author information

A.H.L. and S.R.C. designed and performed the experimental research, analysed the data, drafted the article and prepared figures. L.R.d.l.B. performed experiments, analysed the data and prepared figures. J.T.M. helped with design of experiments and data analysis. K.J.K. and S.N. helped with protein purification. T.Y. provided essential reagents and knowhow. A.S. designed the project, was involved in data analysis and wrote the final version of the manuscript.

Competing interests

The authors declare no competing interests.

Correspondence to Alf Håkon Lystad or Sven R. Carlsson or Anne Simonsen.

Integrated supplementary information

Supplementary Figure 1 Mapping membrane/protein interactions of ATG16L1.

a, Extension of Fig. 1d; Binding of recombinant ATG16L1 aa 28-229 WT, F32A, I35A/I36A or F32A/I35A/I36A and ATG16L1 aa 44-229 to liposomes containing 20% (w/w) Brain PC, 40% (w/w) Brain PS and 40% (w/w) Brain PE in a co-sedimentation assay. S: supernatant, P: pellet (n=3 independent experiments). b, The liposome binding efficiency of the ATG16L1 deletion mutants used in (a) was quantified as percent of total protein in the pellet. Data is presented as mean±SEM from n=3 independent experiments. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. c, Electrostatic surface representation of ATG12-5 together with ATG16L1 helix1 (green) and helix2 (yellow) in solution and in contact with membrane. The model was made with PyMOL using data from Metlagel et al. 20137. d, ATG16L1 KO HEK293 cells rescued or not with HA-tagged ATG16L1 (aa1-249) or ATG16L1 (aa1-249) F32A/I35A/I36A were subjected to HA immunoprecipitation and analyzed by SDS-PAGE and immunoblotting against indicated proteins (n=1 experiment). e, Extension of Fig. 1e; Binding of ATG16L1 aa 78-607, aa 78-319, aa 78-306, aa 78-284, aa 78-265 and aa 311-607 to liposomes containing 20% (w/w) Brain PC, 40% (w/w) Brain PS and 40% (w/w) Brain PE. Note the presence of a contaminating protein*. f, Quantification of the percentage sedimentation of the ATG16L1 deletion mutants used in (e) presented as mean±SEM from n=3 independent experiments. g, EGFP immunoprecipitation of ATG16L1 KO HEK293 cells rescued or not with N-terminal EGFP-tagged ATG16L1β WT (β), ATG16L1β F32A/I35A/I36A (β FII) or ATG16L1α V308A/R309A/V310A (α VRV) followed by immunoblotting for ATG5 and ATG16L1 (n=1 experiment). Unprocessed immunoblots and gels are shown in Supplementary Figure 4. Numerical source data can be found in Source data Supplementary Table 1.

Supplementary Figure 2 Purification of ATG12-5─16L1 complex and functional study of ATG16L1 membrane binding in starvation-induced autophagy and mitophagy.

a, Protocol overview for ATG12-5─16L1 complex purification from HEK-F suspension cells. See methods for a detailed description. b, In vitro lipidation reactions containing 0.5 μM ATG7, 1 μM ATG3, with or without 0.25 μM ATG12-5─16L1β/β FII, 10 mM GABARAPL1/GABARAPL2, 3 mM lipid (10 mol% bl-PI, 50 mol% DOPE and 40 mol% POPC liposomes, sonicated or extruded to 400nm), 1 mM dithiothreitol and 1 mM ATP were incubated at 30°C for 90 min. Reactions were subjected to SDS–PAGE and Coomassie blue stain (n=1 experiment). c, GABARAP lipidation in HEK293 cells, treated for 2h as indicated. Cell lysates were prepared using either 2% SDS or 1% TritonX-100 lysis buffer. Lysates were subjected to SDS-PAGE and immunoblotting against indicated proteins (n=1 experiment). d, LC3B lipidation in WT and ATG16L1 KO HEK293 cells, rescued or not with ATG16L1β WT (β), β F32A/I35A/I36A (β FII) or ATG16L1α V308A/R309A/V310A (α VRV), treated for 2h as indicated. Cell lysates were subjected to SDS-PAGE and immunoblotting against indicated proteins (n=1 experiment). e, Extension of Fig. 4a: Levels of GABARAP-II/GAPDH were quantified from immunoblots in (Fig. 4a) and normalized to fed WT cells based on n=3 independent experiments and presented as mean±SEM. Statistical analyses were performed by Two-way Anova followed by Bonferonis multiple comparison test. f, Immunoblot analysis of WT and ATG16L1 KO U2OS cells with doxycycline inducible expression of mitochondrial matrix localized EGFP-mCHERRY rescued or not with ATG16L1α V308A/R309A/V310A (α VRV), ATG16L1β F32A/I35A/I36A (β FII) or ATG16L1 (aa 1-249) (n=1 experiment). g, Confocal images of U2OS cells presented in (f). Mitophagy was assayed as the appearance of red only structures following treatment with 1 mM DFP for 24 h. Images are representative of n=3 independent experiments. Unprocessed immunoblots and gels are shown in Supplementary Figure 4. Numerical source data can be found in Source data Supplementary Table 1.

Supplementary Figure 3 Further study of ATG16L1 membrane binding in LAP and LC3-lipidation upon various perturbations of the endosomal/lysosomal network.

a, Immunoblot analysis of WT and ATG16L1 KO RAW264.7 cells rescued or not with ATG16L1β WT (β), ATG16L1β F32A/I35A/I36A (β FII) or ATG16L1 (aa 1-249) (n=1 experiment). b, Confocal images of Zymosan-Alexa488 containing phagosomes in ATG16L1 KO RAW264.7 cells immunostained for LC3B in cells rescued with ATG16L1β WT or ATG16L1 aa 1-249. Scale bars: 10μm. Images are representative of n=3 independent experiments. c, LC3B lipidation in ATG16L1 KO HEK293 cells rescued or not with ATG16L1β WT, ATG16L1α VRV or ATG16L1 (aa 1-249). Cells were treated or not with Chloroquine for 6h, monensin for 1h, hypotonic buffer for 1h or NH4Cl for 24h, in the presence of the VPS34 inhibitor VPS34IN1 and the ULK1/2 inhibitor MRT68921. Cell lysates were subjected to SDS-PAGE and immunoblotted against the indicated proteins (n=1 experiment). d, Immunoblot analysis of WT and ATG16L1 KO HEK293 cells rescued or not with N- or C-terminal EGFP tagged ATG16L1β WT (β), ATG16L1β F32A/I35A/I36A (β FII) or ATG16L1α V308A/R309A/V310A (α VRV), treated for 2h as indicated. Cell lysates were subjected to SDS-PAGE and immunoblotting against the indicated proteins (n=1 experiment). Note that samples from treatment 1 in WT cells and KO+ATG16L1β-EGFP were accidently switched*. Unprocessed immunoblots and gels are shown in Supplementary Figure 4. Numerical source data can be found in Source data Supplementary Table 1.

Supplementary Figure 4

Unprocessed images of all gels and blots

Supplementary information

Supplementary Information

Supplementary Figures 1–4 and Supplementary Table legends.

Reporting Summary

Supplementary Table 1

Statistics source data.

Supplementary Table 2

Constructs used in this study.

Supplementary Table 3

Constructs used to produce protein for in vitro lipidation.

Supplementary Table 4

CRISPR–Cas9 guides used in this study.

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Fig. 1: Identification of two membrane-binding regions in ATG16L1.
Fig. 2: The ATG12–5–ATG16L1 complex is required for in vitro LC3B lipidation.
Fig. 3: ATG16L1 membrane binding is required for efficient in vitro LC3 and GABARAP lipidation.
Fig. 4: Membrane binding of ATG16L1 in autophagy and during lipidation on perturbed endosomes reveals isoform-specific functions.
Fig. 5: ATG16L1 membrane binding is not required for recruitment to WIPI2 puncta.
Fig. 6: The C-terminal end of ATG16L1 can compensate for WIPI2b function.
Fig. 7: The β-specific sequence in ATG16L1 is required for recruitment and LC3B lipidation on perturbed endosomes.
Supplementary Figure 1: Mapping membrane/protein interactions of ATG16L1.
Supplementary Figure 2: Purification of ATG12-5─16L1 complex and functional study of ATG16L1 membrane binding in starvation-induced autophagy and mitophagy.
Supplementary Figure 3: Further study of ATG16L1 membrane binding in LAP and LC3-lipidation upon various perturbations of the endosomal/lysosomal network.
Supplementary Figure 4