Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3

Nature Cell Biology volume 16, pages 415–424 (2014)Cite this article

Subjects

An Erratum to this article was published on 01 August 2014

An Erratum to this article was published on 01 July 2014

This article has been updated

Abstract

The components supporting autophagosome growth on the cup-like isolation membrane are likely to be different from those found on closed and maturing autophagosomes. The highly curved rim of the cup may serve as a functionally required surface for transiently associated components of the early acting autophagic machinery. Here we demonstrate that the E2-like enzyme, Atg3, facilitates LC3/GABARAP lipidation only on membranes exhibiting local lipid-packing defects. This activity requires an amino-terminal amphipathic helix similar to motifs found on proteins targeting highly curved intracellular membranes. By tuning the hydrophobicity of this motif, we can promote or inhibit lipidation in vitro and in rescue experiments in Atg3-knockout cells, implying a physiologic role for this stress detection. The need for extensive lipid-packing defects suggests that Atg3 is designed to work at highly curved membranes, perhaps including the limiting edge of the growing phagophore.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: In vitro reconstitution of the lipidation reaction.
Figure 2: Lipidation requires local membrane defects.
Figure 3: The lipidation reaction is membrane curvature dependent.
Figure 4: Atg3 is a membrane curvature sensor.
Figure 5: The N terminus of Atg3 is a curvature-sensing amphipathic helix.
Figure 6: An intact amphipathic helix is necessary for Atg3 function in vivo.

Similar content being viewed by others

Change history

  • 06 June 2014

    In the version of this Article originally published, the x axis labels 'WT' and 'K11L' on the graph in Fig. 5c were swapped. This error has now been corrected in all online versions of the Article.

  • 04 July 2014

    In all previously published versions of this Article, the graph in Fig. 5c was incorrect. This error has now been corrected in all online versions of the Article.

  • 01 August 2014

    Nat. Cell Biol. 16, 415–424 (2014); published online 20 April 2014; corrected after print 4 July 2014 In all previously published versions of this Article, the graph in Fig. 5c was incorrect; the correct graph is shown below. This error has now been corrected in all online versions of the Article.

References

  1. Itakura, E. & Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010).

    Article  CAS  Google Scholar 

  2. Ichimura, Y. et al. A ubiquitin-like system mediates protein lipidation. Nature 408, 488–492 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Nakatogawa, H., Ichimura, Y. & Ohsumi, Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165–178 (2007).

    Article  CAS  Google Scholar 

  5. Weidberg, H. et al. LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis. EMBO J. 29, 1792–1802 (2010).

    Article  CAS  Google Scholar 

  6. Shpilka, T., Weidberg, H., Pietrokovski, S. & Elazar, Z. Atg8: an autophagy-related ubiquitin-like protein family. Genome Biol. 12, 226 (2011).

    Article  CAS  Google Scholar 

  7. Sou, Y. S. et al. The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol. Biol. Cell 19, 4762–4775 (2008).

    Article  CAS  Google Scholar 

  8. Komatsu, M. et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J. Cell Biol. 169, 425–434 (2005).

    Article  CAS  Google Scholar 

  9. Nishida, Y. et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654–658 (2009).

    Article  CAS  Google Scholar 

  10. Mizushima, N. et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J. Cell Biol. 152, 657–668 (2001).

    Article  CAS  Google Scholar 

  11. Hayashi-Nishino, M. et al. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat. Cell Biol. 11, 1433–1437 (2009).

    Article  CAS  Google Scholar 

  12. Fan, W., Nassiri, A. & Zhong, Q. Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L). Proc. Natl Acad. Sci. USA 108, 7769–7774 (2011).

    Article  CAS  Google Scholar 

  13. Drin, G. & Antonny, B. Amphipathic helices and membrane curvature. FEBS Letters 584, 1840–1847 (2010).

    Article  CAS  Google Scholar 

  14. Hayashi-Nishino, M. et al. Electron tomography reveals the endoplasmic reticulum as a membrane source for autophagosome formation. Autophagy 6, 301–303 (2009).

    Article  Google Scholar 

  15. Ichimura, Y. et al. In vivo and in vitro reconstitution of Atg8 conjugation essential for autophagy. J. Biol. Chem. 279, 40584–40592 (2004).

    Article  CAS  Google Scholar 

  16. 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. 281, 3017–3024 (2006).

    Article  CAS  Google Scholar 

  17. Tanida, I., Sou, Y. S., Minematsu-Ikeguchi, N., Ueno, T. & Kominami, E. Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3. FEBS J. 273, 2553–2562 (2006).

    Article  CAS  Google Scholar 

  18. Choy, A. et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. Brownell, J. E. et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol. Cell 37, 102–111 (2010).

    Article  CAS  Google Scholar 

  21. Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2011).

    Article  CAS  Google Scholar 

  22. Zimmerberg, J. & Kozlov, M. M. How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006).

    Article  CAS  Google Scholar 

  23. Hatzakis, N. S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nat. Chem. Biol. 5, 835–841 (2009).

    Article  CAS  Google Scholar 

  24. Vamparys, L. et al. Conical lipids in flat bilayers induce packing defects similar to that induced by positive curvature. Biophys. J. 104, 585–593 (2013).

    Article  CAS  Google Scholar 

  25. Vanni, S. et al. Amphipathic lipid packing sensor motifs: Probing bilayer defects with hydrophobic residues. Biophys. J. 104, 575–584 (2013).

    Article  CAS  Google Scholar 

  26. Kamal, M. M., Mills, D., Grzybek, M. & Howard, J. Measurement of the membrane curvature preference of phospholipids reveals only weak coupling between lipid shape and leaflet curvature. Proc. Natl Acad. Sci. USA 106, 22245–22250 (2009).

    Article  CAS  Google Scholar 

  27. Hope, M. J., Bally, M. B., Webb, G. & Cullis, P. R. Production of large unilamellar vesicles by a rapid extrusion procedure characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta. 812, 55–65 (1985).

    Article  CAS  Google Scholar 

  28. Antonny, B. Mechanisms of membrane curvature sensing. Annu. Rev. Biochem. (2010).

  29. Gautier, R., Douguet, D., Antonny, B. & Drin, G. HELIQUEST: a web server to screen sequences with specific alpha-helical properties. Bioinformatics 24, 2101–2102 (2008).

    Article  CAS  Google Scholar 

  30. Radoshevich, L. et al. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell 142, 590–600 (2010).

    Article  CAS  Google Scholar 

  31. Hanada, T. et al. The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J. Biol. Chem. 282, 37298–37302 (2007).

    Article  CAS  Google Scholar 

  32. Sakoh-Nakatogawa, M. et al. Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site. Nat. Struct. Mol. Biol. 20, 433–439 (2013).

    Article  CAS  Google Scholar 

  33. Noda, N. N., Fujioka, Y., Hanada, T., Ohsumi, Y. & Inagaki, F. Structure of the Atg12-Atg5 conjugate reveals a platform for stimulating Atg8-PE conjugation. EMBO Rep. 14, 206–211 (2013).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).

    Article  CAS  Google Scholar 

  36. Hosokawa, N., Hara, Y. & Mizushima, N. Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett. 580, 2623–2629 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Fujioka, Y. et al. In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy. J. Biol. Chem. 283, 1921–1928 (2008).

    Article  CAS  Google Scholar 

  39. Hanada, T., Satomi, Y., Takao, T. & Ohsumi, Y. The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation. FEBS Lett. 583, 1078–1083 (2009).

    Article  CAS  Google Scholar 

  40. Pranke, I. M. et al. alpha-Synuclein and ALPS motifs are membrane curvature sensors whose contrasting chemistry mediates selective vesicle binding. J. Cell Biol. 194, 89–103 (2011).

    Article  CAS  Google Scholar 

  41. Bigay, J., Casella, J. F., Drin, G., Mesmin, B. & Antonny, B. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24, 2244–2253 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Yu, Z. Q. et al. Dual roles of Atg8-PE deconjugation by Atg4 in autophagy. Autophagy 8 (2012).

    Article  CAS  Google Scholar 

  44. Nakatogawa, H., Ishii, J., Asai, E. & Ohsumi, Y. Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis. Autophagy 8, 177–186 (2012).

    Article  CAS  Google Scholar 

  45. Proikas-Cezanne, T. & Robenek, H. Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes. J. Cell. Mol. Med. 15, 2007–2010 (2011).

    Article  CAS  Google Scholar 

  46. Mizushima, N., Yoshimori, T. & Ohsumi, Y. The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132 (2011).

    Article  CAS  Google Scholar 

  47. Ragusa, M. J., Stanley, R. E. & Hurley, J. H. Architecture of the Atg17 complex as a scaffold for autophagosome biogenesis. Cell 151, 1501–1512 (2012).

    Article  CAS  Google Scholar 

  48. Helfrich, W. Elastic properties of lipid bilayers: theory and possible experiments. Z. Naturforsch. C 28, 693–703 (1973).

    Article  CAS  Google Scholar 

  49. Jotwani, A., Richerson, D. N., Motta, I., Julca-Zevallos, O. & Melia, T. J. Approaches to the study of Atg8-mediated membrane dynamics in vitro. Methods Cell Biol. 108, 93–116 (2012).

    Article  CAS  Google Scholar 

  50. Choy, A. et al. The Legionella effector RavZ inhibits host autophagy through irreversible Atg8 deconjugation. Science 338, 1072–1076 (2012).

    Article  CAS  Google Scholar 

  51. Nair, U. et al. SNARE proteins are required for macroautophagy. Cell 146, 290–302 (2011).

    Article  CAS  Google Scholar 

  52. Jotwani, A., Richerson, D. N., Motta, I., Julca-Zevallos, O. & Melia, T. J. Approaches to the study of Atg8-mediated membrane dynamics in vitro. Methods Cell Biol. 108, 93–116 (2012).

    Article  CAS  Google Scholar 

  53. Melia, T. J. et al. Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins. J. Cell Biol. 158, 929–940 (2002).

    Article  CAS  Google Scholar 

  54. Burnette, W. N. ‘Western blotting’: Electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112, 195–203 (1981).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

Atg3-knockout MEFs were a gift from M. Komatsu, (Tokyo Metropolitan Organization for Medical Research, Japan) and Atg5 knockout MEFs from N. Mizushima (Tokyo University). This work was financially supported by grants from the NIH (GM100930—T.J.M.) and (NS063973—T.J.M. and A.Y.) and the ERC (advanced grant 268888—B.A.) and by Yale fellowships to G.P. (BioSTEP) and T.J.M. (Anderson). We also thank A. Jotwani and B. Mugo for help with electrophoresis experiments, E. Karatekin for help with dynamic light scattering and F. Pincet for helpful comments.

Author information

Author notes

  1. Sangeeta Nath, Julia Dancourt and Vladimir Shteyn: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA

    Sangeeta Nath, Julia Dancourt, Vladimir Shteyn, Gabriella Puente, Shanta Nag, Joerg Bewersdorf & Thomas J. Melia

  2. Departments of Neurology, Pathology and Cell Biology, Columbia University, New York 10032, USA

    Wendy M. Fong & Ai Yamamoto

  3. Institut de Pharmacologie Moléculaire et Cellulaire, CNRS et université de Nice, 06103, France

    Bruno Antonny

Authors
  1. Sangeeta Nath
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Julia Dancourt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vladimir Shteyn
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gabriella Puente
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wendy M. Fong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shanta Nag
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Joerg Bewersdorf
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ai Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bruno Antonny
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Thomas J. Melia
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S. Nath conceived, designed and carried out liposome experiments, analysed the data and assisted with a draft of the manuscript. J.D. conceived, designed and performed cell culture experiments and assisted with statistical analyses. V.S. conceived, designed and performed cell culture experiments and assisted with statistical analyses. G.P. made the initial discovery of curvature sensing in a liposomal system and carried out experiments with lipid composition tests. W.M.F. conceived, designed and carried out experiments testing activity in Atg5 knockout cell lines. S. Nag developed protein expression strategies and generated many of the tools supporting both the liposomal and cell culture studies. J.B. analysed imaging data. A.Y. conceived and designed experiments involved in Atg5 knockout rescues and assisted with the final manuscript. B.A. conceived and designed experiments on liposome curvature studies, assisted with data analysis and assisted with the manuscript. T.J.M. conceived the project, designed experiments, carried out liposome experiments and data analysis and wrote the manuscript.

Corresponding author

Correspondence to Thomas J. Melia.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro observation of lipidation intermediate.

Lipidation of each LC3/GABARAP protein in vitro results in three distinct bands: the starting unlipidated protein, the faster migrating fully-lipidated protein, and a third band with a migration that depends upon the source protein (for LC3 and GRL2 it runs between the unlipidated and lipidated forms (Fig. 1), for GRL1 shown here, it runs slower than either form). To confirm that this additional product is a lipidation intermediate, a lipidation reaction was run as in Fig. 1 but without any final substrate lipid. By 28 minutes, the slowly migrating band can be observed. At this point, liposomes were added and the reaction was run for up to 100 additional minutes. By 120 min, essentially all of the slower migrating species has been consumed, suggesting that it is a reaction intermediate and not an off pathway end product.

Supplementary Figure 2 Mutations result in three classes of helical structures.

Amphipathic wheel representations of each of the mutants tested in Fig. 5 were generated with Heliquest29. Pictures demonstrate how the magnitude and direction of the hydrophobic moment (indicated with vectoral arrow) and the breadth of the hydrophobic surface (blue shading) is altered with each set of mutations.

Supplementary Figure 3 In rescued cells, LC3-II accumulation is increased upon higher Atg3 expression level (viral dose) and in the presence of Bafilomycin A1.

Cells as in Fig. 6a were infected with two different viral loads. The extent of rescue for Atg3 expression and LC3B lipidation is shown. The 5X condition is identical to the panel in Fig. 6a and represents the conditions used throughout the rest of the manuscript. Moreover, in cells rescued with wild-type Atg3 or K11L expression, LC3-II clears upon starvation. This clearance, blocked by Bafilomycin A1, shows that autophagic flux is restored in these cells.

Supplementary Figure 4 Atg16 puncta intensities do not vary significantly with different Atg3 rescues.

a) representative images of Atg16L puncta in cells under full media, images as in Fig. 6b. b) – Average puncta intensity per puncta area is plotted. Intensities and areas were computationally identified with CellProfiler software as described in Methods. Error bars represent standard deviation. c) raw data (from Fig. 6d) of Atg16 puncta area/cell. For each Atg16 area per cell experiment, between seven and 14 images containing 5 to 20cellsper image were analyzed with the CellProfiler image analysis software to identify areas of intensity above a given threshold as described in Methods. N = 3 independent experiments for all samples except V15K for which n = 2. Precise cell numbers for each sample are given in Methods-statistics section.

Supplementary Figure 5 Atg16 and LC3 puncta numbers during starvation.

a) Representative images of starved cells. Both LC3 puncta and Atg16 puncta formation are shown. b) Atg16L puncta numbers do not significantly depend upon Atg3 expression or function during starvation. Cells as in Fig. 6d were starved before being subjected to fixation and immunolabeling with the anti-Atg16L antibody. Atg16L puncta area was obtained as described in Methods. Grey dashed line indicates the value for Atg3-/- cells rescued with wild-type Atg3. Atg3 variants that support lipidation of LC3 family proteins in vitro (Fig. 5) are indicated with black bars, while those that do not support lipidation are indicated with white bars. Error bars represent standard deviation. P-values are shown relative to rescue with WT (dashed line) *P < 0.05; **P < 0.1. c) Raw data (from Fig. 6c) of LC3 puncta in starvation – Bars are color-coded to indicate experiments conducted in parallel across samples. 22-98 cells were counted from at least two separate coverslips. Error bars represent standard deviation. Precise cell numbers for each sample are given in Methods-statistics section.

Supplementary Figure 6 Atg3 overexpression cannot rescue Atg5 deficiency.

A) Exogenous expression of K11V or K11W mutants of Atg3 cannot obviate requirement for Atg5 in LC3B lipidation. Atg3-/- or Atg5-/- MEFs were infected with lentiviruses encoding either the K11D, K11V or K11W Atg3 mutant. 48 hours post-infection, cells were treated with Bafilomycin A1 (Baf), then lysed and subjected to immunoblotting with the anti-LC3B and the anti-Atg3 antibodies. The asterisk indicates a non-specific band recognized by the anti-Atg3 antibody. B) Atg3 overexpression by transient transfection cannot rescue lipidation in Atg5 KO cells. Atg5 KO MEFs were transfected with mock, Atg3 WT or Atg3 K11W constructs, starved for 2 hours in 1X HBSS containing 10 mM HEPES and collected for Immunoblot analysis. Autophagosome maturation was impeded using the lysosome inhibitor chloroquine (10 M). C) Exogenously expressed Atg3 binds to endogenous Atg5 complexes. Overexpressed wild-type Atg3 with a COOH-terminal V5 tag can pull down endogenous Atg5 and Atg16. FT = flow through, T = total, IP = immunoprecipiate. D) V5-tagged Atg3 is functional. Atg3 used in IP (C), is able to rescue LC3-II formation in Atg3 KO cells, but only as long as the amphipathic helix is intact.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1645 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nath, S., Dancourt, J., Shteyn, V. 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). https://doi.org/10.1038/ncb2940

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2940

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing