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Plasma membrane contributes to the formation of pre-autophagosomal structures

Nature Cell Biology volume 12, pages 747–757 (2010)Cite this article

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A Corrigendum to this article was published on 01 October 2010

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Abstract

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which derive from pre-autophagosomal structures. The membrane origins of autophagosomes are unclear and may involve multiple sources, including the endoplasmic reticulum and mitochondria. Here we show in mammalian cells that the heavy chain of clathrin interacts with Atg16L1 and is involved in the formation of Atg16L1-positive early autophagosome precursors. Atg16L1 associated with clathrin-coated structures, and inhibition of clathrin-mediated internalization decreased the formation of both Atg16L1-positive precursors and mature autophagosomes. We tested and demonstrated that the plasma membrane contributes directly to the formation of early Atg16L1-positive autophagosome precursors. This may be particularly important during periods of increased autophagosome formation, because the plasma membrane may serve as a large membrane reservoir that allows cells periods of autophagosome synthesis at levels many-fold higher than under basal conditions, without compromising other processes.

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Figure 1: Atg16L1 interacts with clathrin heavy chain.
Figure 2: Inhibition of clathrin-mediated endocytosis decreases autophagosome formation.
Figure 3: Influence of clathrin-mediated endocytosis on Atg16L1-positive autophagosome precursors.
Figure 4: Atg16L1 vesicles are found close to the plasma membrane.
Figure 5: Atg16L1 vesicles co-localize with vesicles labelled with cholera toxin subunit B.
Figure 6: Analysis of wild-type and deletion mutants of Atg16L1.
Figure 7: Plasma membrane contributes to Atg16L1-positive autophagosome precursors.
Figure 8: Effect of endocytic vesicle scission on phagophore formation.

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Change history

  • 16 September 2010

    In the version of this article initially published online, the cholera toxin in Fig. 8d was placed outside the internalised vesicles instead of inside. This error has been corrected in both the HTML and PDF versions of the article.

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Acknowledgements

We are grateful to P. Luzio, V. Korolchuk, F. Menzies and S. Sarkar, Cambridge Institute for Medical Research, Cambridge for helpful comments, and for funding from a Wellcome Trust Senior Clinical Research Fellowship (D.C.R.), a MRC Programme grant (D.C.R.), Cancer Research UK (C.P.). We thank M. Mizuguchi, Toyama Medical and Pharmaceutical University, Toyama, for hLC3B; R. Tsien, University of California, San Diego, for tdTomato; R. Xavier, Massachusetts General Hospital, Boston, for 3 × Flag–Atg16L1; T. Yoshimori, Osaka University, Osaka, for cyan fluorescent protein (CFP)–LC3 and GFP–Atg5; B. Beaumelle, Université Montpellier 2, France, for dominant-negative dynamin II; T. Meyer, Stanford University Medical Center, Stanford, for PLC(PH); and M. Harbour, Cambridge Institute for Medical Research, Cambridge, for help with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS).

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Author notes

  1. Brinda Ravikumar and Kevin Moreau: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Medical Genetics, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2XY, UK

    Brinda Ravikumar, Kevin Moreau, Luca Jahreiss & David C. Rubinsztein

  2. Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Wellcome/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 2XY, UK

    Claudia Puri

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Contributions

B.R., K.M., L.J. and D.C.R. designed and analysed the experiments. B.R., K.M. and L.J. performed the experiments. C.P. performed all the immunogold electron microscopy analysis. B.R. and D.C.R. wrote the manuscript. D.C.R. supervised the project.

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Correspondence to David C. Rubinsztein.

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Ravikumar, B., Moreau, K., Jahreiss, L. et al. Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat Cell Biol 12, 747–757 (2010). https://doi.org/10.1038/ncb2078

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