1. Biochemistry and Chemical Biology
  2. Cell Biology

Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s

  1. Wenxin Zhang
  2. Taki Nishimura
  3. Deepanshi Gahlot
  4. Chieko Saito
  5. Colin Davis
  6. Harold BJ Jefferies
  7. Anne Schreiber
  8. Lipi Thukral
  9. Sharon A Tooze  Is a corresponding author
  1. The Francis Crick Institute, United Kingdom
  2. University of Tokyo, Japan
  3. CSIR-Institute of Genomics and Integrative Biology, India
https://doi.org/10.7554/eLife.89185
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Cite this article
  1. Wenxin Zhang
  2. Taki Nishimura
  3. Deepanshi Gahlot
  4. Chieko Saito
  5. Colin Davis
  6. Harold BJ Jefferies
  7. Anne Schreiber
  8. Lipi Thukral
  9. Sharon A Tooze
(2023)
Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s
eLife 12:e89185.
https://doi.org/10.7554/eLife.89185
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Abstract

Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1 - 6.

Article and author information

Author details

  1. Wenxin Zhang

    Molecular Cell Biology of Autophagy Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7657-4495

  2. Taki Nishimura

    •Department of Biochemistry and Molecular Biology, University of Tokyo, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4019-5984

  3. Deepanshi Gahlot

    CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2681-8818

  4. Chieko Saito

    Department of Biochemistry and Molecular Biology, University of Tokyo, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.

  5. Colin Davis

    Cellular Degradation Systems Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  6. Harold BJ Jefferies

    Molecular Cell Biology of Autophagy Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  7. Anne Schreiber

    Cellular Degradation Systems Laboratory, The Francis Crick Institute, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.

  8. Lipi Thukral

    CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1961-039X

  9. Sharon A Tooze

    Molecular Cell Biology of Autophagy Laboratory, The Francis Crick Institute, London, United Kingdom
    For correspondence
    sharon.tooze@crick.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2182-3116

Funding

European Research Council (FP7/2007-2013 788708)

  • Wenxin Zhang
  • Sharon A Tooze

Japan Science and Technology, ERATO (JPMJER1702)

  • Chieko Saito

Council of Scientific and Industrial Research, India (OLP1163)

  • Deepanshi Gahlot
  • Lipi Thukral

Wellcome Trust (CC2134)

  • Wenxin Zhang
  • Harold BJ Jefferies
  • Sharon A Tooze

Wellcome Trust (CC2064)

  • Colin Davis
  • Anne Schreiber

Cancer Research UK (CC2134)

  • Wenxin Zhang
  • Harold BJ Jefferies
  • Sharon A Tooze

Cancer Research UK (CC2064)

  • Colin Davis
  • Anne Schreiber

Medical Research Council (CC2134)

  • Wenxin Zhang
  • Harold BJ Jefferies
  • Sharon A Tooze

Medical Research Council (CC2064)

  • Colin Davis
  • Anne Schreiber

Japan Science and Technology, PRESTO (JPMJPR20EC)

  • Taki Nishimura

Grant-in-Aid for Transformative Research Areas (B (grant 21H05146)

  • Taki Nishimura

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Li Yu, Tsinghua University, China

Version history

  1. Preprint posted: June 10, 2022 (view preprint)
  2. Received: May 8, 2023
  3. Accepted: June 3, 2023
  4. Accepted Manuscript published: June 8, 2023 (version 1)
  5. Version of Record published: June 23, 2023 (version 2)

Copyright

© 2023, Zhang et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Wenxin Zhang
  2. Taki Nishimura
  3. Deepanshi Gahlot
  4. Chieko Saito
  5. Colin Davis
  6. Harold BJ Jefferies
  7. Anne Schreiber
  8. Lipi Thukral
  9. Sharon A Tooze
(2023)
Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s
eLife 12:e89185.
https://doi.org/10.7554/eLife.89185

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