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Structural Studies of Mammalian Autophagy Lipidation Complex

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Autophagy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1880))

Abstract

Members of the autophagy-related protein 8 (Atg8) family of ubiquitin-like proteins (ublps), including mammalian LC3 and GABARAP proteins, play crucial roles in autophagosome biogenesis, as well as selective autophagy. Upon induction of autophagy, the autophagic ublps are covalently attached to a phosphatidylethanolamine (PE) molecule of the autophagosomal membrane. This unique lipid conjugation of the autophagic ublps, which is essential for their functions, occurs in a ubiquitination-like reaction cascade consisting of the E1 enzyme ATG7, the E2 ATG3, and the E3 ATG12~ATG5-ATG16L1 complex (~denotes a covalent linkage). These enzymes are structurally unique among those of the canonical ubiquitination cascades, necessitating structural and biochemical studies of these molecules for understanding the molecular mechanisms underlying the lipidation cascade. Here, we will describe methods that were employed in our previous studies (Otomo et al., Nat Struct Mol Biol 20:59–66, 2013; Metlagel et al., Proc Natl Acad Sci U S A 110:18844–18849, 2013; Ohashi and Otomo, Biochem Biophys Res Commun 463:447–452, 2015), including the production of recombinant enzymes, in vitro enzymatic reactions, the crystallization of the E3 complexes, and the NMR-based investigations of E1–E2 and E2–E3 interactions.

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Acknowledgments

This work was supported by US National Institute of Health grant GM092740 (to T.O.) and Japan Society of Promoting Science (JSPS) fellowship (to K.O.).

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Author notes

  1. Kazuto Ohashi

    Present address: Institute for Molecular and Cellular Regulation, Gunma University, Gunma, Japan

  2. Zoltan Metlagel

    Present address: Thermo Fisher Scientific, Hillsboro, OR, USA

Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Chinatsu Otomo & Takanori Otomo

Authors
  1. Kazuto Ohashi
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  2. Chinatsu Otomo
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  3. Zoltan Metlagel
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  4. Takanori Otomo
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Corresponding author

Correspondence to Takanori Otomo .

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Editors and Affiliations

  1. Signalling Programme, Babraham Institute, Cambridge, UK

    Nicholas Ktistakis

  2. Signalling Programme, Babraham Institute, Cambridge, UK

    Oliver Florey

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Ohashi, K., Otomo, C., Metlagel, Z., Otomo, T. (2019). Structural Studies of Mammalian Autophagy Lipidation Complex. In: Ktistakis, N., Florey, O. (eds) Autophagy. Methods in Molecular Biology, vol 1880. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8873-0_3

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  • DOI: https://doi.org/10.1007/978-1-4939-8873-0_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8872-3

  • Online ISBN: 978-1-4939-8873-0

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