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Autophagy pp 57-75 | Cite as

Structural Studies of Mammalian Autophagy Lipidation Complex

  • Kazuto Ohashi
  • Chinatsu Otomo
  • Zoltan Metlagel
  • Takanori Otomo
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

LC3 GABARAP ATG12 ATG5 ATG3 Conjugation Crystallization NMR 

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Kazuto Ohashi
    • 1
  • Chinatsu Otomo
    • 2
  • Zoltan Metlagel
    • 3
  • Takanori Otomo
    • 2
  1. 1.Institute for Molecular and Cellular RegulationGunma UniversityGunmaJapan
  2. 2.Department of Integrative Structural and Computational BiologyThe Scripps Research InstituteLa JollaUSA
  3. 3.Thermo Fisher ScientificHillsboroUSA

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