Structural studies of mammalian autophagy lipidation complex

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.