In Vitro Assays of Lipidation of Mammalian Atg8 Homologs

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<jats:title>Abstract</jats:title><jats:p>Atg8 modifier in yeast is conjugated to phosphatidylethanolamine via ubiquitylation‐like reactions essential for autophagy. Mammalian Atg8 homologs (Atg8s) including LC3, GABARAP, and GATE‐16, are also ubiquitin‐like modifiers. The carboxyl termini of mammalian Atg8 homologs are cleaved by Atg4B, a cysteine protease, to expose carboxyl terminal Gly which is essential for this ubiquitylation‐like reaction. Thereafter, the Atg8 homologs are activated by Atg7, an E1‐like enzyme, to form unstable Atg7‐Atg8 E1‐substrate intermediates via a thioester bond. The activated Atg8 homologs are transferred to mammalian Atg3, an E2‐like enzyme, to form unstable Atg3‐Atg8 E2‐substrate intermediates via a thioester bond. Finally, Atg8 homologs are conjugated to phospholipids, phosphatidylethanolamine, and phosphatidylserine. Here, we describe a protocol for the reconstituted conjugation systems for mammalian Atg8 homologs in vitro using purified recombinant Atg proteins and liposomes. <jats:italic>Curr. Protoc. Cell Biol</jats:italic>. 64:11.20.1‐11.20.13. © 2014 by John Wiley & Sons, Inc.</jats:p>

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