title: In vitro assays of lipidation of Mammalian Atg8 homologs.
abstract: 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.
authors.affiliation: Department of Cell Biology and Biochemistry, National Institute of Infectious Diseases, Tokyo, Japan; Laboratory of Proteomics and Biomolecular Science, Juntendo University Graduate School of Medicine, Tokyo, Japan.
authors.forename: Isei
authors.initials: I
authors.lastname: Tanida
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueno
authors.forename: Eiki
authors.initials: E
authors.lastname: Kominami
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D000071193:Autophagy-Related Protein 7; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D054878:Lipoylation; D008840:Microfilament Proteins / Q000737:chemistry* / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000737:chemistry* / Q000378:metabolism; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D059748:Proteolysis; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; C498698:GABARAPL2 protein, human; D008840:Microfilament Proteins; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; C483858:phosphatidylethanolamine; D044763:Ubiquitin-Conjugating Enzymes; C507524:ATG4B protein, human; D003546:Cysteine Endopeptidases; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes; C455621:ATG3 protein, human
keywords: Atg8; GABARAP; GATE-16; LC3; UBL; autophagy; ubiquitin-like modifier