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.
Keywords:
Atg8; GABARAP; GATE-16; LC3; UBL; autophagy; ubiquitin-like modifier.
Copyright © 2014 John Wiley & Sons, Inc.
MeSH terms
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Adaptor Proteins, Signal Transducing / chemistry*
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Adaptor Proteins, Signal Transducing / metabolism
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Animals
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Autophagy-Related Protein 7
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Autophagy-Related Protein 8 Family
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Autophagy-Related Proteins
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Cysteine Endopeptidases / chemistry
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Cysteine Endopeptidases / metabolism
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Humans
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Lipoylation*
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Microfilament Proteins / chemistry*
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Microfilament Proteins / metabolism
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Phosphatidylethanolamines / chemistry*
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Phosphatidylethanolamines / metabolism
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Phosphatidylserines / chemistry*
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Phosphatidylserines / metabolism
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Proteolysis
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Ubiquitin-Activating Enzymes / chemistry
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Ubiquitin-Activating Enzymes / metabolism
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Ubiquitin-Conjugating Enzymes / chemistry
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Ubiquitin-Conjugating Enzymes / metabolism
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Ubiquitination
Substances
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Adaptor Proteins, Signal Transducing
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Autophagy-Related Protein 8 Family
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Autophagy-Related Proteins
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GABARAPL2 protein, human
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Microfilament Proteins
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Phosphatidylethanolamines
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Phosphatidylserines
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phosphatidylethanolamine
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Ubiquitin-Conjugating Enzymes
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ATG4B protein, human
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Cysteine Endopeptidases
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ATG7 protein, human
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Autophagy-Related Protein 7
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Ubiquitin-Activating Enzymes
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ATG3 protein, human