title: ATG4B contains a C-terminal LIR motif important for binding and efficient cleavage of mammalian orthologs of yeast Atg8.
abstract: The cysteine protease ATG4B cleaves off one or more C-terminal residues of the inactive proform of proteins of the ortholog and paralog LC3 and GABARAP subfamilies of yeast Atg8 to expose a C-terminal glycine that is conjugated to phosphatidylethanolamine during autophagosome formation. We show that ATG4B contains a C-terminal LC3-interacting region (LIR) motif important for efficient binding to and cleavage of LC3 and GABARAP proteins. We solved the crystal structures of the GABARAPL1-ATG4B C-terminal LIR complex. Analyses of the structures and in vitro binding assays, using specific point mutants, clearly showed that the ATG4B LIR binds via electrostatic-, aromatic HP1 and hydrophobic HP2 pocket interactions. Both these interactions and the catalytic site-substrate interaction contribute to binding between LC3s or GABARAPs and ATG4B. We also reveal an unexpected role for ATG4B in stabilizing the unlipidated forms of GABARAP and GABARAPL1. In mouse embryonic fibroblast (MEF) atg4b knockout cells, GABARAP and GABARAPL1 were unstable and degraded by the proteasome. Strikingly, the LIR motif of ATG4B was required for stabilization of the unlipidated forms of GABARAP and GABARAPL1 in cells.
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Mads
authors.initials: M
authors.lastname: Skytte Rasmussen
authors.affiliation: b Structural Biology , The Francis Crick Institute , London , UK.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Mouilleron
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Birendra
authors.initials: B
authors.lastname: Kumar Shrestha
authors.affiliation: c Molecular Cell Biology of Autophagy Laboratory , The Francis Crick Institute , London , UK.
authors.forename: Martina
authors.initials: M
authors.lastname: Wirth
authors.affiliation: b Structural Biology , The Francis Crick Institute , London , UK.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Lee
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Bowitz Larsen
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Yakubu
authors.initials: Y
authors.lastname: Abudu Princely
authors.affiliation: d Peptide Chemistry Science Technology Platform , The Francis Crick Institute , London , UK.
authors.forename: Nicola
authors.initials: N
authors.lastname: O'Reilly
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Eva
authors.initials: E
authors.lastname: Sjøttem
authors.affiliation: c Molecular Cell Biology of Autophagy Laboratory , The Francis Crick Institute , London , UK.
authors.forename: Sharon A
authors.initials: SA
authors.lastname: Tooze
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Trond
authors.initials: T
authors.lastname: Lamark
authors.affiliation: a Molecular Cancer Research Group, Department of Medical Biology , University of Tromsø -The Arctic University of Norway , Tromsø , Norway.
authors.forename: Terje
authors.initials: T
authors.lastname: Johansen
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D000071190:Autophagy-Related Protein 8 Family / Q000378:metabolism*; D000071183:Autophagy-Related Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015003:Yeasts
chemical_list: C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D000071183:Autophagy-Related Proteins; D008840:Microfilament Proteins; D029701:Saccharomyces cerevisiae Proteins; C507524:ATG4B protein, human; C582881:Atg4b protein, mouse; D003546:Cysteine Endopeptidases
keywords: ATG4; GABARAP; GABARAPL1; LC3B; LIR; X-ray structure; autophagy; peptide arrays