title: The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation.
abstract: ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions, ATG12-conjugation and LC3-lipidation. The existence of functional sequences at the amino-terminal region of human ATG7 remains uncertain. Mutational analyses of ATG7 revealed that both mutant ATG7ΔFAP lacking the FAP motif and ATG7FAPtoDDD, in which the Phe15-Ala16-Pro17 sequence was changed to Asp-Asp-Asp, could not complement defects in endogenous ATG12-conjugation and LC3-lipidation when expressed in Atg7-deficient mouse embryonic fibroblasts (MEFs). However, wild-type ATG7 complemented the defects in these cells. Overexpression of GFP-ATG10 and GFP-ATG12 rescued a defect in ATG12-conjugation in Atg7-deficient MEFs expressing mutant ATG7ΔFAP and ATG7FAPtoDDD, whereas overexpression of all ATG proteins related to ATG12-conjugation and LC3-lipidation could not rescue a defect in LC3-lipidation in Atg7-deficient MEFs expressing these ATG7 mutants. Both ATG7ΔFAP and ATG7FAPtoDDD mutants showed severe defects in the formation of an E2-substrate intermediate of ATG3 with LC3 in LC3-lipidation, but were able to form an E1-substrate intermediate of ATG7 with LC3 and the E1- and E2-substrate intermediates in ATG12-conjugation with reduced efficiency. These ATG7 mutants could also form the ATG12-ATG3 conjugate. Co-immunoprecipitation experiments revealed that the FAP motif of ATG7 is essential for the interaction of ATG7 with ATG3, but not for ATG7-homodimerization. These results indicated that the FAP motif of ATG7 is indispensable for formation of the ATG3-LC3 E2-substrate intermediate through the interaction of ATG7 with ATG3.
authors.affiliation: Department of Biochemistry & Cell Biology, National Institute of Infectious Diseases, Tokyo, Japan. tanida@nih.go.jp
authors.forename: Isei
authors.initials: I
authors.lastname: Tanida
authors.forename: Manabu
authors.initials: M
authors.lastname: Yamasaki
authors.forename: Masaaki
authors.initials: M
authors.lastname: Komatsu
authors.forename: Takashi
authors.initials: T
authors.lastname: Ueno
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D000071193:Autophagy-Related Protein 7; D006801:Humans; D050356:Lipid Metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D055503:Protein Multimerization; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*
chemical_list: C466624:ATG12 protein, human; C495773:Atg5 protein, mouse; C502661:Atg7 protein, mouse; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; D050505:Mutant Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D044764:Ubiquitin-Activating Enzymes