title: The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1-E2 complex formation.
abstract: Apg7p/Cvt2p, a protein-activating enzyme, is essential for both the Apg12p-Apg5p conjugation system and the Apg8p membrane targeting in autophagy and cytoplasm-to-vacuole targeting in the yeast Saccharomyces cerevisiae. Similar to the ubiquitin-conjugating system, both Apg12p and Apg8p are activated by Apg7p, an E1-like enzyme. Apg12p is then transferred to Apg10p, an E2-like enzyme, and conjugated with Apg5p, whereas Apg8p is transferred to Apg3p, another E2-like enzyme, followed by conjugation with phosphatidylethanolamine. Evidence is presented here that Apg7p forms a homodimer with two active-site cysteine residues via the C-terminal region. The dimerization of Apg7p is independent of the other Apg proteins and facilitated by overexpressed Apg12p. The C-terminal 123 amino acids of Apg7p (residues 508 to 630 out of 630 amino acids) are sufficient for its dimerization, where there is neither an ATP binding domain nor an active-site cysteine essential for its E1 activity. The deletion of its carboxyl 40 amino acids (residues 591-630 out of 630 amino acids) results in several defects of not only Apg7p dimerization but also interactions with two substrates, Apg12p and Apg8p and Apg12p-Apg5p conjugation, whereas the mutant Apg7p contains both an ATP binding domain and an active-site cysteine. Furthermore, the carboxyl 40 amino acids of Apg7p are also essential for the interaction of Apg7p with Apg3p to form the E1-E2 complex for Apg8p. These results suggest that Apg7p forms a homodimer via the C-terminal region and that the C-terminal region is essential for both the activity of the E1 enzyme for Apg12p and Apg8p as well as the formation of an E1-E2 complex for Apg8p.
authors.affiliation: Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Komatsu
authors.forename: I
authors.initials: I
authors.lastname: Tanida
authors.forename: T
authors.initials: T
authors.lastname: Ueno
authors.forename: M
authors.initials: M
authors.lastname: Ohsumi
authors.forename: Y
authors.initials: Y
authors.lastname: Ohsumi
authors.forename: E
authors.initials: E
authors.lastname: Kominami
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000071193:Autophagy-Related Protein 7; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D015151:Immunoblotting; D008025:Ligases / Q000378:metabolism*; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
chemical_list: C114830:ATG7 protein, S cerevisiae; D000596:Amino Acids; D003432:Cross-Linking Reagents; D005656:Fungal Proteins; D010714:Phosphatidylethanolamines; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D000071193:Autophagy-Related Protein 7; D003545:Cysteine