The Mouse APG10 Homologue, an E2-like Enzyme for Apg12p Conjugation, Facilitates MAP-LC3 Modification

• Published in: The Journal of biological chemistry 2003-10, Vol.278 (41), p.39517-39526
• Main Authors: Nemoto, Takahiro, Tanida, Isei, Tanida-Miyake, Emiko, Minematsu-Ikeguchi, Naoko, Yokota, Masahiro, Ohsumi, Mariko, Ueno, Takashi, Kominami, Eiki
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Protein 7; Autophagy-Related Proteins; Base Sequence; Cell Line; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; Humans; In Vitro Techniques; Ligases - genetics; Ligases - metabolism; Mice; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidoreductases - genetics; Oxidoreductases - metabolism; Proteins - genetics; Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Two-Hybrid System Techniques; Ubiquitin-Conjugating Enzymes
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M300550200

Autophagy is a process for the bulk degradation of cytosolic compartments by lysosomes/vacuoles. The formation of autophagosomes involves a dynamic rearrangement of the membrane for which two ubiquitin-like modifications (the conjugation of Apg12p and the modification of a soluble form of MAP-LC3 to a membrane-bound form) are essential. In yeast, Apg10p is an E2-like enzyme essential for Apg12p conjugation. The isolated mouse APG10 gene product interacts with mammalian Apg12p dependent on mammalian Apg7p (E1-like enzyme), and facilitates Apg12p conjugation. The interaction of Apg10p with Apg12p is dependent on the carboxyl-terminal glycine of Apg12p. Mutational analysis of the predicted active site cysteine (Cys161) within mouse Apg10p shows that mutant Apg10pC161S, which can form a stable intermediate with Apg12p, inhibits Apg12p conjugation even in the presence of Apg7p, while overexpression of Apg7p facilitates formation of an Apg12p-Apg5p conjugate. Furthermore, the coexpression of Apg10p with Apg7p facilitates the modification of a soluble form of MAP-LC3 to a membrane-bound form, a second modification essential for autophagy. Mouse Apg10p interacts with MAP-LC3 in HEK293 cells, while no mutant Apg10pC161S forms any intermediate with MAP-LC3. Direct interaction between Apg10p and MAP-LC3 is also demonstrated by yeast two-hybrid analysis. The inability of mutant Apg10pC161S to form any intermediate with MAP-LC3 has ruled out the possibility that MAP-LC3 interacts with Apg10p as a substrate.