The mouse APG10 homologue, an E2-like enzyme for Apg12p conjugation, facilitates MAP-LC3 modification.
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.
Nemoto
Takahiro
T
Department of Biochemistry, Juntendo University, Medical School, Tokyo 113-8421, Japan.
Tanida
Isei
I
Tanida-Miyake
Emiko
E
Minematsu-Ikeguchi
Naoko
N
Yokota
Masahiro
M
Ohsumi
Mariko
M
Ueno
Takashi
T
Kominami
Eiki
E
D000595:Amino Acid Sequence; D000818:Animals; D001343:Autophagy; D000071192:Autophagy-Related Protein 12; D000071193:Autophagy-Related Protein 7; D000071183:Autophagy-Related Proteins; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D066298:In Vitro Techniques; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes
C114830:ATG7 protein, S cerevisiae; C491971:Atg12 protein, mouse; D000071192:Autophagy-Related Protein 12; D000071183:Autophagy-Related Proteins; D018076:DNA, Complementary; C477012:MAP1LC3A protein, human; C502070:MAP1LC3B protein, human; C502071:MAP1LC3C protein, human; D008869:Microtubule-Associated Proteins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010088:Oxidoreductases; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000071193:Autophagy-Related Protein 7; C455621:ATG3 protein, human