Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method.
Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.
Mizushima
Noboru
N
PRESTO, Japan Science and Technology Corporation, 332-0012, Kawaguchi, Japan.
Yoshimori
Tamotsu
T
Ohsumi
Yoshinori
Y
D000595:Amino Acid Sequence; D000818:Animals; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism* / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D010587:Phagocytosis; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020798:Two-Hybrid System Techniques; D044767:Ubiquitin-Protein Ligases
C491969:ATG12 protein, S cerevisiae; C102268:ATG5 protein, S cerevisiae; C470976:Apg10 protein, S cerevisiae; C491971:Atg12 protein, mouse; D000071192:Autophagy-Related Protein 12; D000071187:Autophagy-Related Protein 5; D018076:DNA, Complementary; D005656:Fungal Proteins; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D044767:Ubiquitin-Protein Ligases