Аннотация: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.
