Mouse Apg10 as an Apg12‐conjugating enzyme: analysis by the conjugation‐mediated yeast two‐hybrid method

FEBS Letters

Ohsumi, Yoshinori¹  Mizushima, Noboru²  Yoshimori, Tamotsu¹ 

[1]Department of Cell Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan

[2]PRESTO, Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan

关键词: Autophagy;    Apg12;    Yeast two-hybrid;    EST;    expressed sequence tag;    PCR;    polymerase chain reaction;    PAGE;    polyacrylamide gel electrophoresis;   

DOI  :  10.1016/S0014-5793(02)03739-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown