Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method
Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method
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DOI：
10.1016/s0014-5793(02)03739-0
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发表时间：
2002-12-18
期刊：
影响因子：
3.5
通讯作者：
Ohsumi, Y
中科院分区：
文献类型：
--
作者：
Mizushima, N;Yoshimori, T;Ohsumi, Y
自噬体的形成是巨噬的中心事件。在这一过程中必不可少的Apg12-Apg5偶联物是由泛素样蛋白偶联系统产生的。在酵母中，Apg12被el样Apg7激活后，与Apg10 （e2样）形成硫酯。Apg12最终通过异肽键与Apg5结合。然而，这种结合可能需要一种e3样蛋白，目前还没有得到证实。Apg12系统在真核生物中是保守的，尽管Apg10的哺乳动物对应物尚未被鉴定出来。在这里，我们报道了小鼠Apg10同源基因的鉴定和表征。以小鼠Apg5 （mApg5）为诱饵进行酵母双杂交筛选，鉴定出与酵母Apg10同源性为19%的新蛋白。我们将该蛋白命名为小鼠Apg10 （mApg10）。我们通过改良酵母双杂交实验证明了mApg10介导mApg12和mApg5的偶联。在HeLa细胞中，mApg12与mApg10的体内相互作用表明mApg10是一种Apg12偶联酶，可能在Apg12系统中作为apg5识别分子。这种新的双杂交方法被我们命名为“偶联介导酵母双杂交”，证明是一种简单而有用的分析蛋白质-蛋白质偶联的技术。(C) 2002年由Elsevier Science B.V.代表欧洲生化学会联合会出版。
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 El-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. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.