Hanada Takao, Ohsumi Yoshinori
Division of Molecular Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
Autophagy. 2005 Jul;1(2):110-8. doi: 10.4161/auto.1.2.1858. Epub 2005 Jul 23.
Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.
自噬相关蛋白12（Atg12）是一种翻译后修饰因子，通过类泛素化偶联系统被激活并与自噬相关蛋白5（Atg5）偶联，尽管它与泛素没有明显的序列同源性。Atg12-Atg5偶联物对于自噬（一种细胞内大量降解过程）至关重要。在此，我们表明Atg12的羧基末端区域预计会折叠成类泛素结构，这对于偶联和自噬都是必要且充分的，这表明自噬的关键结构域位于泛素折叠区域。我们进一步表明泛素折叠区域内的两个疏水残基对自噬很重要：Y149位点的突变会影响类E2酶Atg10催化的偶联物形成，而F154位点的突变对Atg12-Atg5偶联物形成没有影响，但其疏水性对自噬至关重要。针对F154突变，自噬中的另一种类泛素化偶联——Atg8-磷脂乙醇胺（Atg8-PE）偶联会严重减少，自噬体形成失败。凝胶过滤分析表明，F154在自噬体形成所需的功能性Atg12-Atg5.Atg16复合物的组装中起关键作用。
