Yamazaki-Sato Harumi, Tanida Isei, Ueno Takashi, Kominami Eiki
Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, 113-8421 Tokyo, Japan.
FEBS Lett. 2003 Sep 11;551(1-3):71-7. doi: 10.1016/s0014-5793(03)00899-8.
In the yeast, Saccharomyces cerevisiae, two ubiquitin-like modifications, Apg12 conjugation with Apg5 and Apg8 lipidation with phosphatidylethanolamine, are essential for autophagy and the cytoplasm-to-vacuole transport of aminopeptidase I (Cvt pathway). As a unique E1-like enzyme, Apg7 activates two modifiers (Apg12 and Apg8) in an ATP-dependent manner and, for this activity, the carboxyl terminal 40 amino acids are essential. For a better understanding of the function of the carboxyl terminus of Apg7, we performed a sequential deletion of the region. A mutant expressing Apg7DeltaC17 protein, which lacks the carboxyl 17 amino acids of Apg7, showed defects in both the Cvt pathway and autophagy. Apg8 lipidation is inhibited in the mutant, while Apg12 conjugation occurs normally. A mutant expressing Apg7DeltaC13 protein showed a defect in the Cvt pathway, but not autophagy, suggesting that the activity of Apg7 for Apg8 lipidation is more essential for the Cvt pathway than for autophagy. Mutant Apg7DeltaC17 protein is still able to interact with Apg8, Apg12 and Apg3, and forms a homodimer, indicating that the deletion of the carboxyl terminal 17 amino acids has little effect on these interactions and Apg7 dimerization. These results suggest that the carboxyl terminal 17 amino acids of Apg7 play a specific role in Apg8 lipidation indispensable for the Cvt pathway and autophagy.
在酿酒酵母中，两种类泛素修饰，即Apg12与Apg5的缀合以及Apg8与磷脂酰乙醇胺的脂化，对于自噬和氨肽酶I的细胞质到液泡运输（Cvt途径）至关重要。作为一种独特的类E1酶，Apg7以ATP依赖的方式激活两种修饰物（Apg12和Apg8），并且对于该活性而言，羧基末端的40个氨基酸是必不可少的。为了更好地理解Apg7羧基末端的功能，我们对该区域进行了连续缺失。表达缺少Apg7羧基末端17个氨基酸的Apg7DeltaC17蛋白的突变体在Cvt途径和自噬中均表现出缺陷。该突变体中Apg8的脂化受到抑制，而Apg12的缀合正常发生。表达Apg7DeltaC13蛋白的突变体在Cvt途径中表现出缺陷，但在自噬中没有缺陷，这表明Apg7对Apg8脂化的活性对于Cvt途径比对自噬更为重要。突变体Apg7DeltaC17蛋白仍然能够与Apg8、Apg12和Apg3相互作用，并形成同二聚体，这表明羧基末端17个氨基酸的缺失对这些相互作用和Apg7二聚化几乎没有影响。这些结果表明，Apg7的羧基末端17个氨基酸在Cvt途径和自噬所必需的Apg8脂化中起特定作用。
