Tanida I, Tanida-Miyake E, Ueno T, Kominami E
Department of Biochemistry, Juntendo University School of Medicine, Tokyo 113-8421, Japan.
J Biol Chem. 2001 Jan 19;276(3):1701-6. doi: 10.1074/jbc.C000752200. Epub 2000 Nov 28.
Autophagy is a process that involves the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system. In the yeast, Saccharomyces cerevisiae, an autophagosome is formed in the cytosol. The outer membrane of the autophagosome is fused with the vacuole, releasing the inner membrane structure, an autophagic body, into the vacuole. The autophagic body is subsequently degraded by vacuolar hydrolases. Taking advantage of yeast genetics, apg (autophagy-defective) mutants were isolated that are defective in terms of formation of autophagic bodies under nutrient starvation conditions. One of the APG gene products, Apg12p, is covalently attached to Apg5p via the C-terminal Gly of Apg12p as in the case of ubiquitylation, and this conjugation is essential for autophagy. Apg7p is a novel E1 enzyme essential for the Apg12p-conjugation system. In mammalian cells, the human Apg12p homolog (hApg12p) also conjugates with the human Apg5p homolog. In this study, the unique characteristics of hApg7p are shown. A two-hybrid experiment indicated that hApg12p interacts with hApg7p. Site-directed mutagenesis revealed that Cys(572) of hApg7p is an authentic active site cysteine residue essential for the formation of the hApg7p.hApg12p intermediate. Overexpression of hApg7p enhances the formation of the hApg5p.hApg12p conjugate, indicating that hApg7p is an E1-like enzyme essential for the hApg12p conjugation system. Cross-linking experiments and glycerol-gradient centrifugation analysis showed that the mammalian Apg7p homolog forms a homodimer as in yeast Apg7p. Each of three human Apg8p counterparts, i.e. the Golgi-associated ATPase enhancer of 16 kDa, GABA(A) receptor-associated protein, and microtubule-associated protein light chain 3, coimmunoprecipitates with hApg7p and conjugates with mutant hApg7p(C572S) to form a stable intermediate via an ester bond. These results indicate that hApg7p is an authentic protein-activating enzyme for hApg12p and the three Apg8p homologs.
自噬是一个涉及溶酶体/液泡系统对细胞质成分进行大量降解的过程。在酿酒酵母中，自噬体在细胞质中形成。自噬体的外膜与液泡融合，将内膜结构即自噬体释放到液泡中。随后自噬体被液泡水解酶降解。利用酵母遗传学技术，分离出了apg（自噬缺陷型）突变体，这些突变体在营养饥饿条件下自噬体的形成存在缺陷。APG基因产物之一Apg12p通过Apg12p的C末端甘氨酸与Apg5p共价连接，这与泛素化情况类似，并且这种缀合对于自噬至关重要。Apg7p是Apg12p缀合系统所必需的一种新型E1酶。在哺乳动物细胞中，人Apg12p同源物（hApg12p）也与人Apg5p同源物缀合。在本研究中，展示了hApg7p的独特特性。双杂交实验表明hApg12p与hApg7p相互作用。定点诱变显示hApg7p的Cys(572)是形成hApg7p·hApg12p中间体所必需的真实活性位点半胱氨酸残基。hApg7p的过表达增强了hApg5p·hApg12p缀合物的形成，表明hApg7p是hApg12p缀合系统所必需的类似E1的酶。交联实验和甘油梯度离心分析表明，哺乳动物Apg7p同源物如酵母Apg
