The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation
The FAP motif within human ATG7, an autophagy-related E1-like enzyme, is essential for the E2-substrate reaction of LC3 lipidation
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DOI：
10.4161/auto.8.1.18339
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发表时间：
2012-01
期刊：
影响因子：
13.3
通讯作者：
I. Tanida;M. Yamasaki;M. Komatsu;T. Ueno
中科院分区：
文献类型：
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作者：
I. Tanida;M. Yamasaki;M. Komatsu;T. Ueno
ATG 7是一种自噬相关的E1样酶，对于两种泛素化样反应，ATG 12-缀合和LC 3-脂化是必需的。在人ATG 7的氨基末端区域的功能序列的存在仍然不确定。ATG 7的突变分析显示，缺失FAP基序的突变体ATG 7 ΔFAP和其中Phe 15-Ala 16-Pro 17序列改变为Asp-Asp-Asp的ATG 7 FAPtoDDD在Atg 7缺陷型小鼠胚胎成纤维细胞（MEF）中表达时，不能弥补内源性ATG 12缀合和LC 3脂化的缺陷。然而，野生型ATG 7补充了这些细胞中的缺陷。在表达突变型ATG 7 ΔFAP和ATG 7 FAPtoDDD的Atg 7缺陷型MEFs中，GFP-ATG 10和GFP-ATG 12的过表达挽救了ATG 12-缀合缺陷，而与ATG 12-缀合和LC 3-脂化相关的所有ATG蛋白的过表达不能挽救表达这些ATG 7突变型的Atg 7缺陷型MEFs中LC 3-脂化缺陷。ATG 7 ΔFAP和ATG 7 FAPtoDDD突变体在LC 3-脂化中与LC 3形成ATG 3的E2-底物中间体方面均表现出严重缺陷，但能够在ATG 12-缀合中与LC 3以及E1-和E2-底物中间体形成ATG 7的E1-底物中间体，且效率降低。这些ATG 7突变体也可以形成ATG 12-ATG 3缀合物。免疫共沉淀实验表明，ATG 7的FAP基序对于ATG 7与ATG 3的相互作用是必需的，但对于ATG 7-同源二聚化不是必需的。这些结果表明，ATG 7的FAP基序对于通过ATG 7与ATG 3的相互作用形成ATG 3-LC 3 E2-底物中间体是必不可少的。
ATG7 is an autophagy-related E1-like enzyme that is essential for two ubiquitination-like reactions, ATG12-conjugation and LC3-lipidation. The existence of functional sequences at the amino-terminal region of human ATG7 remains uncertain. Mutational analyses of ATG7 revealed that both mutant ATG7ΔFAP lacking the FAP motif and ATG7FAPtoDDD, in which the Phe15-Ala16-Pro17 sequence was changed to Asp-Asp-Asp, could not complement defects in endogenous ATG12-conjugation and LC3-lipidation when expressed in Atg7-deficient mouse embryonic fibroblasts (MEFs). However, wild-type ATG7 complemented the defects in these cells. Overexpression of GFP-ATG10 and GFP-ATG12 rescued a defect in ATG12-conjugation in Atg7-deficient MEFs expressing mutant ATG7ΔFAP and ATG7FAPtoDDD, whereas overexpression of all ATG proteins related to ATG12-conjugation and LC3-lipidation could not rescue a defect in LC3-lipidation in Atg7-deficient MEFs expressing these ATG7 mutants. Both ATG7ΔFAP and ATG7FAPtoDDD mutants showed severe defects in the formation of an E2-substrate intermediate of ATG3 with LC3 in LC3-lipidation, but were able to form an E1-substrate intermediate of ATG7 with LC3 and the E1- and E2-substrate intermediates in ATG12-conjugation with reduced efficiency. These ATG7 mutants could also form the ATG12-ATG3 conjugate. Co-immunoprecipitation experiments revealed that the FAP motif of ATG7 is essential for the interaction of ATG7 with ATG3, but not for ATG7-homodimerization. These results indicated that the FAP motif of ATG7 is indispensable for formation of the ATG3-LC3 E2-substrate intermediate through the interaction of ATG7 with ATG3.