HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3-and GABAA receptor-associated protein-phospholipid conjugates
HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3-and GABAA receptor-associated protein-phospholipid conjugates
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DOI：
10.1074/jbc.m401461200
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发表时间：
2004-08-27
影响因子：
4.8
通讯作者：
Kominami, E
中科院分区：
文献类型：
--
作者：
Tanida, I;Sou, YS;Kominami, E
在酵母中，Atg4/Apg4是一种独特的半胱氨酸蛋白酶，负责切割Atg8/Apg8/Aut7的羧基端，这是自噬体形成过程中脂化所必需的反应。然而，目前尚不清楚四种人类Atg4同源物是否能切割三种人类Atg8同源物微管相关蛋白轻链3 （LC3）、GABARAP和GATE-16的羧基末端。利用无细胞系统，我们发现人类Atg4同源物之一HsAtg4B可以切割这三个Atg4同源物的羧基末端。相比之下，突变体HsAtg4B(C74A)，其中预测的活性位点Cys（74）改变为Ala，缺乏蛋白水解活性，表明Cys（74）对半胱氨酸蛋白酶的裂解活性至关重要。利用磷脂酶D，我们发现内源性LC3和GABARAP的修饰形式被脂化，因此被命名为LC3- pl和GABARAP- pl。将纯化的谷胱甘肽s -转移酶标记的HsAtg4B与富含内源性LC3-PL和GABARAP-PL的膜组分一起体外培养，LC3-PL和GABARAP-PL的迁移率改变为未修饰的蛋白的迁移率。当HsAtg4B的Cys（74）改变为Ala时，没有观察到这些移动性的变化。过表达野生型HsAtg4B可降低HeLa细胞中LC3- pl和GABARAP- pl的数量，增加未修饰的内源性LC3和GABARAP的数量。在饥饿条件下，在HeLa细胞中表达cfp标记的HsAtg4B （CFP-HsAtg4B）和yfp标记的LC3，导致yfp标记的LC3的点状分布显著减少，细胞质分布增加。RNA干扰HsAtg4B可增加HEK293细胞中LC3-PL的数量。综上所述，这些结果表明HsAtg4B通过脱脂作用负调控LC3向膜室的定位。
In yeast, Atg4/Apg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8/Apg8/Aut7, a reaction essential for its lipidation during the formation of autophagosomes. However, it is still unclear whether four human Atg4 homologues cleave the carboxyl termini of the three human Atg8 homologues, microtubule-associated protein light chain 3 (LC3), GABARAP, and GATE-16. Using a cell-free system, we found that HsAtg4B, one of the human Atg4 homologues, cleaves the carboxyl termini of these three Atg8 homologues. In contrast, the mutant HsAtg4B(C74A), in which a predicted active site Cys(74) was changed to Ala, lacked proteolytic activity, indicating that Cys(74) is essential for the cleavage activity of cysteine protease. Using phospholipase D, we showed that the modified forms of endogenous LC3 and GABARAP are lipidated and therefore were designated LC3-PL and GABARAP-PL. When purified glutathione S-transferase-tagged HsAtg4B was incubated in vitro with a membrane fraction enriched with endogenous LC3-PL and GABARAP-PL, the mobility of LC3-PL and GABARAP-PL was changed to those of the unmodified proteins. These mobility shifts were not seen when Cys(74) of HsAtg4B was changed to Ala. Overexpression of wild-type HsAtg4B decreased the amount of LC3-PL and GABARAP-PL and increased the amount of unmodified endogenous LC3 and GABARAP in HeLa cells. Expression of CFP-tagged HsAtg4B (CFP-HsAtg4B) and YFP-tagged LC3 in HeLa cells under starvation conditions resulted in a significant decrease in the punctate pattern of distribution of YFP-tagged LC3 and an increase in its cytoplasmic distribution. RNA interference of HsAtg4B increased the amount of LC3-PL in HEK293 cells. Taken together, these results suggest that HsAtg4B negatively regulates the localization of LC3 to a membrane compartment by delipidation.