在巨自噬过程中，Atg8 蛋白与自噬膜中的磷脂酰乙醇胺 (PE) 结合。在顶复门寄生虫中，两种半胱氨酸蛋白酶 Atg4 和卵巢肿瘤单位 (Otu) 已被鉴定可以使 Atg8 脱脂，从而从膜上释放该蛋白质。在这里，我们研究了半胱氨酸蛋白酶在 Atg8 结合和解结合中的作用，发现疟原虫寄生虫包含这两种活性。我们成功地单独破坏了这些基因；然而，与此同时，它们难以被删除，并且对于寄生虫的生存至关重要。缺乏Atg4和Otu的突变体表现出正常的血液和蚊子阶段发育。所有感染 Otu KO 子孢子的小鼠均获得专利；然而，Atg4 KO 子孢子要么未能建立血液感染，要么表现出延迟的通畅。通过体外和体内分析，我们发现 Atg4 KO 子孢子侵入并正常发育为早期肝脏阶段。然而，核和细胞器分化在后期受到严重阻碍，未能成熟为肝裂殖子。我们发现 Atg4 KO 寄生虫中 Atg8 水平较高，并且 Atg8 的解离受到阻碍。我们确认了 Otu 在 apicoplast 上的定位；然而，缺乏 Otu 的寄生虫没有表现出明显的发育缺陷。我们的数据表明，Atg4 是主要的解偶联酶，Otu 不能完全取代其功能，因为它会裂解甘氨酸 N 末端的肽键，从而在 Atg8 回收过程中不可逆地使其失活。这些发现强调了 Atg8 解结合途径在细胞器生物发生和维持细胞稳态平衡中的作用。© 2023。作者，获得 Springer Nature Switzerland AG 的独家许可。

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance.© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.