Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.

自噬是一种基本的分解代谢过程，它使用独特的翻译后修饰，即 ATG8 蛋白与磷脂酰乙醇胺 (PE) 的结合。 ATG8 脂质化也发生在非典型自噬过程中，这是一种平行途径，涉及 ATG8 在内溶酶体区室中与单膜 (CASM) 结合，在免疫、视觉和神经生物学中具有关键功能。人们普遍认为 CASM 涉及 ATG8 与 PE 的相同缀合，但这尚未经过正式测试。在这里，我们发现所有 ATG8 也可以在 CASM 过程中经历磷脂酰丝氨酸 (PS) 的替代脂化，这是由哺乳动物细胞中 LC3 相关的吞噬作用或甲型流感病毒感染引起的药理学诱导。重要的是，ATG8-PS 和 ATG8-PE 加合物被 ATG4 家族不同程度地脱脂，并具有不同的细胞动力学，表明显着的分子差异。这些结果为自噬信号传导提供了重要的见解，揭示了标志性 ATG8 脂化事件的另一种形式。此外，ATG8-PS 为非规范自噬途径提供了特定的“分子特征”。