AbstractAutophagy is a fundamental catabolic process essential for development, homeostasis and proper immune function1. During autophagy, a cascade of ATG proteins target intracellular cargoes for lysosomal degradation and recycling2. This pathway utilises a unique post-translational modification, the conjugation of ATG8 proteins to phosphatidylethanolamine (PE) at autophagosomes, which modulates cargo selection and maturation. ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving Single Membrane ATG8 Conjugation (SMAC) to endolysosomal compartments, which plays a key role in phagocytosis and other processes3. It has been widely assumed that SMAC involves the same lipidation of ATG8 to PE, but this has yet to be formally tested. Here, we show that ATG8 undergoes alternative lipidation to phosphatidylserine (PS) during non-canonical autophagy/SMAC. Using mass spectrometry, we find that activation of SMAC, by pharmacological agents4,5, or during non-canonical autophagy processes such as LC3-associated phagocytosis6,7and Influenza A virus infection8, induces the covalent conjugation of ATG8 to PS, as well as PE. This alternative lipidation event is dependent on the ATG16L1 WD40 domain, and occurs at PS enriched endolysosomal membranes. Importantly, we find that the ATG8-PS and ATG8-PE adducts are differentially delipidated by isoforms of the ATG4 family, indicating significant molecular distinctions and mechanisms between these two species.Together, these results provide an important new insight into autophagy signalling, revealing an alternative form of the hallmark ATG8-lipidation event, so widely used to define and assay autophagy. Furthermore, ATG8-PS lipidation provides a specific ‘molecular signature’ for non-canonical autophagy, uncovering a novel means of detecting and monitoring this emerging pathway.