Carlsson, S. R. | bioRxiv Science

bioRxiv2026

ATG16L2 creates a VAIL-dedicated heterodimer with ATG16L1

The ATG8 conjugation machinery supports both canonical autophagy, in which ATG8 proteins are lipidated on forming autophagosomes, and CASM, in which ATG8 proteins are conjugated to stressed single-membrane compartments. How cells allocate this shared machinery between these competing membrane programs remains unclear. Here, we identify ATG16L2 as a specialized regulator of V-ATPase-responsive ATG8 lipidation (atg8ylation). ATG16L2 does not form stable homodimers and is inactive in the absence of ATG16L1, but it becomes functional through heterodimerization with ATG16L1. The ATG16L1-ATG16L2 heterodimer constitutes a VAIL-competent E3-like complex that is excluded from WIPI2-positive autophagic membranes but readily recruited to stressed lysosomes. ATG16L2 also confers VAIL activity onto the otherwise VAIL-deficient ATG16L1 isoform. We propose that ATG16L1 homodimers and ATG16L1-ATG16L2 heterodimers represent alternative assemblies of the ATG8 conjugation machinery: ATG16L1 homodimers are dedicated to WIPI2-positive autophagic membranes, whereas ATG16L1-ATG16L2 heterodimers containing either ATG16L1 isoform form a VAIL-selective assembly.

molecular biology↗

bioRxiv2020

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine

Autophagy is a fundamental catabolic process essential for development, homeostasis and proper immune function 1. During autophagy, a cascade of ATG proteins target intracellular cargoes for lysosomal degradation and recycling 2. 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 processes 3. 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 agents 4,5, or during non-canonical autophagy processes such as LC3-associated phagocytosis 6,7 and Influenza A virus infection 8, 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.

cell biology↗