Autophagy involves a core set of autophagy-related (ATG) factors. In humans, there are six ATG8 proteins, divided into the LC3 and GABARAP families, and their covalent conjugation to phosphatidylethanolamine is a key step in autophagosome formation. ATG8s have also been implicated in other processes, but their specific roles remain unclear, as most studies employed overexpression, which may give rise to compensatory effects. In this study, Christian Behrends and colleagues (Eck et al., 2020) use CRISPR/Cas9-based genome editing to tag human ATG8 genes at their natural chromosomal locations in order to study them individually. As proof-of-concept, they then focus on elucidating the role of endogenous GABARAPL2 by mapping its interactome and identify the long-chain-fatty-acid-CoA ligase 3 (ACSL3), a factor involved in lipid droplet (LD) biogenesis, as a new binding partner. Interestingly, ACSL3 is not an autophagy substrate, but stabilises GABARAPL2 at the ER, which is dependent on LC3-interaction regions in ACSL3. Furthermore, the authors show that the interaction between ACSL3 and GABARAPL2 anchors the ubiquitin-like modifier activating enzyme 5 (UBA5), which activates UFM1, at the ER, suggesting that LD biogenesis and ufmylation might be functionally coupled. Taken together, this work thus not only presents a useful tool to dissect the functions of individual ATG8 proteins, but also provides new insights into the regulation of ufmylation.
