Atg4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system Thanh Ngoc Nguyen1, Benjamin Scott Padman1, Susanne Kraft2, Louise Uoselis1, Marvin Skulsuppaisarn1, Christian Behrends2 and Michael Lazarou1 1Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia 2Munich Cluster for System Neurology, Medical Faculty, Ludwig‐Maximilians‐University München, Munich, Germany The mammalian family of Atg8 proteins (LC3/GABARAPs) are crucial for autophagosome-lysosome fusion during starvation-induced autophagy and PINK1/Parkin mitophagy. Although also important for autophagosome expansion, these ubiquitin-like proteins are dispensable for autophagosome biogenesis and cargo sequestration. Atg8s become functional during canonical autophagy upon conjugation to the lipid phosphatidylethanolamine (PE) on autophagosomal membranes, which occurs through a ubiquitin-like conjugation system. However, prior to conjugation, Atg8s must be processed by the Atg4 family of cysteine proteases to expose their C-terminal Glycine which is required for lipidation. The Atg4 family also plays a role in removing Atg8s from PE by a process termed de-lipidation or de-conjugation. In yeast, de-lipidation is proposed to release Atg8 that has been non-specifically conjugated to intracellular membranes in order to supply Atg8 for autophagosome biogenesis. In mammals, the purpose of de-lipidation remains unclear. Nevertheless, it is widely accepted that the role of Atg4s in autophagy is completely dependent on their ability to cleave Atg8 family members. We find that HeLa cells lacking all four human Atg4s (Atg4A, Atg4B, Atg4C and Atg4D) fail to build autophagosomes during PINK1/Parkin mitophagy. This phenotype is unexpected given that autophagosomes can form in both hexa KO cells lacking LC3/GABARAPs, and in Atg5 KO cells lacking LC3/GABARAP lipidation. To address whether Atg4s play an LC3/GABARAP-independent role in autophagosome formation, the Atg4 family was further knocked out in Atg8 hexa KO cells (Atg4/Atg8 deca KO). In contrast to the parental Atg8 hexa KO line, Atg4/Atg8 deca KO cells were incapable of forming mitophagosomes during PINK1/Parkin mitophagy. Our analyses of autophagosome formation revealed that Atg4s drive phagophore growth independently of their protease activity and of Atg8s. Analysis of phagophore ultrastructure using artificial intelligence-directed 3D electron microscopy revealed a key role for the Atg4 family in driving phagophore progression. Collectively, we have uncovered a novel Atg8-independent function of the Atg4 family in regulating autophagosome biogenesis during PINK1/Parkin mitophagy.