ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system

Summary The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step PINK1/Parkin mitophagy. ATG4 family proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 (LC3/GABARAPs). We discover that human ATG4s promote independently their protease activity and processing. proximity networks reveal role for partners, including immune-disease protein LRBA, in ATG9A vesicle trafficking mitochondria. Artificial intelligence-directed 3D electron microscopy phagophores shows phagophore-ER contacts during lipid-transfer phase formation. also show removal maturation does not depend on activity. Instead, can disassemble ATG8-protein conjugates, revealing as deubiquitinating-like enzymes. These findings establish non-canonical roles beyond lipidation axis provide an AI-driven framework rapid microscopy.