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

• Published in: Molecular cell Vol. 81; no. 9; pp. 2013 - 2030.e9
• Main Authors: Nguyen, Thanh Ngoc, Padman, Benjamin Scott, Zellner, Susanne, Khuu, Grace, Uoselis, Louise, Lam, Wai Kit, Skulsuppaisarn, Marvin, Lindblom, Runa S.J., Watts, Emily M., Behrends, Christian, Lazarou, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.05.2021
• Subjects: Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Apoptosis Regulatory Proteins - genetics; Apoptosis Regulatory Proteins - metabolism; Artificial Intelligence; ATG4; ATG9a; autophagosome; Autophagosomes - genetics; Autophagosomes - metabolism; Autophagosomes - ultrastructure; autophagy; Autophagy-Related Protein 8 Family - genetics; Autophagy-Related Protein 8 Family - metabolism; Autophagy-Related Proteins - genetics; Autophagy-Related Proteins - metabolism; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; FIB-SEM; HEK293 Cells; HeLa Cells; Humans; Imaging, Three-Dimensional; Lipid Metabolism; LRBA; Membrane Proteins - genetics; Membrane Proteins - metabolism; Microscopy, Electron, Transmission; Microtubule-Associated Proteins - genetics; Microtubule-Associated Proteins - metabolism; mitochondria; Mitochondria - genetics; Mitochondria - metabolism; Mitochondria - ultrastructure; Mitophagy; Parkin; PINK1; Protein Kinases - genetics; Protein Kinases - metabolism; Protein Transport; Signal Transduction; Ubiquitin-Protein Ligases - genetics; Ubiquitin-Protein Ligases - metabolism; Vesicular Transport Proteins - genetics; Vesicular Transport Proteins - metabolism; FIB-SEM; mitophagy; PINK1; autophagy; LRBA; mitochondria; ATG9a; autophagosome; ATG4; Parkin
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2021.03.001

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy. [Display omitted] •ATG4s promote phagophore growth independently of their protease activity and ATG8s•ATG4s and their proximal partners including LRBA regulate ATG9A vesicle trafficking•AI-directed 3D EM reveals that ATG4s promote phagophore-ER contacts during mitophagy•ATG4s are not crucial for ATG8 removal from autolysosomes but can regulate ATG8ylation Nguyen et al. develop an AI-directed 3D electron microscopy framework, which together with multigene knockouts reveals an unexpected role for ATG4s in promoting phagophore-ER contacts independently of their protease activity and of ATG8s. During PINK1/Parkin mitophagy, ATG4s and their proximity partners, LRBA and ARFIP2, regulate ATG9A vesicle trafficking to mitochondria.