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

Mol Cell. 2021 May 6;81(9):2013-2030.e9. doi: 10.1016/j.molcel.2021.03.001. Epub 2021 Mar 26.

Abstract

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.

Keywords: ATG4; ATG9a; FIB-SEM; LRBA; PINK1; Parkin; autophagosome; autophagy; mitochondria; mitophagy.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Apoptosis Regulatory Proteins / genetics
  • Apoptosis Regulatory Proteins / metabolism*
  • Artificial Intelligence
  • Autophagosomes / genetics
  • Autophagosomes / metabolism*
  • Autophagosomes / ultrastructure
  • 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*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Imaging, Three-Dimensional
  • Lipid Metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Microscopy, Electron, Transmission
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Mitochondria / genetics
  • Mitochondria / metabolism*
  • Mitochondria / ultrastructure
  • Mitophagy
  • 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

Substances

  • ARFIP2 protein, human
  • Adaptor Proteins, Signal Transducing
  • Apoptosis Regulatory Proteins
  • ATG9A protein, human
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • GABARAP protein, human
  • GABARAPL2 protein, human
  • MAP1LC3A protein, human
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • Vesicular Transport Proteins
  • Ubiquitin-Protein Ligases
  • parkin protein
  • Protein Kinases
  • LRBA protein, human
  • PTEN-induced putative kinase
  • ATG4A protein, human
  • ATG4B protein, human
  • ATG4D protein, human
  • Cysteine Endopeptidases