Unique Amphipathicα-helix Drives Membrane Insertion and Enzymatic Activity of ATG3

Abstract Autophagosome biogenesis requires a localized perturbation of lipid membrane dynamics and a unique protein-lipid conjugate. Autophagy-related (ATG) proteins catalyze this biogenesis on cellular membranes, but the underlying molecular mechanism remains unclear. Focusing on the final step of the protein-lipid conjugation reaction, ATG8/LC3 lipidation, we show how membrane association of the conjugation machinery is organized and fine-tuned at the atomistic level. Amphipathic α -helices in ATG3 proteins (AH ATG3 ) are found to have low hydrophobicity and to be less bulky. Molecular dynamics simulations reveal that AH ATG3 regulates the dynamics and accessibility of the thioester bond of the ATG3∼LC3 conjugate to lipids, allowing covalent lipidation of LC3. Live cell imaging shows that the transient membrane association of ATG3 with autophagic membranes is governed by the less bulky- hydrophobic feature of AH ATG3 . Collectively, the unique properties of AH ATG3 facilitate protein- lipid bilayer association leading to the remodeling of the lipid bilayer required for the formation of autophagosomes. Teaser We uncover the unique biophysical property of amphipathic α -helix essential for autophagy