Unique amphipathic α helix drives membrane insertion and enzymatic activity of ATG3

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, the ATG8/LC3 lipidation, we show how the membrane association of the conjugation machinery is organized and fine-tuned at the atomistic level. Amphipathic α helices in ATG3 proteins (AH_ATG3) have low hydrophobicity and contain less bulky residues. Molecular dynamics simulations reveal that AH_ATG3 regulates the dynamics and accessibility of the thioester bond of the ATG3~LC3 conjugate to lipids, enabling the 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. 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.