Identification of ATG7 as a protein ATG8ylation substrate

Protein ATG8ylation is a post-translational modification where ubiquitin-like protein LC3/ATG8 forms covalent conjugation with cellular proteins, a process reversed by Atg4. In contrast to the well-characterized ATG8 lipidation/membrane ATG8ylation, research on protein ATG8ylation remains limited. In this study, we identify deconjugation-resistant LC3B Q116A and F80A/L82A mutants as tools for protein ATG8ylation. We demonstrate that protein ATG8ylation depends exclusively on Atg4, ATG3, and Atg7. Tandem affinity purification-mass spectrometry reveals Atg7 as a substrate of protein ATG8ylation with K140 as its modification site. We show that protein ATG8ylation of Atg7 forms a mono-LC3B conjugate, while ATG3 undergoes lysine-dependent, mixed-linkage poly-LC3B chains. Atg7 and ATG3 function as E1 and E2 Enzymes in protein ATG8ylation, potentially cooperating with E3 Ligases. Notably, endogenous Atg7 ATG8ylation attenuates Autophagy by disrupting its interaction with ATG3. These findings highlight Atg7 as both a central catalytic enzyme and key substrate in Autophagy regulation through protein ATG8ylation.