The dual role of core autophagy E1 enzyme ATG7: revealing a new negative feedback mechanism as a substrate for protein ATG8ylation

The conjugation of mammalian Atg8 (ATG8)-family proteins to membrane components is a fundamental process in membrane ATG8ylation (lipidation). While membrane ATG8ylation is well-characterized, protein ATG8ylation, the direct conjugation of ATG8 to cellular proteins, remains enigmatic. In this study, we demonstrate that protein ATG8ylation depends exclusively on ATG4, ATG3, and ATG7. We discovered that the core macroautophagy/autophagy E1 enzyme ATG7 serves a dual role: it is not only the essential E1 enzyme for protein ATG8ylation but also a key substrate. We determined that ATG7 K140 is the modification site and show that protein ATG8ylation of ATG7 forms a mono-LC3B conjugate. We demonstrated that this self-modification creates a negative-feedback loop by hindering the ATG7-ATG3 interaction, thereby attenuating autophagic flux. Our findings redefine ATG7 as a central player and regulator in the protein ATG8ylation cascade, revealing a new mechanism of autophagy regulation.<b>Abbreviation</b>: ATG: autophagy related; GABARAP: GABA type A receptor-associated protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PE: phosphatidylethanolamine; ULK1: unc-51 like autophagy activating kinase 1.