The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy

In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism of the ULK complex formation remain poorly understood. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 binds to the base of the two UBL domains in the FIP200 dimer using two phenylalanines and to the tandem MIT domain of ULK1, allowing for the 1:1:2 stoichiometry of the ULK1–ATG13–FIP200 complex. We confirmed the predicted interactions by point mutations and revealed the existence of direct triad interactions among ULK1, ATG13, and FIP200 in vitro and in cells, in which each interaction was additively important for autophagic flux. These results indicate that the ULK1–ATG13–FIP200 triadic interaction is crucial for autophagosome formation and provide a structural basis and insights into the regulation mechanism of autophagy initiation in mammals.