Atg23 interacts with both the N- and C-termini of Atg9 via a hydrophobic binding pocket.

Macroautophagy is a cellular process where cytosolic material is captured in double membrane vesicles, termed autophagosomes, which fuse with the vacuole or lysosomes leading to the degradation of the captured contents. The biogenesis of autophagosomes is initiated by the fusion of a few small vesicles which contain the integral membrane protein Atg9. Atg9 vesicle trafficking is, in part, regulated by the peripheral membrane protein Atg23. However, the structure of Atg23 and the mechanism by which Atg23 interacts with Atg9 are currently unknown. Therefore, we determined the crystal structure for a monomeric form of Atg23 and characterized the interaction between Atg23 and Atg9. This work reveals that Atg23 contains a novel fold, which is consistent with the AlphaFold 3 prediction except that the helices running toward the dimerization region have a bend, giving a more curved global architecture than the prediction. In addition, we demonstrate that conserved sequences at the very distal regions of the N and C-terminal disordered regions of Atg9 bind to a hydrophobic cavity on Atg23. These Atg23 binding regions are distinct from the previously identified Atg11 binding region within the disordered N-terminus of Atg9, suggesting that Atg9 contains multiple different protein interaction regions within its disordered termini.