Coiled-coil-mediated dimerization of Atg16 is required for binding to the PROPPIN Atg21.

PROPPINs/WIPIs are ß-propeller proteins that bind phosphoinositides and contribute to the recruitment of protein complexes involved in membrane remodelling processes such as autophagosome formation and endosomal trafficking. Yeast Atg21 and mammalian WIPI2 interact with Atg16/ATG16L1 to mediate recruitment of the lipidation machinery to the autophagosomal membrane. Here, we used the reverse double two-hybrid method (RD2H) to identify residues in Atg21 and Atg16 critical for protein-protein binding. Although our results are generally consistent with the crystal structure of the Atg21-Atg16 complex reported previously, they also reveal that dimerization of the Atg16 coiled-coil domain is required for Atg21 binding. Furthermore, most of the residues identified in Atg21 are conserved in WIPI2 and we showed that these residues also mediate ATG16L1 binding. Strikingly, these residues occupy the same position in the ß-propeller structure as residues in PROPPINs/WIPIs Hsv2 and WIPI4 that mediate Atg2/ATG2A binding, supporting the idea that these proteins use different amino acids at the same position to interact with different autophagic proteins. Finally, our findings demonstrate the effectiveness of the RD2H system to identify critical residues for protein-protein interactions and the utility of this method to generate combinatory mutants with a complete loss of binding capacity.

A conserved ATG2 binding site in WIPI4 and yeast Hsv2 is disrupted by mutations causing ß-propeller protein-associated neurodegeneration.

PROPPINs are phosphoinositide-binding ß-propeller proteins that mediate membrane recruitment of other proteins and are involved in different membrane remodeling processes. The main role of PROPPINs is their function in autophagy, where they act at different steps in phagophore formation. The human PROPPIN WIPI4 (WDR45) forms a complex with ATG2 involved in phagophore elongation, and mutations in this gene cause ß-propeller protein-associated neurodegeneration (BPAN). The yeast functional counterpart of WIPI4 is Atg18, although its closest sequence homolog is another member of the PROPPIN family, Hsv2, whose function remains largely undefined. Here, we provide evidence that Hsv2, like WIPI4 and Atg18, interacts with Atg2. We show that Hsv2 and a pool of Atg2 colocalize on endosomes under basal conditions and at the pre-autophagosomal structure (PAS) upon autophagy induction. We further show that Hsv2 drives the recruitment of Atg2 to endosomes while Atg2 mediates Hsv2 recruitment to the PAS. HSV2 overexpression results in mis-sorting and secretion of carboxypeptidase CPY, suggesting that the endosomal function of this protein is related to the endosome-to-Golgi recycling pathway. Furthermore, we show that the Atg2 binding site is conserved in Hsv2 and WIPI4 but not in Atg18. Notably, two WIPI4 residues involved in ATG2 binding are mutated in patients with BPAN, and there is a correlation between the inhibitory effect of these mutations on ATG2 binding and the severity of the disease.