Molecular mechanisms of the interaction between PROPPIN Atg21 and Atg16

Abstract

Atg8/LC3 lipidation is essential for phagophore expansion during autophagy. WIPI2 in mammals and its yeast homolog Atg21, mediate the recruitment of the lipidation machinery to the phagophore membrane through their interaction with PI3P and ATG16L1/Atg16. Our work aims to further study the molecular mechanism of the Atg21- Atg16 interaction in yeast and the differences and similarities with the mammalian homologs. For this purpose, we used two-hybrid techniques to identify the residues in both Atg16 and Atg21 that mediate this interaction. Additionally, we used the reporters Pho8Δ60, GFP-Atg8, and Ape1 to analyze the effect of mutations of these residues on bulk and selective autophagy. We show that disruption of Atg21-Atg16 binding blocks selective autophagy, but also strongly impairs bulk autophagy. We find that the Atg16 binding site on Atg21 is conserved in human WIPI2 and involves the same binding surface identified in yeast WIPI4 or Hsv2 as responsible for Atg2 binding, thus indicating that WIPI proteins use the same mechanism to interact with different proteins of the autophagic machinery. Furthermore, our data indicate that Atg16 dimerization is required for Atg21 binding, and that residues in both Atg16 monomers mediate Atg21 binding. Finally, our findings support the idea that the WIPI2 binding site in ATG16L1 has been lost in yeast Atg16, and has been replaced by a site in the coiled-coil domain involving residues in both Atg16 monomers.