Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy

Membrane targeting of autophagy‐related complexes is an important step that regulates their activities and prevents their aberrant engagement on non‐autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre‐autophagosomal structure (PAS) and showed that it requires sequences within its coiled‐coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3‐phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation. Synopsis Recruitment of core autophagy protein ATG16L1 to the phagophore, essential for autophagosome biogenesis, involves it intrinsic ability to bind phosphoinositides on pre‐autophagosomal compartments, to promote LC3 lipidation and autophagosome maturation. The coiled‐coil domain (CCD) of ATG16L1 interacts with phosphatidylinositol‐3‐phosphate and other phosphoinositides. CCD‐lipid interaction is required for efficient recruitment of ATG16L1 to the phagophore. Disrupting the interaction between ATG16L1 and phosphoinositides inhibits LC3 lipidation. Graphical Abstract The core autophagy protein ATG16L1 binds phosphoinositides on pre‐autophagosomal structures via its coiled‐coil domain to promote LC3 lipidation and autophagosome maturation.