Molecular Mechanism of Autophagic Membrane-Scaffold Assembly and Disassembly

Summary Autophagy is a catabolic pathway that sequesters undesired cellular material into autophagosomes for delivery to lysosomes for degradation. A key step in the pathway is the covalent conjugation of the ubiquitin-related protein Atg8 to phosphatidylethanolamine (Atg8–PE) in autophagic membranes by a complex consisting of Atg16 and the Atg12–Atg5 conjugate. Atg8 controls the expansion of autophagic precursor membranes, but the underlying mechanism remains unclear. Here, we reconstitute Atg8 conjugation on giant unilamellar vesicles and supported lipid bilayers. We found that Atg8–PE associates with Atg12–Atg5-Atg16 into a membrane scaffold. By contrast, scaffold formation is counteracted by the mitochondrial cargo adaptor Atg32 through competition with Atg12–Atg5 for Atg8 binding. Atg4, previously known to recycle Atg8 from membranes, disassembles the scaffold. Importantly, mutants of Atg12 and Atg16 deficient in scaffold formation in vitro impair autophagy in vivo. This suggests that autophagic scaffolds are critical for phagophore biogenesis and thus autophagy.