Autophagosome membrane size correlates with levels of Atg8 conjugated to phosphatidylethanolamine (Atg8-PE). Conjugation and association of Atg8-PE with autophagic membranes is mediated by Atg5-modified Atg12 (Atg12-Atg5), which also associates with Atg16. The Atg12-Atg5–Atg16 complex localizes to the convex face of the sequestering membrane, called the phagophore, and is required for autophagosome biogenesis. Kaufmann et al. reconstitute these interactions in vitro and reveal that the Atg12-Atg5–Atg16 complexes form a mesh-like scaffold on phagophore membranes (Cell 156, 469–481; 2014).

The authors found that Atg12-Atg5 and Atg16 associated with giant unilamellar vesicles (GUVs) containing Atg8-PE, suggesting that lipidated Atg8 recruits and retains Atg12-Atg5 at membranes. Intriguingly, Atg16 caused deformation of GUV membranes reminiscent of protein clustering, raising the possibility that Atg16 immobilizes Atg12-Atg5–Atg8-PE complexes on GUVs. Further analyses revealed that Atg16 acts as a scaffold for generating a flat meshwork of Atg12-Atg5–Atg8-PE complexes. The addition of the cargo adaptor Atg32 caused the meshwork to dissemble by displacing Atg12-Atg5 from Atg8-PE. Lipidated Atg8 was then cleaved in an Atg4-dependent manner, and dissociated from the membrane. Importantly, mutations in Atg16 or Atg12 that blocked scaffold formation but did not affect Atg8 lipidation inhibited autophagic flux in vivo.