Autophagy is a fundamental cellular recycling pathway that captures cytoplasmic material by a phagophore membrane and delivers it to lysosomes for degradation. Nonselective phagophores are generated at specialized ER-domains termed omegasomes. Mechanistically, this process is not well understood. Here, we reconstituted the formation of phagophores from purified components on supported lipid bilayers and by ectopic induction of nonselective autophagy at the plasma membrane. We found that enzymatic conjugation of LC3B to model membranes induces the formation of phagophore-like membrane cups. LC3B functions as membrane anchor, ensuring a sustained interaction of the E3-like ligase complex ATG12¬–ATG5-ATG16L1 with membranes by forming extended membrane coats. ATG16L1 is the major membrane remodeling factor that induces positive membrane curvature, promotes cup formation and stabilizes the rim of membrane cups. Redirecting WIPI2 to the plasma membrane by expressing WIPI2-CAAX induced the formation of omegasome-like membrane cisternae to which LC3B was conjugated. Membrane cups, identical to those observed in vitro, emerged from these cisternae. ATG16L1 variants that did not induce the formation of cups in vitro also failed to promote cup formation in vivo and inhibited the biogenesis of nonselective autophagosomes in cells. We thus propose that ATG16L1 drives the formation of nonselective phagophores by shaping flat donor membranes into membrane cups.
