ABSTRACT
Autophagy is an enigmatic cellular process in which double-membrane autophagic compartments form de novo adjacent to the ER, sequestering cytoplasmic contents within and delivering them to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each tip of ATG2A. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle with another PI3P-free vesicle. These data suggest that ATG2A mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, prerequisites for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.