Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. Autophagy-related proteins (ATGs) 2A and 9A have an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-electron microscopy structures of human ATG2A in complex with WD-repeat protein interacting with phosphoinositides 4 (WIPI4) at 3.2 Å and the ATG2A–WIPI4–ATG9A complex at 7 Å global resolution. On the basis of molecular dynamics simulations, we propose a mechanism of lipid extraction from the donor membranes. Our analysis revealed 3:1 stoichiometry of the ATG9A–ATG2A complex, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, and an interaction of the ATG9A trimer with both the N-terminal and the C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A liposome-binding states showed that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.

自噬的特征是形成称为自噬体的双膜囊泡。自噬相关蛋白 (ATG) 2A 和 9A 通过介导脂质转移和膜间重新平衡以形成自噬体，在自噬中发挥重要作用。在这里，我们报告了人 ATG2A 与 WD 重复蛋白复合物与磷酸肌醇 4 (WIPI4) 相互作用的复合物的冷冻电子显微镜结构，分辨率为 3.2 Å，以及 ATG2A-WIPI4-ATG9A 复合物的全局分辨率为 7 Å。在分子动力学模拟的基础上，我们提出了一种从供体膜中提取脂质的机制。我们的分析揭示了 ATG9A-ATG2A 复合物的 3:1 化学计量比，直接将 ATG9A 侧孔与 ATG2A 脂质转移腔对齐，以及 ATG9A 三聚体与杆状 ATG2A 的 N 端和 C 端尖端的相互作用。 ATG2A 脂质体结合状态的冷冻电子断层扫描表明 ATG2A 以不同方向束缚脂质囊泡。总之，这项研究为吞噬细胞膜的生长提供了分子基础，并为自噬体形成过程中空间耦合的脂质运输和重新平衡提供了结构见解。