De novo formation of the double-membrane compartment autophagosome is seeded by small vesicles carrying membrane protein autophagy-related 9 (ATG9), the function of which remains unknown. Here we find that ATG9A scrambles phospholipids of membranes in vitro. Cryo-EM structures of human ATG9A reveal a trimer with a solvated central pore, which is connected laterally to the cytosol through the cavity within each protomer. Similarities to ABC exporters suggest that ATG9A could be a transporter that uses the central pore to function. Moreover, molecular dynamics simulation suggests that the central pore opens laterally to accommodate lipid headgroups, thereby enabling lipids to flip. Mutations in the pore reduce scrambling activity and yield markedly smaller autophagosomes, indicating that lipid scrambling by ATG9A is essential for membrane expansion. We propose ATG9A acts as a membrane-embedded funnel to facilitate lipid flipping and to redistribute lipids added to the outer leaflet of ATG9 vesicles, thereby enabling growth into autophagosomes.

双膜室自噬体的从头形成是由携带膜蛋白自噬相关 9 (ATG9) 的小囊泡播种的，其功能尚不清楚。在这里我们发现 ATG9A 在体外扰乱细胞膜的磷脂。人 ATG9A 的冷冻电镜结构揭示了一个带有溶剂化中心孔的三聚体，它通过每个原体内的空腔横向连接到细胞质。与 ABC 出口商的相似之处表明 ATG9A 可能是一种使用中央孔发挥功能的转运蛋白。此外，分子动力学模拟表明，中央孔横向打开以容纳脂质头部基团，从而使脂质翻转。孔中的突变降低了加扰活动并产生明显更小的自噬体，表明 ATG9A 对脂质的加扰对于膜扩张至关重要。