The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9’s ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.

Atg9（自噬体形成机制中唯一的跨膜蛋白）的分子功能仍不清楚。 Atg9 与 Atg2 共定位于隔离膜 (IM) 的扩张边缘，Atg2 在此接收来自内质网 (ER) 的磷脂。在这里，我们报道酵母和人类 Atg9 是脂质扰乱酶，可在体外将磷脂在脂质体的外叶和内叶之间易位。裂殖酵母 Atg9 的冷冻电镜显示出同源三聚体，其中两个相连的孔在两个膜小叶之间形成路径：一个孔位于原聚体处，横向通向细胞质小叶；另一个孔位于原聚体处，向细胞质小叶侧向开口；另一个位于三聚体中心，垂直穿过膜。孔内残基的突变损害了酵母中的 IM 扩张和自噬活性，并消除了 Atg9 在脂质体小叶之间运输磷脂的能力。这些结果表明，Atg2 递送的磷脂通过 Atg9 从细胞质转移到管腔小叶，从而驱动自噬体膜扩张。