The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12–ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12–ATG5–ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.

非选择性自噬的标志是形成捕获大量细胞质的杯形吞噬泡。该过程伴随着 LC3B 通过包含 ATG12-ATG5 和 ATG16L1 的 E3 连接酶复合物与吞噬细胞的结合。在这里，我们结合了两种互补的重建方法来揭示 LC3B 及其连接酶复合物在吞噬细胞扩张过程中的功能。我们发现 LC3B 与 ATG12–ATG5–ATG16L1 一起形成一层膜涂层，将扁平膜重塑成与吞噬细胞非常相似的杯状结构。从机制上讲，我们揭示了杯的形成严格取决于 LC3B 和 ATG16L1 之间的密切合作。此外，只有 LC3B 能够促进杯的形成，而 ATG8 蛋白家族的其他成员则没有。缺乏 C 端膜结合结构域的 ATG16L1 截短可催化 LC3B 脂化，但无法组装外壳，不促进杯形成并抑制非选择性自噬体的生物发生。因此，我们的结果表明 ATG16L1 和 LC3B 诱导并稳定了吞噬细胞的特征性杯状形状。