Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are ATP-driven proton pumps comprised of a cytoplasmic V₁ complex for ATP hydrolysis and a membrane-embedded V_o complex for proton transfer. They play important roles in acidification of intracellular vesicles, organelles, and the extracellular milieu in eukaryotes. Here, we report cryoelectron microscopy structures of human V-ATPase in three rotational states at up to 2.9-Å resolution. Aided by mass spectrometry, we build all known protein subunits with associated N-linked glycans and identify glycolipids and phospholipids in the V_o complex. We define ATP6AP1 as a structural hub for V_o complex assembly because it connects to multiple V_o subunits and phospholipids in the c-ring. The glycolipids and the glycosylated V_o subunits form a luminal glycan coat critical for V-ATPase folding, localization, and stability. This study identifies mechanisms of V-ATPase assembly and biogenesis that rely on the integrated roles of ATP6AP1, glycans, and lipids.

囊泡型或液泡型三磷酸腺苷酶 (V-ATPases) 是 ATP 驱动的质子泵，由用于 ATP 水解的细胞质 V ₁复合物和用于质子转移的膜包埋 V _o复合物组成。它们在真核生物的细胞内囊泡、细胞器和细胞外环境的酸化中发挥重要作用。在这里，我们报告了三种旋转状态下人类 V-ATPase 的低温电子显微镜结构，分辨率高达 2.9-Å。在质谱分析的帮助下，我们构建了所有已知的蛋白质亚基与相关的 N-连接聚糖，并鉴定了 V _o复合物中的糖脂和磷脂。我们将 ATP6AP1 定义为 V _o复杂装配的结构枢纽，因为它连接到多个 V _oc环中的亚基和磷脂。糖脂和糖基化的 V _o亚基形成对 V-ATP 酶折叠、定位和稳定性至关重要的管腔聚糖涂层。本研究确定了 V-ATPase 组装和生物发生的机制，这些机制依赖于 ATP6AP1、聚糖和脂质的综合作用。