Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
Physical and Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3QZ, UK.
Mol Cell. 2020 Nov 5;80(3):501-511.e3. doi: 10.1016/j.molcel.2020.09.029. Epub 2020 Oct 15.
Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are ATP-driven proton pumps comprised of a cytoplasmic V complex for ATP hydrolysis and a membrane-embedded V 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 complex. We define ATP6AP1 as a structural hub for V complex assembly because it connects to multiple V subunits and phospholipids in the c-ring. The glycolipids and the glycosylated V 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.
囊泡型或空泡型三磷酸腺苷酶（V-ATPases）是由细胞质 V 复合物进行 ATP 水解和膜嵌入 V 复合物进行质子传递组成的 ATP 驱动质子泵。它们在真核生物的细胞内囊泡、细胞器和细胞外环境酸化中发挥重要作用。在这里，我们报道了高达 2.9-Å 分辨率的人类 V-ATPase 在三种旋转状态下的冷冻电子显微镜结构。借助质谱分析，我们构建了所有已知的蛋白亚基及其相关的 N 连接糖链，并在 V 复合物中鉴定了糖脂和磷脂。我们将 ATP6AP1 定义为 V 复合物组装的结构枢纽，因为它与 c 环中的多个 V 亚基和磷脂相连。糖脂和糖基化的 V 亚基形成一个内腔糖被，对于 V-ATPase 的折叠、定位和稳定性至关重要。这项研究确定了 V-ATPase 组装和生物发生的机制，这些机制依赖于 ATP6AP1、糖和脂质的综合作用。
