Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly

Abstract

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.