Cryo-EM structure of the Arabidopsis thaliana V-type ATPase

V-ATPases couple ATP hydrolysis in their cytosolic V₁ region to proton translocation through their membrane-embedded V_O region to acidify the lumen of organelles. Functional diversity in the pump is enabled by tissue- and organelle-specific expression of paralogous genes for its subunits. Interactions between V-ATPase and TLDc domain-containing proteins regulate the enzyme in yeast and mammals but their relevance in plants is unclear. We isolated V-ATPase from Arabidopsis thaliana and determined its structure by electron cryomicroscopy. Mass spectrometry showed that most of the enzyme originated from the tonoplast. The structural analysis revealed three main rotational states, and structural and biochemical experiments showed S-acylation of subunits AP1 and the tonoplast-specific subunit a3 isoform. A subpopulation of complexes derived from the trans-Golgi network/early endosome was bound to the TLDc protein OXR5. These findings reveal plant-specific V-ATPase features and suggest organelle-specific interactions with TLDc proteins, pointing to conserved but context-dependent V-ATPase regulation in eukaryotes.