Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization

Abstract

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptors or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The ctive V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abudance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subuits were associated with detergent resistant membrane (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cell with U18666A, a drug that leads tothe accumulation of cholesterol in late endosomes, affected acidification of late endosomes. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments. © 2008 Lafourcade et al.