Abstract
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 ± 0.3 and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 and 21.7 ± 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of membrane and close to each other as visualized with antibody and gold nanoparticles against 6□His tag on C-ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 4.2 ± 1.4 nm. Thus our study is the first to provide the structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.