Abstract
The vacuolar H+-ATPases (V-ATPases) acidify intracellular acidic-organella of eukaryotic cells. The V-ATPase activity is known to be regulated by redox state of the cytoplasm and Forgac et al. proposed that a disulfide bond formation at the substrate-binding site of the catalytic A subunit regulates the activity (J. Exp. Biol. 203: 71-80).
We measured H+ pump activity of V-ATPase in vacuolar membranes of S. cerevisiae by patch clamp method and confirmed redox-regulation of the wild-type enzyme. Then we examined the oxidation-resistant enzyme containing the mutant of A subunit (C261V). Interestingly, H+ pump activity of the vacuole containing this mutant V-ATPase drastically diminished when the vacuole was exposed to CuSO4. The activity was partially recovered after the addition of DTT. These results suggest the presence of the additional redox-sensitive cysteines in yeast V-ATPase.
