Electrophysiological Analysis of the Yeast V-Type Proton Pump: Variable Coupling Ratio and Proton Shunt

Abstract

Isolated vacuoles from the yeast Saccharomyces cerevisiae were examined in the whole-vacuole mode of patch recording, to get a detailed functional description of the vacuolar proton pump, the V-ATPase. Functioning of the V-ATPase was characterized by its current-voltage (I-V) relationship, obtained for various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as the difference between I-V curves obtained with the pump switched on (ATP, ADP, and P i present) or off (no ATP). These difference current-voltage relationships usually crossed the voltage axis within the experimental range (from −80 to +80 mV), thus measuring the reversal voltage ( E R) for the V-ATPase, which could be compared with the standing ion gradients and free energy of ATP hydrolysis, to calculate the apparent pump stoichiometry or coupling ratio: the number of protons transported for each ATP molecule hydrolyzed. This ratio was found to depend strongly upon the pH difference (ΔpH) across the vacuolar membrane, being ∼2H +/ATP at high ΔpH (4 pH units) and increasing to >4H +/ATP for small or zero ΔpH. That result is in quantitative agreement with previous determinations on plant vacuoles. Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases, led to a linear equivalent circuit—which quantitatively accounts for all observations of variable coupling ratios in fungal and plant V-ATPases by variations of the conductance for bona fide proton pumping ( G P) through the ATPase relative to independent proton shunting ( G S) through the same protein.