Journal
BIOPHYSICAL JOURNAL
Volume 85, Issue 6, Pages 3730-3738Publisher
BIOPHYSICAL SOCIETY
DOI: 10.1016/S0006-3495(03)74789-4
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Funding
- NIGMS NIH HHS [R01 GM060696, GM-60696] Funding Source: Medline
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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 (DeltapH) across the vacuolar membrane, being similar to2H(+)/ATP at high DeltapH (4 pH units) and increasing to >4H(+)/ATP for small or zero DeltapH. 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. de proton pumping (G(P)) through the ATPase relative to independent proton shunting (G(S)) through the same protein.
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