Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt.
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 Pi 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 (ER) 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 approximately 2H+/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 fide proton pumping (GP) through the ATPase relative to independent proton shunting (GS) through the same protein.
Kettner
Carsten
C
Botanisches Institut I, Universität Karlsruhe, Karlsruhe, Germany.
Bertl
Adam
A
Obermeyer
Gerhard
G
Slayman
Clifford
C
Bihler
Hermann
H
D000255:Adenosine Triphosphate / Q000737:chemistry; D055592:Biophysical Phenomena; D001703:Biophysics; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D017494:Proton Pumps; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
D007477:Ions; D017494:Proton Pumps; D011522:Protons; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases