Poster: Late and Moved Abstracts

Abs # 963: Electrophysiological analysis of the yeast V-type proton pump: variable coupling ratio and proton shunt

Presenter:	Bertl, Adam , adam.bertl@chem-bio.uni-karlsruhe.de
Authors	Bihler, Hermann  (A)   Kettner, Carsten  (A)   Obermeyer, Gerhard  (B)   Slayman, Clifford L. (C)   Bertl, Adam  (A)
Affiliations:	(A): Botany I, University of Karlsruhe (B): Plant Physiology, University of Salzburg (C): Medical School, Yale University

Isolated vacuoles from the yeast S. cerevisiae were examined in the whole-vacuole mode of patch recording, to obtain a functional description of the vacuolar proton pump, the V-ATPase, via its current voltage (I-V) characteristic determined at various levels of vacuolar and cytosolic pH. I-V curves for the V-ATPase were computed as differences between corresponding curves obtained with the pump switched on (ATP, ADP, & P_i present), then off (ATP absent). These difference I-V curves usually crossed the voltage axis within the experimentally accessible voltage range (-80 mV to +80 mV), and therefore yielded a measure of the reversal voltage (ER) for the V-ATPase, for comparison with the standing ion gradients and free energy of ATP hydrolysis. This comparison allowed calculation of the apparent pump stoichiometry, or coupling ratio (n) for the ATPase: the number of protons transported for each ATP molecule hydrolyzed. n was found to depend strongly upon the pH difference (ΔpH) across the vacuolar membrane, being ~2H⁺/ATP at high ΔpH (4 units), but increasing to >4H⁺/ATP for small or zero ΔpH, a result in complete agreement with previous determinations on plant vacuoles (Davies et al., 1994). Considerations of purely electrical behavior, together with the physical properties of a recent detailed structural model for V-ATPases (Grabe et al., 2000), led to a linear equivalent circuit which quantitatively accounts for all observations of variable coupling ratio 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