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J Biol Chem, Vol. 274, Issue 52, 37270-37279, December 24, 1999

Alternative Mechanisms of Vacuolar Acidification in H+-ATPase-deficient Yeast*

Pamela J. PlantDagger §, Morris F. ManolsonDagger §, Sergio GrinsteinDagger §, and Nicolas Demaurex∥**

From the Dagger  Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, the § Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 2Z9, Canada, and the ∥ Department of Physiology, University of Geneva Medical Center, 1211 Geneva 4, Switzerland

Acidification of the endosomal/lysosomal pathway by the vacuolar-type proton translocating ATPase (V-ATPase) is necessary for a variety of essential eukaryotic cellular functions. Nevertheless, yeasts lacking V-ATPase activity (Delta vma) are viable when grown at low pH, suggesting alternative methods of organellar acidification. This was confirmed by directly measuring the vacuolar pH by ratio fluorescence imaging. When Delta vma yeasts were cultured and tested in the acidic conditions required for growth of V-ATPase-deficient mutants, the vacuolar pH was 5.9. Fluid-phase pinocytosis of acidic extracellular medium cannot account for these observations, because the V-ATPase-independent vacuolar acidification was unaffected in mutants deficient in endocytosis. Similarly, internalization of the plasmalemmal H+-ATPase (Pma1p) was ruled out, because overexpression of Pma1p failed to complement the Delta vma phenotype and did not potentiate the vacuolar acidification. To test whether weak electrolytes present in the culture medium could ferry acid equivalents to the vacuole, wild-type and the Delta vma yeasts were subjected to sudden changes in extracellular pH. In both cell types, the vacuoles rapidly alkalinized when external pH was raised from 5.5 (the approximate pH of the culture medium) to 7.5 and re-acidified when the yeasts were returned to a medium of pH 5.5. Importantly, these rapid pH changes were only observed when NH4+, routinely added as a nitrogen source, was present. The NH4+-dependent acidification was not due to efflux of NH3 from the vacuole, as cells equilibrated to pH 7.5 in the absence of weak electrolytes rapidly acidified when challenged with an acidic medium containing NH4+. These findings suggest that although NH3 can act as a cell-permeant proton scavenger, NH4+ may function as a protonophore, facilitating equilibration of the pH across the plasma and vacuolar membranes of yeast. The high concentration of NH4+ frequently added as a nitrogen source to yeast culture media together with effective NH4+ transporters thereby facilitate vacuolar acidification when cells are suspended in acidic solutions.

* This work was supported by operating grants from the Medical Research Council of Canada (to S. G. and M. M.) and by Grant 31-46859.96 from the Swiss National Science Foundation (to N. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current holder of the Pitblado Chair in Cell Biology and is an International Scholar of the Howard Hughes Medical Institute.

** A fellow from the Dr. Max Cloetta Foundation. To whom correspondence should be addressed: Dept. of Physiology, University of Geneva Medical Center, 1, Michel-Servet, CH-1211 Geneva 4, Switzerland. Tel.: 4122-702-5399; Fax: 4122-702-5402; E-mail: Nicolas.Demaurex@medecine.unige.ch.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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