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J. Biol. Chem., Vol. 283, Issue 29, 20309-20319, July 18, 2008

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Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast^*

From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K⁺ ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65–75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.

Received for publication, December 22, 2007 , and in revised form, April 17, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM50322 (to P. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Current e-mail address: gloria_martinez2006{at}yahoo.com.mx.

² To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210; Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}upstate.edu.

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