Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic ph homeostasis in yeast

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 283, NO. 29, pp. 20309 –20319, July 18, 2008 © 2008 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast*
Received for publication, December 22, 2007, and in revised form, April 17, 2008 Published, JBC Papers inPress, May 23, 2008, DOI 10.1074/jbc.M710470200

Gloria A. Martınez-Munoz1 and Patricia Kane2 ´ ˜ 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 andcytosolic 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. Incontrast, 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 membranefractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65–75% lower than in fractions from wildtype 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 allconditions 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.

The importance of V-ATPases3 for acidification of the vacuole/lysosomes, Golgi apparatus, andendosomes of eukaryotic cells is well established (1, 2). Multiple cellular processes, including secondary transport of ions and metabolites, maturation of iron transporters, endocytic and biosynthetic protein sorting, and zymogen activation depend on compartment acid-

* This work was supported, in whole or in part, by National Institutes of Health
Grant GM50322 (to P. M. K.). The costs ofpublication 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@yahoo.com.mx. 2 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, 750 EastAdams St., Syracuse, NY 13210; Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm@upstate.edu. 3 The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; BCECF-AM, 2 ,7 -bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester; MES, 2-[N-morpholino]ethanesulfonic acid; MOPS, 2-[N-morpholino]propanesulfonic acid; TEA, triethanolamine.

ification and have beenlinked to V-ATPase activity (1, 3). In some cells such as macrophages, V-ATPases play specialized roles that clearly include regulation of cytosolic pH (4, 5). However, although V-ATPases pump protons from the cytosol into organelles in all cells, they are not generally believed to play a major role in cytosolic pH regulation. The yeast Saccharomyces cerevisiae has emerged as a major model system...