Vacuolar and Plasma Membrane Proton Pumps Collaborate to Achieve Cytosolic pH Homeostasis in Yeast
2008; Elsevier BV; Volume: 283; Issue: 29 Linguagem: Inglês
10.1074/jbc.m710470200
ISSN1083-351X
AutoresGloria A. Martínez‐Muñoz, Patricia M. Kane,
Tópico(s)Mitochondrial Function and Pathology
ResumoVacuolar 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. 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. The importance of V-ATPases 3The 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. 3The 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. for acidification of the vacuole/lysosomes, Golgi apparatus, and endosomes of eukaryotic cells is well established (1Forgac M. Nat. Rev. Mol. Cell Biol. 2007; 8: 917-929Crossref PubMed Scopus (1049) Google Scholar, 2Kane P.M. Microbiol. Mol. Biol. Rev. 2006; 70: 177-191Crossref PubMed Scopus (323) Google Scholar). 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 acidification and have been linked to V-ATPase activity (1Forgac M. Nat. Rev. Mol. Cell Biol. 2007; 8: 917-929Crossref PubMed Scopus (1049) Google Scholar, 3Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar). In some cells such as macrophages, V-ATPases play specialized roles that clearly include regulation of cytosolic pH (4Swallow C.J. Grinstein S. Sudsbury R.A. Rotstein O.D. J. Cell. Physiol. 1993; 157: 453-460Crossref PubMed Scopus (60) Google Scholar, 5Swallow C.J. Grinstein S. Rotstein O.D. J. Biol. Chem. 1990; 265: 7645-7654Abstract Full Text PDF PubMed Google Scholar). 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 for eukaryotic V-ATPases. One reason for this is that yeast mutants lacking all V-ATPase activity (vma mutants) are viable, but loss of V-ATPase activity in eukaryotes other than fungi is lethal (6Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar, 7Bowman E.J. Kendle R. Bowman B.J. J. Biol. Chem. 2000; 275: 167-176Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 8Davies S.A. Goodwin S.F. Kelly D.C. Wang Z. Sozen M.A. Kaiser K. Dow J.A. J. Biol. Chem. 1996; 271: 30677-30684Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Sun-Wada G. Murata Y. Yamamoto A. Kanazawa H. Wada Y. Futai M. Dev. Biol. 2000; 228: 315-325Crossref PubMed Scopus (98) Google Scholar). Yeast vma mutants do exhibit a set of distinctive phenotypes, however, that includes the inability to grow at pH values lower than 3 or higher than 7 and sensitivity to high extracellular calcium concentrations (2Kane P.M. Microbiol. Mol. Biol. Rev. 2006; 70: 177-191Crossref PubMed Scopus (323) Google Scholar). This Vma- phenotype suggests a perturbation of pH homeostasis in these cells that is not fully understood. It has been suggested that vma mutants survive at low extracellular pH (pH 5) by endocytosis of acidic extracellular fluid and transport to the vacuole (6Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar, 10Munn A.L. Riezman H. J. Cell Biol. 1994; 127: 373-386Crossref PubMed Scopus (229) Google Scholar) or that they acidify the vacuole through diffusion of permeant acids (11Plant P.J. Manolson M.F. Grinstein S. Demaurex N. J. Biol. Chem. 1999; 274: 37270-37279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). There have been few direct measurements of cytosolic or vacuolar pH in the vma mutants under different extracellular conditions, however (11Plant P.J. Manolson M.F. Grinstein S. Demaurex N. J. Biol. Chem. 1999; 274: 37270-37279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar).pH homeostasis is critical for survival of yeast cells, as it is for all eukaryotic cells. V-ATPases function in tandem with Pma1p, an essential P-type proton pump localized to the plasma membrane, to help control pH (12Serrano R. Kielland-Brandt M.C. Fink G.R. Nature. 1986; 319: 689-693Crossref PubMed Scopus (572) Google Scholar, 13Serrano, R. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthese, and Energetics (Broach, J. R., Pringle, J. R., and Jones, E. W., eds) Vol. 1, pp. 523-585, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar). Glucose, the preferred carbon source for S. cerevisiae, is metabolized through fermentation and respiration. Enormous amounts of carbonic and organic acids are produced by energy metabolism, and these constitute the main source of protons in yeast (13Serrano, R. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthese, and Energetics (Broach, J. R., Pringle, J. R., and Jones, E. W., eds) Vol. 1, pp. 523-585, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar, 14Conway E.J. Brady T.G. Biochem. J. 1950; 47: 360-369Crossref PubMed Scopus (45) Google Scholar). Despite major differences in localization and structure, there are a number of similarities in regulation and function between Pma1p and V-ATPases. Both Pma1p and the V-ATPase use energy from ATP hydrolysis to pump protons out of the cytosol (13Serrano, R. (1991) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthese, and Energetics (Broach, J. R., Pringle, J. R., and Jones, E. W., eds) Vol. 1, pp. 523-585, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar). Both pumps are regulated by extracellular glucose, although their regulatory mechanisms are distinct. Glucose stimulates Pma1 H+-ATPase activity by inducing phosphorylation (15Lecchi S. Nelson C.J. Allen K.E. Swaney D.L. Thompson K.L. Coon J.J. Sussman M.R. Slayman C.W. J. Biol. Chem. 2007; 282: 35471-35481Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), resulting in a decreased Km for ATP and an increased Vmax (16Eraso P. Mazon M.J. Portillo F. Biochim. Biophys. Acta. 2006; 1758: 164-170Crossref PubMed Scopus (45) Google Scholar, 17Portillo F. Eraso P. Serrano R. FEBS Lett. 1991; 287: 71-74Crossref PubMed Scopus (87) Google Scholar). V-ATPases are also activated by glucose, but they reversibly disassemble in response to glucose deprivation and readdition, resulting in higher levels of assembled pumps and ATPase activity at the vacuole in the presence of glucose (18Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar, 19Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). The activity of both pumps is sensitive to pH, indicating that they may be tuned to respond to changes in cytosolic pH in vivo (20Padilla-Lopez S. Pearce D.A. J. Biol. Chem. 2006; 281: 10273-10280Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 21Eraso P. Gancedo C. FEBS Lett. 1987; 224: 187-192Crossref PubMed Scopus (130) Google Scholar). Pma1p, in particular, is activated in response to cytosolic acidification (21Eraso P. Gancedo C. FEBS Lett. 1987; 224: 187-192Crossref PubMed Scopus (130) Google Scholar), and this mode of regulation is likely to be important for maintenance of cytosolic pH in a narrow range, relatively independent of extracellular pH. Taken together, these data suggest that in addition to their parallel roles in removal of protons from the cytosol, these two pumps are responsive to similar metabolic stimuli.Both Pma1p and the V-ATPase are also electrogenic pumps. Each establishes a pH gradient and membrane potential (ΔΨ) critical for operation of other transporters in their respective membranes but also requires mechanisms for balancing the generation of membrane potential (22Seto-Young D. Perlin D.S. J. Biol. Chem. 1991; 266: 1383-1389Abstract Full Text PDF PubMed Google Scholar, 23Grabe M. Oster G. J. Gen. Physiol. 2001; 117: 329-344Crossref PubMed Scopus (238) Google Scholar). Potassium ion is primarily responsible for balancing the plasma membrane potential in yeast (24Madrid R. Gomez M.J. Ramos J. Rodriguez-Navarro A. J. Biol. Chem. 1998; 273: 14838-14844Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Transport of K+ ion into the cell results in a depolarization of the plasma membrane, allowing stimulation of Pma1p (22Seto-Young D. Perlin D.S. J. Biol. Chem. 1991; 266: 1383-1389Abstract Full Text PDF PubMed Google Scholar) and consequent cytosolic alkalinization (25Pena A. Cinco G. Gomez-Puyou A. Tuena M. Arch. Biochem. Biophys. 1972; 153: 413-425Crossref PubMed Scopus (59) Google Scholar, 26Calahorra M. Martinez G.A. Hernandez-Cruz A. Pena A. Yeast. 1998; 14: 501-515Crossref PubMed Scopus (28) Google Scholar), and pH-responsive regulation of the Trk1p potassium transporter via phosphorylation provides additional means of activating Pma1p when cytosolic pH drops. The exact mechanisms for balancing the membrane potential at the vacuolar membrane are less clear; many anion channels and other transporters are present in the vacuolar membrane that could contribute to the final potential, but their individual contributions to membrane potential are not well defined.In addition to the primary proton pumps, multiple transporters and buffers combine to determine the final cytosolic and vacuolar pH under various conditions. Both the plasma membrane and organellar membranes contain alkali cation/H+ exchangers that contribute to overall pH homeostasis (27Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (199) Google Scholar, 28Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In mammalian cells where there is no primary proton pump comparable with Pma1p; the plasma membrane NHE (Na+/H+) antiporters play a central role in pH homeostasis (29Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). Yeast cells also have Na+/H+ exchangers (Nha1p) at the plasma membrane, but their function in pH homeostasis is clearly secondary to that of Pma1p (27Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (199) Google Scholar). In addition, both mammalian and yeast cells contain organellar K+(Na+)/H+ exchangers. The founding member of this class of exchangers, yeast Nhx1p (28Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), localizes to the yeast endosome and has clearly been shown to play a role in regulating both vacuolar and cytosolic pH (30Ali R. Brett C.L. Mukherjee S. Rao R. J. Biol. Chem. 2004; 279: 4498-4506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). The existence of a distinct vacuolar K+(Na+)/H+ exchanger was inferred from physiological studies (32Martinez-Munoz G.A. Pena A. Yeast. 2005; 22: 689-704Crossref PubMed Scopus (12) Google Scholar) and was recently proposed to be the VNX1 gene product (33Cagnac O. Leterrier M. Yeager M. Blumwald E. J. Biol. Chem. 2007; 282: 24284-24293Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Both small molecule and protein buffers are also likely to contribute to pH homeostasis in the cytosol and vacuole. In addition to the potential role for permeant acids in the vma mutants, highlighted by Plant et al. (11Plant P.J. Manolson M.F. Grinstein S. Demaurex N. J. Biol. Chem. 1999; 274: 37270-37279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), it is notable that the vacuole contains very high concentrations of polyphosphate that can provide buffering capacity (34Freimoser F.M. Hurlimann H.C. Jakob C.A. Werner T.P. Amrhein N. Genome Biology. 2006; 7: R109Crossref PubMed Google Scholar, 35Pick U. Bental M. Chitlaru E. Weiss M. FEBS Lett. 1990; 274: 15-18Crossref PubMed Scopus (66) Google Scholar). It has also been proposed that vacuolar proteins are well suited to buffer the vacuole to its typical pH range (36Brett C.L. Donowitz M. Rao R. FEBS Lett. 2006; 580: 717-719Crossref PubMed Scopus (25) Google Scholar).Several pieces of data suggest that the plasma membrane and vacuolar proton pumps Pma1 and V-ATPase are functionally interdependent. Bowman and Bowman (37Bowman E.J. Bowman B.J. J. Exp. Biol. 2000; 203: 97-106Crossref PubMed Google Scholar) isolated mutants in Neurospora crassa that were resistant to the specific V-ATPase inhibitor concanamycin A and discovered that they contained mutations in Pma1p. Mislocalization of Pma1 from the plasma membrane to the endoplasmic reticulum (38Perzov N. Nelson H. Nelson N. J. Biol. Chem. 2000; 275: 40088-40095Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) or the vacuole (39Hirata R. Takatsuki A. RIKEN Review. 2001; 41: 90-91Google Scholar) in the vma mutants has been reported, but the physiological implications of the altered localization have not been clarified. To address a potential coordination of function between the V-ATPase and Pma1p, we examined pH homeostasis under varied extracellular conditions in two vma mutants, vma2Δ and vma3Δ. Both mutants lack all V-ATPase activity, but the vma2Δ mutation deletes a subunit of the peripheral V1 sector of the enzyme, whereas the vma3Δ mutation deletes the major proteolipid subunit of the proton-pore containing the Vo sector (2Kane P.M. Microbiol. Mol. Biol. Rev. 2006; 70: 177-191Crossref PubMed Scopus (323) Google Scholar). Measurements of cytosolic and vacuolar pH indicate that wild-type cells readjust pH in response to the addition of glucose and K+ ion, as expected but that the vma mutations have dramatically perturbed pH homeostasis in both the vacuole and the cytosol. Mislocalization of Pma1p may account for some of the defects in the vma mutants, but even an acute loss of V-ATPase activity in the presence of concanamycin A abolishes vacuolar pH responses and perturbs cytosolic pH homeostasis. These results suggest an unexpectedly large role for the V-ATPase in cellular pH homeostasis.EXPERIMENTAL PROCEDURESMaterials—2′,7′-Bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM) was purchased from Invitrogen, and the yeast pHluorin plasmid was a generous gift from Dr. Rajini Rao (Johns Hopkins University). Polyclonal anti-Pma1p was a generous gift from Dr. Ramón Serrano. Monoclonal antibody against Pma1p (40B7) was obtained from Abcam, and monoclonal antibodies against Pep12p and alkaline phosphatase were purchased from Invitrogen. Alexa Fluor 488 goat anti-mouse IgG used for immunofluorescence was purchased from Invitrogen. Vanadate was purchased from Sigma and activated by heating at alkaline pH as described (40Gordon J.A. Methods Enzymol. 1991; 201: 477-482Crossref PubMed Scopus (527) Google Scholar). Concanamycin A and other chemicals were purchased from Sigma.Strains and Culture Conditions—Cells were grown in YEPD medium (1% yeast extract, 1% bactopeptone, 2% glucose) buffered to pH 5 or 7.5 with 50 mm potassium phosphate, 50 mm potassium succinate as described (41Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (145) Google Scholar). The BY4741 wild-type strain (MATα his3Δ1 leu2Δ0 met15Δ0 ura3Δ0; Open Biosystems) and the congenic vma2Δ::kanMX and vma3Δ::kanMX mutants were used throughout, except for those experiments requiring a pep4 mutants strain. For these experiments the SF838-1Dα wild-type (MATα ura3-53 leu2-3, 112 his4-519 pep4-3 ade6) and the congenic vma2Δ::LEU2 and vma3Δ::URA3 mutants were used (42Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar).Cytosolic and Vacuolar pH Measurements—Cytosolic pH was measured using a pH-sensitive green fluorescent protein, yeast pHLuorin, as described by Brett et al. (31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). BY4741 wild-type and vma mutant cells were transformed with the yeast pHLuorin-containing plasmid (31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar), and transformants were selected in fully supplemented minimal medium lacking leucine (SD-leucine) and confirmed by fluorescence microscopy (31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). Cells containing the pHLuorin plasmid were grown to log phase (A600 = 0.5–0.6) in YEPD buffered to the desired pH. Cells were collected by centrifugation and washed 2–3 times in YEP (YEPD medium without glucose) buffered at pH 5 and suspended at 1 g of wet cell mass/ml in the same medium. For pH shift experiments, cells were grown to log phase as described above, but cells were harvested at an absorbance of 0.5, shifted to YEPD, pH 7.5, and incubated for 3–4 more hours. The cells were washed and resuspended in YEP pH 7.5. For pH measurements, 25 μl of cell suspension was added to 2 ml of 1 mm MES/triethanolamine (TEA), pH 5, or MOPS/TEA, pH 7.5, and the mixture was stirred in a cuvette. Cytosolic pH responses were recorded at 30 °C (a) after 5 min of stirring, (b) after the addition of glucose to 50 mm final concentration and an additional 5 min stirring, and (c) after the addition of KCl to 50 mm final and an additional 3 min of incubation. Fluorescence intensity at excitation wavelengths 405 and 485 nm was measured in triplicate for each sample at a constant emission wavelength of 508 nm in a SPEX Fluorolog-3–21 fluorometer. Calibration of fluorescence with pH was carried out for each strain in each experiment as described (31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). Calibration curves were constructed for every strain and included buffers titrated to 5, 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.5.Vacuolar pH measurement used the pH-sensitive ratiometric dye BCECF-AM as described (11Plant P.J. Manolson M.F. Grinstein S. Demaurex N. J. Biol. Chem. 1999; 274: 37270-37279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Ali R. Brett C.L. Mukherjee S. Rao R. J. Biol. Chem. 2004; 279: 4498-4506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). Briefly, cells were grown to log phase in YEPD, pH 5 media, and collected by centrifugation, and 100 mg of cells mass were resuspended in 100 μl of the same medium. For experiments at extracellular pH 7.5, cells were washed and resuspended in YEP pH 7.5. Cells were incubated in 50 μm BCECF-AM for 30 min at 30 °C with shaking. The cells were then washed 2–3 times with YEP, pH 5 or 7.5 (depending on the experiment), to remove the dye and resuspended at the same density in the same media. For vacuolar pH measurements, 25 μl of cell suspension was added to 2 ml of 1 mm MES/TEA, pH 5, or MOPS/TEA, pH 7.5, and the mixture was stirred in a cuvette. Response of vacuolar pH was recorded at 30 °C with stirring as described above, except that fluorescence intensity at excitation wavelengths 450 and 490 nm was measured in triplicate for each sample at a constant emission wavelength of 535 nm. Calibration of fluorescence with pH was carried out for each strain as described (30Ali R. Brett C.L. Mukherjee S. Rao R. J. Biol. Chem. 2004; 279: 4498-4506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 31Brett C.L. Tukaye D.N. Mukherjee S. Rao R. Mol. Biol. Cell. 2005; 16: 1396-1405Crossref PubMed Scopus (236) Google Scholar). Values are expressed as the mean ± S.E. for each condition, and statistic significance was taken as p ≤ 0.05 of identical means in the two-sample t test for paired means.Proton Export—Proton pumping across the plasma membrane was measured by recording extracellular pH changes with a pH meter (Beckman Selection 2000) (43Pena A. Arch Biochem. Biophys. 1975; 167: 397-409Crossref PubMed Scopus (81) Google Scholar). Cells were grown to log phase in YEPD, pH 5 medium, washed, and resuspended as described above. 25 μl of the cell suspension was incubated in 15 ml of buffer (1 mm MES/TEA, pH 5), and the extracellular pH was monitored for 16 min at 30 °C with shaking. Extracellular pH was recorded manually every 30 s, and glucose (to 40 mm final concentration) and KCl (to 40 mm final) were added after 3 and 8 min of incubation, respectively.Isolation of Plasma Membrane—Preparation of total extract and plasma membrane from yeast was done according to Panaretou and Piper (44Panaretou B. Piper P. Methods Mol. Biol. 2006; 313: 27-32PubMed Google Scholar) using wild-type and vma cells from the SF838-1Dα strain background. One liter of cells in log phase (absorbance 0.6–0.8) were harvested by centrifugation (5 min, 5000 × g), then resuspended in 80 ml of 0.4 m sucrose in buffer A (25 mm imidazole-HCl, pH 7, containing 1 μg/ml pepstatin A, 2 μg/ml chymostatin, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 1 μg/ml leupeptin). The cell suspension was divided between two 50-ml centrifuge tubes and pelleted by centrifugation (5000 × g for 10 min). Glass beads were added to twice the pellet volume of cells followed by enough 0.4 m sucrose in buffer A to cover the cells and glass beads. Cells were subjected to vortex mixing for three periods of 2.5 min each, with pauses of 30 s on ice. To pellet unbroken cells and glass beads, suspension was diluted three times in 0.4 m sucrose buffer A and centrifuged at 530 × g for 20 min. Supernatant from this step was then centrifuged at 22,000 × g for 30 min. The pellet was resuspended in 2 ml of buffer A by gentle vortexing (30 s), and 1-ml aliquots of resuspended membranes were loaded onto a discontinuous sucrose gradient (prepared by overlaying three 4-ml layers of 2.25, 1.65, and 1.1 m sucrose, all in buffer A) and centrifuged overnight (14 h) at 80,000 × g in the Beckman SW40Ti rotor. Membranes banding at the 2.25/1.65 m are enriched for plasma membrane. This band was collected, diluted 4 times with buffer A, and pelleted at 30,000 × g for 40 min. The pellet was resuspended in buffer B (25 mm imidazole-HCl, pH 7, 50% (v/v) glycerol) containing the same protease inhibitors and stored at -20 °C.Plasma membrane H+-ATPase activity was assayed in membrane fractions with or without a 5-min preincubation with 50 μm vanadate at room temperature with gentle shaking. ATPase activity was determined using the coupled enzyme assay (45Lotscher H.R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4140-4143Crossref PubMed Scopus (99) Google Scholar), and vanadate-sensitive activity is reported. Typically 80% of the total ATPase activity in the plasma membrane fraction was inhibited by vanadate, and there was very little concanamycin A-sensitive ATPase activity, suggesting minimal contamination with vacuoles or endosomes. Protein concentrations were determined by the Lowry method (46Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar).For assessment of protein levels of Pma1p, Pep12p, and alkaline phosphatase, plasma membrane fractions were solubilized, separated by SDS-PAGE, and transferred to nitrocellulose as described (42Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar), except that a portion of the samples to be blotted for Pma1p were solubilized in 100 mm Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, and 10% β-mercaptoethanol rather than cracking buffer. Blots were probed with mouse monoclonal antibodies against Pep12p and alkaline phosphatase and rabbit polyclonal antibodies against Pma1p. Western blot signals were revealed with alkaline phosphatase-conjugated second antibody (anti-mouse or anti-rabbit as appropriate), and the signals were quantitated using ImageJ (National Institutes of Health). Equal protein concentrations were loaded for each strain; 40 μg was loaded for detection of Pma1p, and 30 μg was loaded for detection of alkaline phosphatase and Pep12p.Immunolocalization—Immunofluorescence staining of cells was done essentially as described (47Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). For Pma1p staining, cells were grown to a density of 1 A600/ml, rapidly fixed by direct addition of formaldehyde for 30 min, and then harvested and fixed overnight with 4.4% formaldehyde in 0.1 m potassium phosphate, pH 6.5. Before staining, fixed cells were permeabilized with 2% SDS. They were then stained with mouse monoclonal anti-Pma1p (Abcam, 40B7 antibody) followed by incubation with goat anti-mouse IgG conjugated to Alexa Fluor 488 (Invitrogen). Stained cells were visualized under fluorescein fluorescence optics on a Zeiss Axioplan 2 fluorescence microscope.RESULTSvma Mutants Poorly Regulate Both Vacuolar and Cytosolic pH—One of the defining phenotypes of vma mutants is their sensitivity to external pH, but relatively little is known about how cytosolic and vacuolar pH are regulated in response to changes in extracellular pH, even in wild-type cells. Ratiometric fluorescent methods in living yeast cells have allowed measurements of vacuolar and cytosolic pH in cells grown under different conditions and in mutants lacking proteins implicated in pH homeostasis. We introduced the fluorescent dye BCECF-AM, which measures vacuolar pH in yeast (11Plant P.J. Manolson M.F. Grinstein S. Demaurex N. J. Biol. Chem. 1999; 274: 37270-37279Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 30Ali R. Brett C.L. Mukherjee S. Rao R. J. Biol. Chem. 2004; 279: 4498-4506Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar), or yeast pHluorin, a green fluorescent protein analog that measures cytoso
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