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• W2000977339 abstract "Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Δ strain also exhibits a severe imbalance in intracellular Ca2+ homeostasis when grown under these conditions. In the present study, we examined how the pgm2Δ mutation alters yeast Ca2+ homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca2+ uptake in wild-type cells, whereas Ca2+ uptake increased 8-fold in the pgm2Δ mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca2+-ATPase Pmc1p, suppressed the Ca2+-related phenotypes observed in the pgm2Δ strain. This suggests that excessive vacuolar Ca2+ uptake is tightly coupled to these defects in Ca2+ homeostasis. An in vitro assay designed to measure Ca2+ sequestration into intracellular compartments confirmed that the pgm2Δ mutant contained a higher level of Pmc1p-dependent Ca2+ transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca2+ uptake also coincided with a large induction of the unfolded protein response in the pgm2Δ mutant, suggesting that Ca2+ uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca2+ uptake and accumulation previously shown to be associated with the pgm2Δ mutation are due to a severe imbalance in the distribution of cellular Ca2+ into different intracellular compartments. Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2Δ strain also exhibits a severe imbalance in intracellular Ca2+ homeostasis when grown under these conditions. In the present study, we examined how the pgm2Δ mutation alters yeast Ca2+ homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca2+ uptake in wild-type cells, whereas Ca2+ uptake increased 8-fold in the pgm2Δ mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca2+-ATPase Pmc1p, suppressed the Ca2+-related phenotypes observed in the pgm2Δ strain. This suggests that excessive vacuolar Ca2+ uptake is tightly coupled to these defects in Ca2+ homeostasis. An in vitro assay designed to measure Ca2+ sequestration into intracellular compartments confirmed that the pgm2Δ mutant contained a higher level of Pmc1p-dependent Ca2+ transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca2+ uptake also coincided with a large induction of the unfolded protein response in the pgm2Δ mutant, suggesting that Ca2+ uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca2+ uptake and accumulation previously shown to be associated with the pgm2Δ mutation are due to a severe imbalance in the distribution of cellular Ca2+ into different intracellular compartments. The regulation of intracellular Ca2+ homeostasis in eukaryotic cells is a remarkably intricate process. Ca2+ transport across the plasma membrane and its intracellular sequestration is tightly regulated such that the resting cytosolic Ca2+ concentration is maintained in a range of 50–200 nm (1Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar, 2Nakajima-Shimada J. Iida H. Tsuji F.I. Anraku Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6878-6882Crossref PubMed Scopus (100) Google Scholar, 3Miseta A. Fu L. Kellermayer R. Buckley J. Bedwell D.M. J. Biol. Chem. 1999; 274: 5939-5947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 4Miseta A. Kellermayer R. Aiello D.P. Fu L. Bedwell D.M. FEBS Lett. 1999; 451: 132-136Crossref PubMed Scopus (120) Google Scholar). Small variations in cytosolic Ca2+ that occur in response to a number of stimuli are sufficient to activate a variety of Ca2+-sensing proteins, such as calmodulin and calcineurin. This then leads to the induction of various downstream signal transduction pathways (5Putney Jr., J.W. Adv. Second Messenger Phosphoprotein Res. 1992; 26: 143-160PubMed Google Scholar). Equally as important, the Ca2+ concentrations within the lumen of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; PGM, phosphoglucomutase; wt, wild-type strain; UPR, unfolded protein response; Glc-6-P, glucose 6-phosphate; Glc-1-P, glucose 1-phosphate; YP, yeast extract/peptone; SM, synthetic medium; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol.1The abbreviations used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; PGM, phosphoglucomutase; wt, wild-type strain; UPR, unfolded protein response; Glc-6-P, glucose 6-phosphate; Glc-1-P, glucose 1-phosphate; YP, yeast extract/peptone; SM, synthetic medium; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol. and Golgi apparatus are carefully maintained to ensure the proper folding and processing of proteins transported through the secretory pathway (6Michalak M. Robert Parker J.M. Opas M. Cell Calcium. 2002; 32: 269-278Crossref PubMed Scopus (366) Google Scholar). In the budding yeast Saccharomyces cerevisiae, the vacuole is the major cellular Ca2+ storage compartment and contains >95% of total cellular Ca2+ (7Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar). This large store of Ca2+ is maintained through the action of two transporters, the Ca2+-ATPase Pmc1p and the Ca2+/H+ exchanger Vcx1p (8Cunningham K.W. Fink G.R. J. Exp. Biol. 1994; 196: 157-166Crossref PubMed Google Scholar, 9Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (129) Google Scholar). Once thought to be relatively static by virtue of its association with inorganic polyphosphate (7Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar), the vacuolar Ca2+ store has recently been suggested to be more dynamic in nature. The recently identified yeast transient receptor potential channel homologue, Yvc1p, was shown to localize to the vacuolar membrane and mediate Ca2+ efflux out of the vacuole (10Palmer C.P. Zhou X.L. Lin J. Loukin S.H. Kung C. Saimi Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7801-7805Crossref PubMed Scopus (171) Google Scholar). Additional reports have shown that vacuolar Ca2+ efflux by Yvc1p can be specifically induced by hypotonic shock, which may be mediated by a mechano-sensitive mechanism (11Denis V. Cyert M.S. J. Cell Biol. 2002; 156: 29-34Crossref PubMed Scopus (236) Google Scholar, 12Zhou X.L. Batiza A.F. Loukin S.H. Palmer C.P. Kung C. Saimi Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 7105-7110Crossref PubMed Scopus (117) Google Scholar). In addition to the vacuole, the ER and Golgi apparatus are also important for maintaining proper intracellular Ca2+ homeostasis in yeast. The transporters responsible for maintaining proper Ca2+ levels in the secretory pathway include the Ca2+-ATPases Pmr1p (3Miseta A. Fu L. Kellermayer R. Buckley J. Bedwell D.M. J. Biol. Chem. 1999; 274: 5939-5947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 13Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J.I. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 14Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar, 15Halachmi D. Eilam Y. FEBS Lett. 1996; 392: 194-200Crossref PubMed Scopus (60) Google Scholar, 16Marchi V. Sorin A. Wei Y. Rao R. FEBS Lett. 1999; 454: 181-186Crossref PubMed Scopus (55) Google Scholar) and Cod1p/Spf1p (17Suzuki C. Shimma Y.I. Mol. Microbiol. 1999; 32: 813-823Crossref PubMed Scopus (75) Google Scholar, 18Cronin S.R. Khoury A. Ferry D.K. Hampton R.Y. J. Cell Biol. 2000; 148: 915-924Crossref PubMed Scopus (90) Google Scholar, 19Cronin S.R. Rao R. Hampton R.Y. J. Cell Biol. 2002; 157: 1017-1028Crossref PubMed Scopus (131) Google Scholar, 20Vashist S. Frank C.G. Jakob C.A. Ng D.T. Mol. Biol. Cell. 2002; 13: 3955-3966Crossref PubMed Scopus (78) Google Scholar). Pmr1p is localized primarily to the Golgi apparatus, where it plays an essential role in maintaining the lumenal Ca2+ concentration required for the proper glycosylation and processing of proteins in this compartment (13Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J.I. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 14Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar). The loss of Pmr1p results in a number of alterations in Ca2+ homeostasis, including an increased rate of cellular Ca2+ uptake from the extracellular environment and a greater sensitivity to elevated extracellular Ca2+ levels (15Halachmi D. Eilam Y. FEBS Lett. 1996; 392: 194-200Crossref PubMed Scopus (60) Google Scholar). The elevated Ca2+ uptake observed in the pmr1Δ mutant is mediated by the MID1 and CCH1 gene products and is reminiscent of the mammalian capacitative Ca2+ entry (CCE) response (21Locke E.G. Bonilla M. Liang L. Takita Y. Cunningham K.W. Mol. Cell. Biol. 2000; 20: 6686-6694Crossref PubMed Scopus (180) Google Scholar). The depletion of secretory pathway Ca2+ stores caused by the pmr1Δ mutation also leads to improper folding and processing of proteins that transit through the ER and Golgi (14Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar, 22Dürr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (345) Google Scholar). It was recently reported that this lumenal Ca2+ depletion induces the unfolded protein response (UPR) (23Bonilla M. Nastase K.K. Cunningham K.W. EMBO J. 2002; 21: 2343-2353Crossref PubMed Scopus (211) Google Scholar). The UPR is activated by the presence of unfolded proteins in the ER and results in the increased expression of molecular chaperones that aid in protein folding in this compartment (24Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (363) Google Scholar). This increased expression is mediated by the transcription factor Hac1p/Ern4p (25Mori K. Sant A. Kohno K. Normington K. Gething M.J. Sambrook J.F. EMBO J. 1992; 11: 2583-2593Crossref PubMed Scopus (309) Google Scholar, 26Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (303) Google Scholar, 27Kawahara T. Yanagi H. Yura T. Mori K. Mol. Biol. Cell. 1997; 8: 1845-1862Crossref PubMed Scopus (232) Google Scholar). Recent evidence suggests that some products of carbohydrate metabolism also influence intracellular Ca2+ homeostasis in yeast cells. In particular, the sugar phosphates Glc-6-P and Glc-1-P have been proposed to play a role in modulating intracellular Ca2+ homeostasis (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The enzyme phosphoglucomutase (PGM) interconverts Glc-1-P and Glc-6-P and is required for the metabolism of galactose. Yeast strains lacking the major isoform of this enzyme (pgm2Δ) accumulate a high level of intracellular Glc-1-P when galactose is utilized as the carbon source due to a metabolic bottleneck in the conversion of Glc-1-P to Glc-6-P. This strain also exhibits alterations in cellular Ca2+ homeostasis under these conditions, including dramatically increased Ca2+ uptake and accumulation, sensitivity to high extracellular Ca2+ concentrations, and increased sensitivity to the calcineurin inhibitor cyclosporin A (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Most recently, it was demonstrated that the simultaneous overproduction of both Glc-1-P and Glc-6-P restored normal Ca2+ homeostasis, suggesting that the ratio of these glucose metabolites plays an important role in controlling this process (29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In the present study our results indicate that the pgm2Δ strain experiences elevated vacuolar Ca2+ uptake and reduced ER Ca2+ sequestration when grown with galactose as the carbon source. Furthermore, we show that these phenotypes are suppressed by the deletion of the MC1 gene. These results demonstrate that the relative levels of Glc-1-P and Glc-6-P play an important role in regulating the distribution of Ca2+ into different intracellular compartments. Strains and Plasmids Used—Yeast strain YDB0316 is a wild-type strain described previously (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The PGM2 gene in YDB0316 was disrupted by insertion of a previously described fragment containing the LEU2 gene (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) to generate YDB0473. To generate strains YDB0484 (pmc1Δ) and YDB0483 (pgm2Δ/pmc1Δ), the PMC1 gene was disrupted using standard methods (30Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar) in YDB0316 (wt) and YDB0473 (pgm2Δ) using the S. cerevisiae HIS3 open reading frame that was PCR-amplified from pRS313 (31Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The primers used to generate this product were DB1158 (5′-TAGAAAAGTG GTTCTAAAAA AAAAAAACTG TGTGCGTAAC AAAAAAAATA GCTTGGTGAG CGCTAGGAGT-3′) and DB1159 (5′-CAATTTTGAA AATATAACTA TTACACACAT CTTTTCATTT GGTCACTTAC CTGTTCGTAT ACATACTTAC TGAC-3′). To generate strains YDB0475 (vcx1Δ) and YDB0474 (pgm2Δ/vcx1Δ), the VCX1 gene was disrupted using standard methods (30Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar) in YDB0316 (wt) and YDB0473 (pgm2Δ) with a previously described knockout construct (3Miseta A. Fu L. Kellermayer R. Buckley J. Bedwell D.M. J. Biol. Chem. 1999; 274: 5939-5947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). For each strain, gene disruptions were confirmed by PCR, Southern blotting, and/or PGM enzymatic assays (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Finally, strain BY4741 (wild type) and the isogenic yvc1Δ strain BY4741 (clone 1863) were purchased form Open Biosystems. A pgm2::LEU2 disruption construct (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) was used to disrupt the PGM2 gene in BY4741 to generate the isogenic pgm2Δ strain YDB0626 and in BY4741 (clone 1863) to generate the isogenic pgm2Δ/yvc1Δ strain YDB0627. The induction of UPR signaling was monitored using the reporter plasmid pMCZ-Y (a kind gift from Kazutoshi Mori) that contains lacZ under the control of the yeast CYC1 promoter containing an unfolded protein response element (26Mori K. Kawahara T. Yoshida H. Yanagi H. Yura T. Genes Cells. 1996; 1: 803-817Crossref PubMed Scopus (303) Google Scholar). Culture Media—Bacterial strains used for cloning and plasmid maintenance were grown in standard media as described (32Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). Similarly, yeast media were prepared as described (30Adams A. Gottschling D.E. Kaiser C.A. Stearns T. Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997Google Scholar). Yeast extract/peptone (YP) medium and synthetic medium (SM) were supplemented with 2% glucose (dextrose) (YPD or SMD) or 2% galactose (YPGal or SMGal). YPD and YPGal media were routinely buffered to pH 5.5 with 40 mm MES-Tris. To prepare membranes for in vitro45Ca2+ uptake assays, cells were grown in Yeast Mito medium containing 6.7 g of yeast nitrogen base (Difco) and 0.3% yeast extract (Difco)/liter in the presence of 2% glucose or 2% galactose (YMMG) (33Koh J.Y. Hajek P. Bedwell D.M. Mol. Cell. Biol. 2001; 21: 7576-7586Crossref PubMed Scopus (14) Google Scholar). Liquid cultures were grown for a minimum of five generations to ≤1.0 A600 units/ml before harvesting. Measurement of Whole Cell Ca2+Uptake, Total Cellular Ca2+, and Exchangeable Ca2+Pools—Whole cell Ca2+ uptake measurements were performed as described previously (15Halachmi D. Eilam Y. FEBS Lett. 1996; 392: 194-200Crossref PubMed Scopus (60) Google Scholar, 29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Measurement of total cell Ca2+ by flame photometry was also carried out as previously described (3Miseta A. Fu L. Kellermayer R. Buckley J. Bedwell D.M. J. Biol. Chem. 1999; 274: 5939-5947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cells for Ca2+ exchange experiments were grown in YPGal medium to a density of 0.05 A600. The medium was then supplemented with 45Ca2+, and growth was continued to a cell density of 0.5–1 A600/ml. The cells were then harvested by centrifugation at 4000 × g for 5 min, washed, and resuspended in fresh YPGal supplemented with 50 mm CaCl2. At the indicated times, aliquots were removed, filtered, washed, and processed for scintillation counting as previously described (3Miseta A. Fu L. Kellermayer R. Buckley J. Bedwell D.M. J. Biol. Chem. 1999; 274: 5939-5947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). β-Galactosidase Assays—Yeast strains transformed with plasmid pMCZ-Y were grown to mid-log phase in SMGal medium as indicated. Cells were then harvested by centrifugation and permeabilized by repeated freeze-thawing in liquid nitrogen (34Schneider S. Buchert M. Hovens C.M. Biotechniques. 1996; 20: 960-962Crossref PubMed Scopus (72) Google Scholar). β-Galactosidase activity was assayed using the colorimetric substrate 2-nitrophenyl-β-d-galactopyranoside according to a previously described protocol (35Guarente L. Methods Enzymol. 1983; 101: 181-191Crossref PubMed Scopus (871) Google Scholar). To examine the affect of extracellular Ca2+ and dithiothreitol (DTT), cells were treated with 20 mm CaCl2 or 5 mm DTT, respectively, for 4 h before harvest. Units of β-galactosidase activity are defined as the absorbance at 420 nm × 103/min/A600 unit of cells. Isolation and Subcellular Fractionation of Total Cell Membranes— Total cell membranes were isolated from yeast using a protocol based on previous publications (14Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar, 16Marchi V. Sorin A. Wei Y. Rao R. FEBS Lett. 1999; 454: 181-186Crossref PubMed Scopus (55) Google Scholar, 36Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Yeast strains were grown at 30 °Cin the indicated media to 0.6–0.8 A600 units/ml. Before harvest, NaN3 was added to a final concentration of 10 mm, and the cells were rapidly chilled in ice water for 10 min. Cells were then harvested by centrifugation at 4 °C for 5 min at 6000 rpm (6000 × g) in a Sorvall SLA-3000 rotor. Cell pellets were resuspended in spheroplasting buffer (1.4 m sorbitol, 50 mm Tris-HCl, pH 7.5, 10 mm NaN3, 40 mm β-mercaptoethanol, 0.5 mm phenylmethylsulfonyl fluoride (Sigma), and 0.3 mg/ml yeast lytic enzyme (ICN). Cells were converted to spheroplasts by incubation for 30–45 min at 37 °C. The spheroplasts were then harvested by centrifugation at 4 °C for 5 min at 2500 rpm (750 × g) in a Sorvall SS-34 rotor and gently resuspended in lysis buffer (0.3 m sorbitol, 20 mm triethanolamine acetate, pH 7.2, 1 mm EDTA, and protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 2 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin; all from Sigma). The spheroplasts were then disrupted mechanically using 20 strokes of a Wheaton Type A (tight) Dounce homogenizer. The cell lysates were then cleared of unbroken cells by centrifugation twice at 4 °C for 5 min at 2000 rpm (450 × g) in a Sorvall SS-34 rotor. To fractionate membranes, 3 ml of lysate was loaded onto a 10-step (18–54% in 4% increments) sucrose gradient in 10 mm HEPES, pH 7.5, 1 mm MgCl2. Gradients were centrifuged at 4 °C for 2 h at 27,000 rpm in a Beckman SW-28 rotor. After centrifugation, gradient fractions were collected manually in 3-ml aliquots from top to bottom. Individual fractions from multiple gradients were then pooled and stored at –80 °C in 1-ml aliquots. Protein concentrations in each gradient fraction were determined by the method of Bradford using bovine serum albumin to generate a standard curve (37Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Ca2+Uptake Assays in Isolated Membranes—To assay for Ca2+ transport activity in sucrose gradient fractions, 0.7 ml of O-Buffer (10 mm HEPES-NaOH, pH 6.7, 150 mm KCl, 5 mm MgCl2, 0.5 mm ATP (pre-buffered to pH 6.7), 5 mm NaN3, 0.5 μCi/ml 45CaCl2 (9.6 mCi/mg)) was added to 0.3 ml of gradient fraction and incubated for 12 min at 25 °C (36Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). The entire 1-ml sample was then collected by filtration through a pre-washed nitrocellulose filter (Millipore, HAWP02500) and washed twice with 5 ml of ice-cold wash buffer (10 mm HEPES-NaOH, pH 7.5, 150 mm KCl) (36Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Membrane-sequestered 45Ca2+ was then determined by liquid scintillation counting. Where indicated, Ca2+ transport by Vcx1p was inhibited by the addition of 25 μm carbonyl cyanide m-chlorophenylhydrazone (Sigma). To test for the effect of sugar phosphates on Ca2+ uptake, pre-buffered (pH 6.7) glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, or mannose 6-phosphate was added to the O-Buffer mix at the indicated concentrations as indicated. To test for the effect on Ca2+ uptake by free phosphate, 5 mm sodium phosphate was added to the O-Buffer mix as indicated. A Sustained Increase in Cellular Ca2+Uptake Occurs upon Carbon Source Shift—Previous studies show that the pgm2Δ mutation causes increased cellular Ca2+ uptake and accumulation when galactose is utilized as the carbon source (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 38Tökés-Füzesi M. Bedwell D.M. Repa I. Sipos K. Sümegi B. Rab A. Miseta A. Mol. Microbiol. 2002; 44: 1299-1308Crossref PubMed Scopus (32) Google Scholar). This increase in Ca2+ uptake coincides with a large increase in the level of intracellular Glc-1-P and a resulting alteration in the Glc-1-P/Glc-6-P ratio. It has been proposed that the altered ratio of these glucose metabolites induces a mechanism that normally couples their relative abundance to intracellular Ca2+ homeostasis (28Fu L. Miseta A. Hunton D. Marchase R.B. Bedwell D.M. J. Biol. Chem. 2000; 275: 5431-5440Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 29Aiello D.P. Fu L. Miseta A. Bedwell D.M. J. Biol. Chem. 2002; 277: 45751-45758Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 38Tökés-Füzesi M. Bedwell D.M. Repa I. Sipos K. Sümegi B. Rab A. Miseta A. Mol. Microbiol. 2002; 44: 1299-1308Crossref PubMed Scopus (32) Google Scholar). This model predicts that wild-type cells shifted from the metabolism of glucose to galactose should also experience a significant increase in Ca2+ uptake. To test this hypothesis, we measured 45Ca2+ uptake in the wild-type and pgm2Δ strains after a carbon source shift from glucose to galactose (Fig. 1). The rates of 45Ca2+ uptake in the wild-type and pgm2Δ strains were similar before the shift. We found that the rate of 45Ca2+ uptake in the wild-type strain increased 2-fold within 6 h of re-suspending the cells in medium containing galactose as the carbon source and remained constant thereafter. In contrast, the pgm2Δ mutant exhibited a 4-fold increase in 45Ca2+ uptake 6 h after the shift, and uptake increased to 8-fold higher than the pre-shift level after 12 h. These results support the hypothesis that a component of the normal adaptive response of yeast cells to the utilization of galactose as the carbon source is an increase in Ca2+ transport across the plasma membrane. Furthermore, these findings suggest that the defect in Ca2+ homeostasis observed in the pgm2Δ strain may result from an inability to properly regulate this normal physiological response due to the overproduction of Glc-1-P in this strain. Disruption of the PMC1 Gene Partially Suppresses Phenotypes Associated With the pgm2Δ Mutation—The vacuole serves as the major Ca2+ storage compartment in yeast (7Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar, 39Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (363) Google Scholar). Given the high rate of cellular Ca2+ uptake observed when the pgm2Δ strain utilizes galactose as carbon source, we reasoned that efficient vacuolar Ca2+ sequestration might be critical for the viability of this strain. To determine the consequences of reducing the level of vacuolar Ca2+ sequestration in the pgm2Δ mutant, we disrupted the genes encoding the vacuolar Ca2+ ATPase Pmc1p and the vacuolar Ca2+/H+ exchanger Vcx1p both independently and together in the pgm2Δ strain. The loss of Vcx1p activity had no effect on the growth of the pgm2Δ mutant (data not shown). Surprisingly, the pmc1Δ mutation partially suppressed the slow growth and Ca2+ sensitivity phenotypes of the pgm2Δ mutant on both standard YPGal plates and on YPGal plates supplemented with 50 mm CaCl2 (Fig. 2, A and B). Combining both the pmc1Δ and vcx1Δ mutations together in the pgm2Δ mutant provided no greater suppression than that observed for the pmc1Δ mutation alone (data not shown). These results suggest that vacuolar Ca2+ sequestration mediated by Pmc1p may be detrimental to growth of the pgm2Δ strain. To determine whether this partial suppression of the pgm2Δ growth defect by the pmc1Δ mutation correlated with changes in cellular Ca2+ homeostasis, we next measured the level of total cellular Ca2+ accumulation in strains grown with galactose as the carbon source (Fig. 3). As reported previously, the pgm2Δ mutant exhibited a 4-fold higher level of total cell Ca2+ than the wild-type strain. Consistent with the observed growth phenotypes, the pgm2Δ/vcx1Δ double mutant had a level of total cell Ca2+ that was similar to the level measured in the pgm2Δ mutant. In contrast, the pgm2Δ/pmc1Δ strain had a level of total cell Ca2+ that was only 1.5-fold higher than that found in the wild-type strain. These results demonstrate that the introduction of the pmc1Δ mutation (and presumably a reduction in vacuolar Ca2+ sequestration) coincides with the suppression of the Ca2+ homeostasis phenotypes observed in the pgm2Δ strain. Intracellular Ca2+ in yeast has been shown to exist in two distinct states termed the exchangeable and non-exchangeable pools (7Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar). The exchangeable pool was so named because it was rapidly released from yeast cells when they were introduced into medium containing a limiting level of extracellular Ca2+. In contrast, the non-exchangeable pool of cellular Ca2+ was released from the cell under these conditions at a much slower rate. Until recently, the non-exchangeable pool of cellular Ca2+ was thought to be largely synonymous with the vacuole pool, where it is thought to exist in complex with polyphosphate (7Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar). In contrast, the exchangeable pool was thought to represent Ca2+ located primarily within the ER and Golgi compartments. Thus, our finding that the exchangeable Ca2+ pool in the pgm2Δ strain was not reduced when grown with galactose as the carbon source suggested that the Ca2+ level was not depleted in the compartments of the secretory pathway. However, Cyert and Denis (11Denis V. Cyert M.S. J. Cell Biol. 2002; 156: 29-34Crossref PubMed Scopus (236) Google Scholar) recently found that a fraction of the vacuolar Ca2+ store is more dynamic than previously thought and can be rapidly released" @default.
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• W2000977339 title "The Ca2+ Homeostasis Defects in a pgm2Δ Strain of Saccharomyces cerevisiae Are Caused by Excessive Vacuolar Ca2+ Uptake Mediated by the Ca2+-ATPase Pmc1p" @default.
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