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• W2007459574 abstract "The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range. In this study, we examined how cellular Ca2+ homeostasis is maintained in a vps33Δ vacuolar biogenesis mutant. We found that growth of the vps33Δ strain was sensitive to high or low extracellular Ca2+. This strain could not properly regulate cytosolic Ca2+ levels and was able to retain only a small fraction of its total cellular Ca2+ in a nonexchangeable intracellular pool. Surprisingly, thevps33Δ strain contained more total cellular Ca2+ than the wild type strain. Because most cellular Ca2+ is normally found within the vacuole, this suggested that other intracellular compartments compensated for the reduced capacity to store Ca2+ within the vacuole of this strain. To test this hypothesis, we examined the contribution of the Golgi-localized Ca2+ ATPase Pmr1p in the maintenance of cellular Ca2+ homeostasis. We found that avps33Δ/pmr1Δ strain was hypersensitive to high extracellular Ca2+. In addition, certain combinations of mutations effecting both vacuolar and Golgi Ca2+ transport resulted in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca2+homeostasis when vacuolar biogenesis is compromised. The vacuole is the major site of intracellular Ca2+ storage in yeast and functions to maintain cytosolic Ca2+ levels within a narrow physiological range. In this study, we examined how cellular Ca2+ homeostasis is maintained in a vps33Δ vacuolar biogenesis mutant. We found that growth of the vps33Δ strain was sensitive to high or low extracellular Ca2+. This strain could not properly regulate cytosolic Ca2+ levels and was able to retain only a small fraction of its total cellular Ca2+ in a nonexchangeable intracellular pool. Surprisingly, thevps33Δ strain contained more total cellular Ca2+ than the wild type strain. Because most cellular Ca2+ is normally found within the vacuole, this suggested that other intracellular compartments compensated for the reduced capacity to store Ca2+ within the vacuole of this strain. To test this hypothesis, we examined the contribution of the Golgi-localized Ca2+ ATPase Pmr1p in the maintenance of cellular Ca2+ homeostasis. We found that avps33Δ/pmr1Δ strain was hypersensitive to high extracellular Ca2+. In addition, certain combinations of mutations effecting both vacuolar and Golgi Ca2+ transport resulted in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca2+homeostasis when vacuolar biogenesis is compromised. cyclosporin A kilobase 4-morpholineethanesulfonic acid light emission maximum light emission Like all eukaryotes, the yeast Saccharomyces cerevisiaenormally maintains a resting cytosolic Ca2+ concentration of 50–200 nm (1Batiza A.F. Schulz T. Masson P.H. J. Biol. Chem. 1996; 271: 23357-23362Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 2Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar, 3Nakajima-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). This tight regulation of intracellular Ca2+ is required to control the complex signaling pathways mediated by cytosolic Ca2+-sensing proteins such as calmodulin. Remarkably, yeast cells can maintain intracellular Ca2+ homeostasis in the presence of environmental Ca2+ concentrations ranging from <1 μm to >100 mm (4Cunningham K.W. Fink G.R. J. Exp. Biol. 1994; 196: 157-166Crossref PubMed Google Scholar). The vacuole is thought to play a key role in maintaining Ca2+ tolerance over this wide range because it contains >90% of the total cellular Ca2+ (5Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar, 6Eilam Y. Lavi H. Grossowicz N. J. Gen. Microbiol. 1985; 131: 623-629Google Scholar). Accordingly, many different vacuolar mutations result in an inability to grow in the presence of high concentrations of extracellular Ca2+ (7Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (364) Google Scholar, 8Dunn T. Gable K. Beeler T. J. Biol. Chem. 1994; 269: 7273-7278Abstract Full Text PDF PubMed Google Scholar, 9Garrett-Engele P. Moilanen B. Cyert M.S. Mol. Cell. Biol. 1995; 15: 4103-4114Crossref PubMed Scopus (164) Google Scholar, 10Hemenway C.S. Dolinski K. Cardenas M.E. Hiller M.A. Jones E.W. Heitman J. Genetics. 1995; 141: 833-844Crossref PubMed Google Scholar, 11Kitamoto K. Yoshizawa K. Ohsumi Y. Anraku Y. J. Bacteriol. 1988; 170: 2687-2691Crossref PubMed Google Scholar, 12Ohya Y. Miyamoto S. Ohsumi Y. Anraku Y. J. Bacteriol. 1986; 165: 28-33Crossref PubMed Google Scholar, 13Ohya Y. Umemoto N. Tanida I. Ohta A. Iida H. Anraku Y. J. Biol. Chem. 1991; 266: 13971-13977Abstract Full Text PDF PubMed Google Scholar). Currently, two Ca2+ transporters have been described which act to sequester Ca2+ in the vacuole. The first of these is the vacuolar Ca2+ ATPase encoded by the PMC1gene, a homolog of the mammalian PMCA plasma membrane family of Ca2+ ATPases. The loss of Pmc1p results in an inability to grow in the presence of high environmental Ca2+ (7Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (364) Google Scholar). The second protein known to be involved in vacuolar Ca2+ transport is the H+/Ca2+ exchanger encoded by theVCX1 (HUM1) gene (14Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (381) Google Scholar, 15Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (129) Google Scholar). Although mutants that do not express Vcx1p show little or no decrease in Ca2+tolerance, the combination of pmc1Δ and vcx1Δmutations leads to a more severe Ca2+-sensitive phenotype than the loss of either transporter alone. Both the expression and function of these two vacuolar Ca2+ transporters are regulated by calcineurin, a highly conserved protein phosphatase that is activated by Ca2+/calmodulin. As in mammalian cells, the activation of yeast calcineurin can be blocked by the immunosupressant drugs cyclosporin A (CsA)1and FK506 (16Breuder T. Hemenway C.S. Movva N.R. Cardenas M.E. Heitman J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5372-5376Crossref PubMed Scopus (125) Google Scholar, 17Foor F. Parent S.A. Morin N. Dahl A.M. Ramadan N. Chrebet G. Bostian K.A. Nielsen J.B. Nature. 1992; 360: 682-684Crossref PubMed Scopus (181) Google Scholar). Although the functional relationship between these two vacuolar Ca2+ transporters is complex, it has been reported that calcineurin activation stimulates Pmc1p function and inhibits Vcx1p function (14Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (381) Google Scholar, 15Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (129) Google Scholar). Several other genes encoding potential Ca2+ ATPases have been identified within the yeast genome (18Catty P. Goffeau A. Biosci. Rep. 1996; 16: 75-85Crossref PubMed Scopus (21) Google Scholar); however, the only member of this group demonstrated to play a role in Ca2+ transport is encoded by the PMR1 gene. Pmr1p is related to the SERCA family of Ca2+ ATPases and has been shown to reside in the Golgi apparatus of S. cerevisiae (19Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (377) Google Scholar, 20Halachmi D. Eilam Y. FEBS Lett. 1996; 392: 194-200Crossref PubMed Scopus (60) Google Scholar, 21Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J.A. Davidow L.S. Mao J. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 22Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Although Pmr1p and Pmc1p both act to partition Ca2+ into distinct cellular compartments, their roles in Ca2+ homeostasis do not appear to be equivalent. First, cells lacking Pmc1p are sensitive to high environmental Ca2+, whereas cells lacking Pmr1p cannot grow under low Ca2+ conditions. In addition, the total cellular Ca2+ level in a pmc1Δ strain is 2–3-fold lower than normal, but the total cellular Ca2+ level in thepmr1Δ mutant is 4–5-fold higher than normal. These different phenotypes suggest that the vacuole and the Golgi apparatus normally carry out distinct roles in Ca2+ homeostasis. Genetic screens have identified at least 60 different genes involved in vacuolar protein localization (23Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar). Among these, the class C vacuolar protein sorting mutants (which include the vps11,vps16, vps18, and vps33 mutants) result in the most severe defects in vacuolar biogenesis. For example, strains carrying the vps33Δ mutation lack a morphologically distinguishable vacuole but instead accumulate small vesicular and Golgi-like structures (24Banta L.M. Robinson J.S. Klionsky D.J. Emr S.D. J. Cell Biol. 1988; 107: 1369-1383Crossref PubMed Scopus (299) Google Scholar, 25Banta L.M. Vida T.A. Herman P.K. Emr S.D. Mol. Cell. Biol. 1990; 10: 4638-4649Crossref PubMed Scopus (92) Google Scholar, 26Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (736) Google Scholar). These anomalous compartments may result from the inability to dock and/or fuse late transport vesicles from the biosynthetic, endocytic, and autophagic pathways with the vacuole (27Rieder S.E. Emr S.D. Mol. Biol. Cell. 1997; 8: 2307-2327Crossref PubMed Scopus (255) Google Scholar). A vps33Δ strain was also found to secrete >90% of soluble vacuolar proteins such as carboxypeptidase Y and to mislocalize nearly 50% of the vacuolar membrane protein α-mannosidase to the cell surface (24Banta L.M. Robinson J.S. Klionsky D.J. Emr S.D. J. Cell Biol. 1988; 107: 1369-1383Crossref PubMed Scopus (299) Google Scholar). In this study we asked how the severe defects in vacuolar biogenesis associated with the vps33Δ mutation affect cellular Ca2+ homeostasis. We found that the vps33Δstrain was sensitive to both high and low levels of environmental Ca2+ and was unable to regulate cytosolic Ca2+levels properly when exposed to a sudden, large increase in environmental Ca2+. Despite its defect in vacuolar biogenesis, we found that the vps33Δ strain contains more total cellular Ca2+ than a wild type strain. To determine whether other intracellular compartments compensate for reduced vacuolar Ca2+ storage, we examined whether the Golgi-localized Ca2+ ATPase Pmr1p plays a significant role in Ca2+ homeostasis in the vps33Δ strain. We found that PMR1 expression is elevated in thevps33Δ strain. We also found that avps33Δ/pmr1Δ strain is hypersensitive to high extracellular Ca2+, and the combination of certain mutations effecting both vacuolar and Golgi Ca2+ transport results in synthetic lethality. These results indicate that the Golgi apparatus plays a significant role in maintaining Ca2+homeostasis when vacuolar biogenesis is compromised. Strains used in this study are listed in TableI. The PMC1 andVCX1 genes were disrupted using the one-step gene replacement method (28Rothstein R.J. Methods Enzymol. 1983; 101: 202-209Crossref PubMed Scopus (2026) Google Scholar). A 1.62-kb fragment of the PMC1 gene was generated by PCR using wild type yeast genomic DNA as template. The forward primer used was 5′-ATCGGTACCA CTTGGATTGC AT-3′, and the reverse primer was 5′-CATGGATCCT GCCATCCTCA-3′. These primers contained KpnI and BamHI restriction endonuclease sites respectively (underlined). The PCR product was digested with KpnI and BamHI and cloned into a pBluescript II KS (+) plasmid. The 1.06-kb segment of thePMC1 gene was then removed by digestion withAflIII and EcoRI and replaced by theTRP1 gene taken from pJJ280 plasmid (29Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar). AKpnI/NotI fragment containing the disruptedpmc1Δ::TRP1 fragment was then used to transform yeast. Trp+ colonies were selected, and the correct gene replacement was confirmed by PCR.Table IYeast strains used in this studyStrainRelevant genotypeComplete genotypeSourceSEY6210Wild typeMATα, ura3–52, leu2–3 112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9S. EmrYDB224pmc1ΔMATα, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9, pmc1Δ::TRP1This studyYDB225vcx1ΔMATα, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9, vcx1Δ::URA3This studyYDB254pmc1Δ/vcx1ΔMATα, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9, pmc1Δ::TRP1, vcx1Δ::URA3This studyYDB279pmr1ΔMATa, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9?, pmr1Δ1::LEU2This studyLBY317vps33ΔMATα, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9, vps33Δ::HIS3S. EmrYDB282vps33Δ/pmr1ΔMATa, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, ade2; suc2-Δ9, vps33Δ::HIS3, pmr1-Δ1::LEU2This studyYDB255vps33Δ/pmc1Δ/vcx1ΔMATα, ura3–52, leu2–3,112, his3-Δ200, trp1-Δ901, lys2–801, suc2-Δ9, vps33Δ::HIS3, pmc1Δ::TRP1, vcx1Δ::URA3This study Open table in a new tab Similarly, a 2.04-kb fragment of the VCX1 gene was generated by PCR using genomic DNA as template. The forward primer used was 5′-CGTGGTACCT TGTCATCCTCAC-3′, and the reverse primer was 5′-GCTAGGATCC GCTAAAATAG G-3′. Again, these primers contained KpnI and BamHI restriction endonuclease sites, respectively (underlined). The fragment was digested with these enzymes and cloned into a pBluescript II KS (+) plasmid. A 1.56-kb fragment was removed from the VCX1 DNA by digestion withHincII and HindIII endonucleases and replaced with a fragment containing the URA3 gene obtained from pJJ244 (29Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar). A KpnI/BamHI fragment containing the disrupted vcx1Δ::URA3 fragment from this plasmid was used to transform yeast. The replacement of wild typeVCX1 was confirmed by PCR analysis. Other genetic manipulations were carried out by standard methods (30Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Bacterial strains were grown on standard media (31Miller J.H. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992Google Scholar). Yeast strains were maintained on YP medium containing 2%d-glucose (YPD) or synthetic minimal medium containing 2%d-glucose (SMD) and other supplements as required (30Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Growth media were routinely buffered with 40 mm MES-Tris, pH 5.5. EGTA was used to reduce the Ca2+ concentration of buffered media. Because YPD and SMD media contain divalent cations other than Ca2+, the effective concentrations of Mg2+, Mn2+, Fe2+, K+, and Na+ were considered when calculating free Ca2+ concentrations. Known quantities of CaCl2stock solutions were added, and the resulting free Ca2+concentrations were calculated based on the total concentration of Ca2+ as well as other cations, pH, and temperature of the medium. These calculations were done using the Maxchelator 1.2 program. 50–100 A 600 units of yeast growing in YPD supplemented with CaCl2 or EGTA were harvested by centrifugation at 5,000 × g for 5 min. The cell pellets were washed with fresh YP and transferred to microcentrifuge tubes whose mass had previously been determined gravimetrically to an accuracy of 0.1 mg on an analytical balance. The tubes were centrifuged at 15,000 × g for 5 min, and the supernatants were removed carefully. The tubes were then respun, and any remaining supernatant was again removed. The tubes containing the pellets were weighed to determine the wet weight of the pellet, and the pellets were then dried to completion in a Savant SpeedVac system. The tubes were then weighed again to determine the dry weight of the pellet. 1m HCl was added to the dry pellets, and the capped microcentrifuge tubes were vortexed and incubated on a rocker for at least 24 h. Thereafter each sample was centrifuged briefly in a microcentrifuge, and multiple aliquots of each supernatant were taken for ion measurements. Ca2+, Na+, and K+ measurements of aliquots were carried out with an Eppendorf EFOX-5070 flame photometer; Mg2+ levels of aliquots were determined using a Varian AA-20 atomic absorption spectrophotometer. Cellular ion concentrations were then calculated based on the dry weight of the samples and dilution factors. Total combined orthophosphate and polyphosphate levels (referred to as total inorganic phosphate) were determined in the 1 m HCl hydrolysate described above using an acid molybdate-based diagnostic kit (Sigma). The phosphorus levels measured represent the sum of the acid-hydrolyzed polyphosphate and the inorganic phosphate present (32Stanton M.G. Anal. Biochem. 1968; 22: 27-34Crossref PubMed Scopus (161) Google Scholar). A pEVP11-based plasmid containing a functional apoaequorin gene (pAEQ) was transformed into yeast using theLEU2 gene as selectable marker (1Batiza A.F. Schulz T. Masson P.H. J. Biol. Chem. 1996; 271: 23357-23362Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). This plasmid was a gift from Patrick Masson. Cells containing the pAEQ plasmid were grown in SMD medium containing other necessary supplements and were harvested in the logarithmic growth phase. 10 A 600 units of cells were resuspended in 0.2 ml of aequorin test medium, which consists of SMD medium (which contains 1 mmCa2+) supplemented with 2 mm EGTA and 20 mm MES-Tris, pH 6.5. The free Ca2+concentration of this medium was calculated to be 6 μm. To convert the apoaequorin to aequorin, 10 μl of 590 μmcoelenterazine (dissolved in methanol) was added, and the cells were incubated for 20 min at room temperature. They were then centrifuged briefly in a microcentrifuge, and the supernatant containing excess coelenterazine was removed. The cells were washed again in 0.5 ml of aequorin test medium, and the cells were then resuspended in test medium and incubated at room temperature for 20 min before initiating the experiment. A Berthold Lumat 9050 luminometer was used to collect aequorin light emission data at 200-ms intervals. The data were downloaded directly to a computer using the MS Windows Terminal software and transferred to Microsoft Excel 5.0 for analysis. To determine the concentration of cytosolic Ca2+ using the aequorin reporter system, it was necessary to determine: 1) the total amount of reconstituted aequorin available for light emission and 2) the relationship between Ca2+ concentration and light emission (33Allen D.G. Blinks J.R. Prendergast F.G. Science. 1977; 195: 996-998Crossref PubMed Scopus (250) Google Scholar). The total amount of reconstituted aequorin was determined routinely in a crude extract of each strain by measuring the maximum light emission (L max) value in the presence of a saturating concentration of Ca2+. To prepare the crude extract, 2 A 600 units of cells in 0.2 ml of aequorin standard buffer (100 mm MES-Tris, pH 6.5; 150 mm KCl; 20 mm NaCl; 5 mmMgCl2; and 2 mm phenylmethylsulfonyl fluoride) were lysed by agitation with glass beads at 4 °C. A 25-μl aliquot was placed in the luminometer, and the L max of this sample was induced by injecting 25 μl of a 50 mmCaCl2 solution. The L max value was generally between 0.5 and 1.0 × 107 relative light units/s. The protein concentration of cell lysates was also measured using a Bio-Rad protein assay kit. A correction factor based upon theL max value/unit of protein was determined for each strain, and this value was used to correct for minor differences in the concentration of aequorin in different strains. To determine the relationship between the free Ca2+concentration and aequorin-based light emission, a standard curve was prepared using a cell lysate as described (33Allen D.G. Blinks J.R. Prendergast F.G. Science. 1977; 195: 996-998Crossref PubMed Scopus (250) Google Scholar). Briefly, increasing concentrations of CaCl2 were added to a crude extract of wild type cells prepared in aequorin standard buffer. To determine the cytosolic Ca2+ concentration within intact cells, both theL observed in intact cells and theL max emission observed in a crude extract of the same cells were determined. The ratio between these values (L:L max) was then used to estimate the cytosolic free Ca2+ concentration from our standard curve. In no case was the L value in an experiment greater than 2–3% of the L max value. Thus, the absolute amount of reconstituted aequorin was not limiting in any of these experiments. To determine the rate of Ca2+ uptake by different mutant strains, cells were grown in SMD medium to approximately 1.0A 600/ml. Cells were harvested and resuspended in a buffer containing 40 mm MES-Tris, pH 6.5, and 20 mmd-glucose. An aliquot of45Ca2+ (NEN Life Science Products) was then added, and aliquots were filtered through 0.45-μm Millipore filters on a 12-position Millipore vacuum manifold at the indicated times. The filtered cells were washed immediately with two 5-ml aliquots of ice-cold blocking solution (150 mm NaCl, 20 mmMgCl2, and 2 mm LaCl3). The cell-associated counts on the filter were then determined by scintillation counting. To calculate absolute Ca2+ levels, cpm were converted to mmol of Ca2+/kg dry mass based upon total cellular Ca2+ measurements as determined by flame photometry under identical growth conditions. Cells for Ca2+ exchange experiments were grown in YPD medium to a density of 0.05 A 600. The medium was then supplemented with 45Ca2+, and the cells were grown to a cell density of 0.5–1 A 600/ml. The cells were then harvested by centrifugation at 4,000 ×g for 5 min, washed, and resuspended in fresh YPD supplemented with 50 mm CaCl2. At the indicated times, aliquots were removed, filtered, washed, and processed for scintillation counting as described above. RNA extraction and Northern analysis were carried out as described previously (34Bonetti B. Fu L. Moon J. Bedwell D.M. J. Mol. Biol. 1995; 251: 334-345Crossref PubMed Scopus (233) Google Scholar). Strains were grown in YPD medium in the presence of 1 mm EGTA (estimated to result in 0.01 mm free Ca2+) or 50 mm calcium to 1 A 600/ml. A 0.56-kb region of thePMR1 gene was amplified by PCR using the primers DB-483 (5′-GGCCCCAATGAAATAACCGT AG-3′) and DB-484 (5′-CCTGTTCCTAC GACGATACCC T-3′). The ACT1 probe was prepared by PCR amplification using the primers DB-154 (5′-GCGCG GAATT CAACG TTCCA GCCTT CTAC-3′) and DB-155 (5′-GGATG GAACA AAGCT TCTGG-3′). All probes were labeled with [α-32P]dATP using the random hexamer method. Radioactivity in specific hybrids was quantitated using a PhosphorImager (Molecular Dynamics). After quantitating the radioactivity associated with PMR1 mRNA, the membranes were hybridized with the ACT1 probe. After background correction, the PMR1 signal of each sample was corrected with the ACT1 mRNA control. These corrected values were then normalized to the wild type strain grown under low Ca2+ conditions. We initially compared the Ca2+ tolerance of yeast strains containing knockouts of genes involved in vacuolar Ca2+ transport (pmc1Δ, vcx1Δ, or pmc1Δ/vcx1Δ) vacuolar biogenesis (vps33Δ) or a combination of both classes (vps33Δ/pmc1Δ/vcx1Δ). Each strain was streaked onto YPD plates supplemented with increasing concentrations of CaCl2 or with 10 mm EGTA and incubated at 30 °C for 48 h. The wild type, pmc1Δ, vcx1Δ, andpmc1Δ/vcx1Δ strains grew similarly on standard YPD plates (buffered to pH 5.5) containing 0.3 mmCa2+ (Fig. 1 A), whereas the colony size of the vps33Δ andvps33Δ/pmc1Δ/vcx1Δ strains was slightly smaller. The wild type, pmc1Δ, and vcx1Δ strains also grew similarly on YPD medium supplemented with 100 mmCaCl2, whereas the growth rate of thepmc1Δ/vcx1Δ double mutant was reduced significantly on this medium (Fig. 1 B). In contrast, neither thevps33Δ strain nor the vps33Δ/pmc1Δ/vcx1Δstrain was able to form visible colonies under these growth conditions during the 48-h incubation period. When the YPD plates were supplemented with 200 mmCaCl2, both the wild type and vcx1Δ strains grew somewhat more slowly than on YPD plates supplemented with 100 mm CaCl2. The pmc1Δ/vcx1Δ double mutant was unable to grow under these conditions, whereas thepmc1Δ strain grew much more slowly than the wild type strain (Fig. 1 C). A further doubling of the Ca2+concentration in the YPD plate to 400 mm completely inhibited growth of the pmc1Δ strain but not the growth of the wild type and vcx1Δ strains (not shown). None of these strains was inhibited by the addition of either 400 mm NaCl or 400 mm KCl to the YPD plates, indicating that the increased osmolarity associated with 200 mmCaCl2 did not cause the growth sensitivity described above. We conclude that strains harboring the vps33Δ mutation show greater sensitivity to high extracellular Ca2+ than strains carrying the pmc1Δ mutation, thevcx1Δ mutation, or both mutations together. Overall, the rank order of Ca2+ sensitivity observed for these strains was: vps33Δ/pmc1Δ/vcx1Δ and vps33Δstrains > pmc1Δ/vcx1Δ strain >pmc1Δ strain > vcx1Δ and wild type strains. We also examined whether the growth of these strains was sensitive to inhibition by the chelating agent EGTA. We found thatpmc1Δ, vcx1Δ, and pmc1Δ/vcx1Δstrains grew similarly to the wild type strain on YPD plates buffered to pH 5.5 and supplemented with 10 mm EGTA (Fig.1 D). In contrast, the growth of the vps33Δ andvps33Δ/pmc1Δ/vcx1Δ strains was severely inhibited under these conditions, suggesting that they require a higher minimal level of environmental Ca2+ for efficient growth than the other strains. However, not only Ca2+ but other cations such as Zn2+, Fe2+, and Mn2+ are also complexed effectively by EGTA. To confirm that low environmental Ca2+ was responsible for EGTA sensitivity, we supplemented EGTA-pretreated media with different divalent cations to determine the component(s) required for growth of the vps33Δ strain. We found that the addition of Ca2+ could restore a significant amount of growth in YPD medium treated with EGTA, whereas several other cations (Mg2+, Mn2+, Fe2+, Zn2+, and Cu2+) could not (data not shown). These results lead us to conclude that the vps33Δ andvps33Δ/pmc1Δ/vcx1Δ strains are more sensitive to either high or low levels of environmental Ca2+ than the wild type, pmc1Δ, vcx1Δ, andpmc1Δ/vcx1Δ strains. Yeast cells, like mammalian cells, have been reported to maintain cytosolic free Ca2+ levels in the range of 50–200 nm(1Batiza A.F. Schulz T. Masson P.H. J. Biol. Chem. 1996; 271: 23357-23362Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 2Iida H. Yagawa Y. Anraku Y. J. Biol. Chem. 1990; 265: 13391-13399Abstract Full Text PDF PubMed Google Scholar, 3Nakajima-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). To determine how the above mutations affect the ability of yeast to maintain cytosolic Ca2+ homeostasis, we introduced a plasmid encoding a cytosolic form of apoaequorin into each strain (1Batiza A.F. Schulz T. Masson P.H. J. Biol. Chem. 1996; 271: 23357-23362Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Apoaequorin can be converted to aequorin by incubating the strains with the membrane-permeant cofactor coelenterazine. Once active aequorin is generated, it is capable of emitting light as a function of the free Ca2+ concentration present in the cytosol (33Allen D.G. Blinks J.R. Prendergast F.G. Science. 1977; 195: 996-998Crossref PubMed Scopus (250) Google Scholar). In the experiments described here, the aequorin-dependent light emission of each strain was sampled throughout the experiment at 200-ms intervals. To determine the cytosolic Ca2+concentration as a function of light emission, a standard curve was prepared using crude extracts from the wild type strain where the light emission at each Ca2+ concentration was correlated to theL max each sample was capable of discharging (Fig. 2 A). Using this method, the relative light units/s emitted from the wild type strain routinely corresponded to a resting free cytosolic Ca2+ concentration of ∼75 nm when cells were incubated in a medium containing low (∼6 μm) free Ca2+ (for further details, see “Materials and Methods”). To determine how various mutations affect the ability of these strains to respond to a sudden increase in extracellular Ca2+, 50 mm CaCl2 was injected rapidly into the cell suspension while the cytosolic aequorin-dependent light emission was continuously monitored. We found that the light emission of the wild type strain increased rapidly and reached a peak level corresponding to ∼300 nm cytosolic Ca2+within 5 s (Fig. 2 B). The Ca2+concentration decreased rapidly thereafter and returned to a new steady-state free cytosolic Ca2+ concentration of ∼80–85 nm within 90 s. The light emission measured in the pmc1Δ/vcx1Δ strain corresponded to a basal cytosolic Ca2+ concentration of 75–80 nm. When 50 mm CaCl2 was injected, the light emission reached a peak value corresponding to ∼385 nm cytosolic free Ca2+, which was somewhat higher than the peak observed with the wild type strain. The recovery phase of the pmc1Δ/vcx1Δ strain was also much weaker than the wild type control. The post-shock steady-state cytosolic Ca2+ concentration was ∼310 nm, which was 4-fold higher than the steady-state cytosolic Ca2+ concentration observed in the wild type strain after the same Ca2+ shock. This sugg" @default.
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