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• W2000615534 abstract "Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca2+ pumps, requires micromolar Ca2+ for growth. In medium containing 100 μm Ca2+, K616 can withstand osmotic stress (750 mm sorbitol) and ionic stress (300 mm KCl) but not hypersodic stress (300 mm NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca2+-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca2+-depleted medium. All stresses tested generated transient elevation of cytosolic Ca2+ in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca2+ in K616. Both the Ca2+ transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na+ predominantly by pumping it out across the plasma membrane, the transformant sequestered Na+ in internal organelles. This sequestration requires the presence of an endomembrane Na+/H+-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na+-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca2+ pump results in the rapid depletion of cytosolic Ca2+ and the activation of an alternate mechanism for surviving saline stress. Stress responses in both plants and yeast utilize calcium-mediated signaling. A yeast strain, K616, which lacks Ca2+ pumps, requires micromolar Ca2+ for growth. In medium containing 100 μm Ca2+, K616 can withstand osmotic stress (750 mm sorbitol) and ionic stress (300 mm KCl) but not hypersodic stress (300 mm NaCl). Heterologous expression of the endoplasmic reticulum-located Arabidopsis thaliana Ca2+-ATPase, ACA2, permits K616 to grow under NaCl stress even in Ca2+-depleted medium. All stresses tested generated transient elevation of cytosolic Ca2+ in wild type yeast, K601, whereas NaCl alone induced prolonged elevation of cytosolic Ca2+ in K616. Both the Ca2+ transient and survival of cultures subjected to NaCl stress was similar for the ACA2 transformant and K601. However, whereas K601 maintained low cytosolic Na+ predominantly by pumping it out across the plasma membrane, the transformant sequestered Na+ in internal organelles. This sequestration requires the presence of an endomembrane Na+/H+-antiporter, NHX1, which does not play a significant role in salt tolerance of wild type yeast except at acidic pH. Transcript levels of the plasma membrane Na+-ATPase, ENA1, were strongly induced only in K601, whereas NHX1 was strongly induced in both K601 and the ACA2 transformant. The calmodulin kinase inhibitor KN62 significantly reduced the salt tolerance of the ACA2 transformant and the transcriptional induction of NHX1. Thus, the heterologous expression of a plant endomembrane Ca2+ pump results in the rapid depletion of cytosolic Ca2+ and the activation of an alternate mechanism for surviving saline stress. Eukaryotic cells regulate a variety of cellular processes, including responses to abiotic stresses using calcium-mediated processes. Soil salinity adversely affects plant yields worldwide (1Maas E.V. Tanji K. Agricultural Salinity Assessment and Management. New York American Society of Civil Engineers, New York1990: 262Google Scholar), salt-tolerant cultivars being reported to utilize signal transduction systems involving calcium (2Anil V.S. Krishnamurthy H. Mathew M.K. Proc. Indian Nat. Sci. Acad. 2007; 73: 43-50Google Scholar, 3Anil V.S. Krishnamurthy H. Mathew M.K. Physiol. Plant. 2007; 129: 607-621Crossref Scopus (41) Google Scholar, 4Epstein E. Science. 1998; 280: 1906-1907Crossref PubMed Scopus (118) Google Scholar). NaCl-induced [Ca2+]cyt transients have been observed in plants (2Anil V.S. Krishnamurthy H. Mathew M.K. Proc. Indian Nat. Sci. Acad. 2007; 73: 43-50Google Scholar, 5Knight H. Trewavas A.J. Knight M.R. Plant J. 1997; 12: 1067-1078Crossref PubMed Scopus (693) Google Scholar), and the model plant Arabidopsis utilizes a calcineurin-like Ca2+-sensing protein in the salt overly sensitive (SOS) 2The abbreviations used are: CaMcalmodulinSOSsalt overly sensitiveERendoplasmic reticulumRTreverse transcriptionACAN-(p-amylcinnamoyl) anthranilic acidMES4-morpholineethanesulfonic acid. 2The abbreviations used are: CaMcalmodulinSOSsalt overly sensitiveERendoplasmic reticulumRTreverse transcriptionACAN-(p-amylcinnamoyl) anthranilic acidMES4-morpholineethanesulfonic acid. stress signaling pathway (6Zhu J.K. Annu. Rev. Plant. Biol. 2002; 53: 247-273Crossref PubMed Scopus (4216) Google Scholar). Other calcium-sensing proteins, such as calmodulin (CaM) (7Phean O.P.S. Punteeranurak P. Buaboocha T. J. Biochem. Mol. Biol. 2005; 38: 432-439PubMed Google Scholar) and Ca2+-dependent protein kinase may also be recruited in regulating Na+ homeostasis under saline stress (8Kawasaki S. Borchert C. Deyholos M. Wang H. Brazille S. Kawai K. Galbraith D. Bohnert H.J. Plant Cell. 2001; 13: 889-905Crossref PubMed Scopus (777) Google Scholar, 9Saijo Y. Hata S. Kyozuka J. Shimamoto K. Izui K. Plant. J. 2000; 23: 319-327Crossref PubMed Google Scholar). calmodulin salt overly sensitive endoplasmic reticulum reverse transcription N-(p-amylcinnamoyl) anthranilic acid 4-morpholineethanesulfonic acid. calmodulin salt overly sensitive endoplasmic reticulum reverse transcription N-(p-amylcinnamoyl) anthranilic acid 4-morpholineethanesulfonic acid. Cells have evolved mechanisms for maintaining low resting levels of Ca2+ in their cytoplasm (10Sanders D. Pelloux J. Brownlee C. Harper J.F. Plant Cell. 2002; 14: 401-417Crossref PubMed Scopus (962) Google Scholar). These involve regulated entry and active removal of Ca2+, the latter being mediated by high affinity Ca2+-ATPases and low affinity H+/Ca2+ antiporters (11Sze H. Liang F. Hwang I. Curran A.C. Harper J.F. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 2000; 51: 433-462Crossref PubMed Scopus (247) Google Scholar). The sensitivity and response of the cell to various stresses, including salinity, is dependent on its ability to adequately sequester and use Ca2+ from internal stores (12Hirschi K. Trends Plant. Sci. 2001; 6: 100-104Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Cessna S.G. Chandra S. Low P.S. J. Biol. Chem. 1998; 273: 27286-27291Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Ca2+ is stored in the cell wall and organelles with the vacuole serving as the main Ca2+ sequestration site in plant cells, whereas the ER has also been suggested to play an important role in regulating Ca2+ homeostasis (14Klusener B. Boheim G. Liss H. Engelberth J. Weiler E.W. EMBO J. 1995; 14: 2708-2714Crossref PubMed Scopus (129) Google Scholar, 15Allen G.J. Muir S.R. Sanders D. Science. 1995; 268: 735-737Crossref PubMed Scopus (299) Google Scholar). Plants, like animals, produce a range of distinct [Ca2+]cyt“signatures,” such as spikes, waves, and oscillations (16Evans N.H. McAinsh M.R. Hetherington A.M. Curr. Opin. Plant Biol. 2001; 4: 415-420Crossref PubMed Scopus (181) Google Scholar, 17Anil V.S. Rao K.S. Plant Physiol. 2000; 123: 1301-1312Crossref PubMed Scopus (83) Google Scholar, 18Malho R. Trewavas A.J. Plant Cell. 1996; 8: 1935-1949Crossref PubMed Scopus (251) Google Scholar), to different stimuli, which couple to a correspondingly wide array of responses (10Sanders D. Pelloux J. Brownlee C. Harper J.F. Plant Cell. 2002; 14: 401-417Crossref PubMed Scopus (962) Google Scholar, 40Elbe R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar, 19Anil V.S. Rao K.S. J. Plant Physiol. 2001; 158: 1237-1256Crossref Scopus (49) Google Scholar). Sculpting the Ca2+ signature may be expected to require modulation of both Ca2+ entry into the cytosol and of transporters affecting reuptake/efflux. Several lines of evidence, such as the increase in transcript levels of plant endoplasmic reticulum (ER) Ca2+-ATPases in tomato (20Wimmers L.E. Ewing N.N. Bennett A.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9205-9209Crossref PubMed Scopus (142) Google Scholar) and tobacco (21Perez-Prat E. Narasimhan M.L. Binzel M.L. Botella M.A. Chen Z. Valpuesta V. Bressan R.A. Hasegawa P.M. Plant. Physiol. 1992; 100: 1471-1478Crossref PubMed Scopus (74) Google Scholar), suggest the involvement of Ca2+ pumps in salt tolerance. The presence of multiple isoforms amenable to a range of regulation (11Sze H. Liang F. Hwang I. Curran A.C. Harper J.F. Annu. Rev. Plant. Physiol. Plant. Mol. Biol. 2000; 51: 433-462Crossref PubMed Scopus (247) Google Scholar) also supports the hypothesis that Ca2+-ATPases may play a more direct role in signal transduction (22Hwang I. Sze H. Harper J.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6224-6229Crossref PubMed Scopus (140) Google Scholar, 23Niu X. Bressan R.A. Hasegawa P.M. Pardo J.M. Plant Physiol. 1995; 109: 735-742Crossref PubMed Scopus (705) Google Scholar). However, this hypothesis has not thus far been tested. Higher plants and the yeast Saccharomyces cerevisiae share several conserved elements in the cellular mechanisms of Na+ homeostasis (24Serrano R. Rodriguez-Navarro A. Curr. Opin. Cell Biol. 2001; 13: 399-404Crossref PubMed Scopus (223) Google Scholar). Multiple transport pathways mediate cellular Na+ homeostasis in S. cerevisiae.Na+ is believed to enter the yeast cell via the K+ transporters TRK1 and TRK2 (25Haro R. Rodriguez-Navarro A. Biochim. Biophys. Acta. 2002; 1564: 114-122Crossref PubMed Scopus (57) Google Scholar). The primary route of Na+ extrusion across the plasma membrane is through the P-type ion pump ENA1 (26Hirata D. Harada S. Namba H. Miyakawa T. Mol. Gen. Genet. 1995; 249: 257-264Crossref PubMed Scopus (63) Google Scholar). Na+/H+-antiporters in the plasma membrane (NHA1), the late endosomal/prevacuolar membrane (NHX1), and the vacuolar VNX1 (27Cagnac O. Leterrier M. Yeager M. Blumwald E. J. Biol. Chem. 2007; 282: 24284-24293Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), a monovalant cation/H+ antiporter, could also serve to deplete [Na+]cyt. NHA1 has been shown to extrude Na+ into acidic medium (28Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (199) Google Scholar). No significant role in tolerance responses has been demonstrated for NHX1 at neutral pH, but it contributes to endosomal/vacuolar Na+ sequestration when stressed in acidic medium (29Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 30Nass R. Rao R. Microbiology. 1999; 145: 3221-3228Crossref PubMed Scopus (86) Google Scholar). These multiple transport pathways regulating yeast Na+ homeostasis are under complex regulation; ENA1 is transcriptionally activated by several distinct pathways (26Hirata D. Harada S. Namba H. Miyakawa T. Mol. Gen. Genet. 1995; 249: 257-264Crossref PubMed Scopus (63) Google Scholar, 31Proft M. Serrano R. Mol. Cell. Biol. 1999; 19: 537-546Crossref PubMed Scopus (158) Google Scholar), of which the HOG1 pathway has also been proposed to enhance activity of the plasma membrane-located NHA1 (32Proft M. Struhl K. Cell. 2004; 118: 351-361Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). A substantive understanding of the mechanisms of Ca2+ homeostasis makes yeast an ideal system to study how Ca2+ pumps could influence Na+ homeostasis and salt tolerance. Under normal conditions, extracellular Ca2+ enters the yeast cytosol through an as yet unidentified transporter (33Cui J. Kaandorp J.A. Cell Calcium. 2006; 39: 337-348Crossref PubMed Scopus (31) Google Scholar). The Ca2+-ATPase PMR1 pumps [Ca2+]cyt into the ER and Golgi, whereas the high affinity Ca2+-ATPase, PMC1, and low affinity Ca2+/H+ exchanger, VCX1, serve to sequester it in the vacuole (33Cui J. Kaandorp J.A. Cell Calcium. 2006; 39: 337-348Crossref PubMed Scopus (31) Google Scholar). Hypertonic shock, induced by extracellular NaCl, induces release of vacuolar Ca2+ into the cytosol through the tonoplast-located Ca2+ channel YVC1 (34Denis V. Cyert M.S. J. Cell Biol. 2002; 156: 29-34Crossref PubMed Scopus (228) Google Scholar) and possibly influx of external Ca2+ via the CCH1/MID1 Ca2+ channel on the plasma membrane (35Matsumoto T.K. Ellsmore A.J. Cessna S.G. Low P.S. Pardo J.M. Bressan R.A. Hasegawa P.M. J. Biol. Chem. 2002; 277: 33075-33080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Expression and function of PMC1, PMR1, and VCX1 are regulated by calcineurin (36Matheos D.P. Kingsbury T.J. Ahsan U.S. Cunningham K.W. Genes Dev. 1997; 11: 3445-3458Crossref PubMed Scopus (272) Google Scholar), a highly conserved protein phosphatase that is activated by Ca2+/CaM. In contrast to plants, there are no Ca2+-ATPases or exchangers detected on the yeast plasma membrane (33Cui J. Kaandorp J.A. Cell Calcium. 2006; 39: 337-348Crossref PubMed Scopus (31) Google Scholar) and consequently little or no active efflux of Ca2+ across the plasma membrane. The ER-located ACA2 and vacuolar ACA4 are Arabidopsis Ca2+-ATPases that have previously been expressed in yeast (37Harper J.F. Hong B. Hwang I. Guo H.Q. Stoddard R. Huang J.F. Palmgren M.G. Sze H. J. Biol. Chem. 1998; 273: 1099-1106Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 38Geisler M. Frangne N. Gomes E. Martinoia E. Palmgren M.G. Plant. Physiol. 2000; 124: 1814-1827Crossref PubMed Scopus (160) Google Scholar), and the latter has been shown to ameliorate the salt hypersensitivity of the host strain (38Geisler M. Frangne N. Gomes E. Martinoia E. Palmgren M.G. Plant. Physiol. 2000; 124: 1814-1827Crossref PubMed Scopus (160) Google Scholar). This investigation was carried out to determine whether an ER-located plant Ca2+ pump could facilitate a salt tolerance response and, if so, to understand the underlying cellular mechanism involved. Here we demonstrate that the ER-located ACA2 relieves the hypersensitivity of the salt-sensitive triple mutant yeast strain K616, in which genes encoding the Ca2+ pumps (PMC1, PMR1, and the regulatory subunit of calcineurin, CNB1) have been deleted (39Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (354) Google Scholar). This strain requires 10-4 m Ca2+ to grow and millimolar Ca2+ to survive hypertonic stress. Heterologous expression of ACA2 in this strain alleviates the calcium requirement in both cases. ACA2-expressing cells sequester Na+ into internal stores rather than extrude it across the plasma membrane, as does wild type yeast. Using the aequorin reporter system, we demonstrate that ACA2 alters the [Ca2+]cyt transient induced by NaCl stress in K616. We propose that this action of the pump contributes to the triggering of an alternative pathway, which activates the endomembrane Na+/H+-antiporter, NHX1, resulting in enhanced salt tolerance. Yeast Strains and Growth MediaS. cerevisiae wild type strain K601 (MATa, leu2, his3, ade2, trp1, and ura3) and mutant strain K616 (MATa pmr1::HIS3 pmc1::TRP1 cnb1::LEU2, ura3) were grown in complete synthetic medium (SC) and SC minus Trp, His, and Leu, respectively, throughout this study. For the K616 strain, the medium was supplemented with 10 mm CaCl2. SC medium consisted of 1.7 g/liter yeast nitrogen base without amino acids, 5 g/liter (NH4)2SO4, 1.3 g/liter drop-out mix without Ura, Trp, His, and Leu (these amino acids were added from separate stock solutions when required), and 2% (w/w) dextrose as a carbon source. Medium pH was adjusted to 6 (buffered with 5 mm MES) prior to the addition of agar and sterilization. Buffering by MES was found to be efficient, since autoclaving did not introduce significant change in medium pH. In all experiments under hyperosmotic stress, a Ca-EGTA buffer was used to maintain the free Ca2+ of SC medium at 100 μm, unless otherwise mentioned. Yeast TransformationFor transformations with pYX112 constructs, K616 was transformed by the lithium acetate/polyethylene glycol method (40Elbe R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar), and transformants were selected for their ability to grow in the absence of uracil on plates containing SC medium minus Trp, Leu, His, and Ura. For complementation studies, Ura-positive colonies were streaked on complete SC plates containing 10 mm EGTA and incubated for 3 days at 30 °C. For transformations with yeast vector pKC147/AEQ, K601 and K616 were transformed and selected for Ura autotrophy as mentioned above. A Ura-positive K616 colony was then subjected to a second transformation with pYX112-ACA2. Transformants were then selected based on their ability to complement on low calcium medium by spreading on SC minus Trp, Leu, His, and Ura, containing 5 mm EGTA. Controls for this selection included a transformation with the same volume of water and a vector alone, both of which showed no growth on the EGTA selection plate. Double transformants were analyzed for the expression of apoaequorin by monitoring luminescence before and after lysis with 5% Triton X-100 and 2 m CaCl2 and by colony PCR to confirm the presence of the ACA2 gene. They were also tested for salt tolerance in SC-medium containing 400 mm NaCl. NHX1 Knock-out Mutants in K616 BackgroundA forward primer (5′-AAACGTGATAGCAAGGAACTG-3′) that hybridizes 200 bp upstream of the start codon of NHX1 and a reverse primer (5′-GGCGTTGAGTAAGAGAGAATG-3′) that hybridizes 80 bp downstream of the NHX1 stop codon were used to amplify the NHX1::KanMX cassette from the appropriate deletion strain in the background of the yeast strain, BY4742 (a gift from Dr. R. Rao). The 1.82-kb PCR product thus obtained was gel-eluted and used to transform K616. This strategy allows specific gene disruption by homologous recombination and chromosomal integration. Recombinants were selected on SC medium containing 1 mg/ml G418. A positive transformant was then subjected to a second round of transformation with pYX112-ACA2 and selected on SC minus Ura, Trp, Leu, and His plates containing 1 mg/ml G418. Genomic DNA was isolated from these colonies and analyzed for the presence of ACA2 and the KAN-NHX1 sequences by PCR using specific primers followed by DNA sequencing. Monitoring Expression of ENA1 and NHX1 by RT-PCRTotal RNA was extracted from yeast cells exposed to 400 mm NaCl for a range of time periods (0-50 h). The RNA was first treated with RNase-free DNase (Promega, Madison, WI) to eliminate contaminating DNA. 500 ng of RNA each was then subject to a reverse transcriptase reaction using 100 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) for 1 h at 37 °C in buffer containing 50 mm Tris-HCl, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol, and 0.5 mm each of dNTPs. The cDNA thus obtained was subjected to a 30-cycle PCR reaction using standard conditions with primers specific for ENA1 (forward primer, 5′-ATG GGC GAA GGA ACT ACT AA-3′; reverse primer, 5′-CAC ACA AAT GGC ATA GAT AGC-3′) amplifying a 960-bp product and for NHX1 (forward primer, 5′-ATG CTA TCC AAG GTA TTG CTG-3′; reverse primer, 5′-AAC CGA CGA AAA TGT TGC-3′) amplifying a 759-bp product. The constitutively expressed ACT1 (actin) was amplified under identical PCR conditions using specific primers (forward primer, 5′-GAG GTT GCT GCT TTG GTT AT-3′; reverse primer, 5′-GCG GTT TGC ATT TCT TGT-3′) amplifying a 680-bp product. Stress ConditionsFor drop tests, midlog yeast cultures were washed twice with water, and A600 was adjusted to 0.5 with sterile double-distilled water. Cells were serially diluted with water to obtain 10-, 102-, and 103-fold dilutions. Five microliters of each dilution was then spotted on SC medium buffered at 20 nm, 100 μm, or 5 mm Ca2+ and supplemented with 300 mm KCl, 400/800 mm NaCl, or 750 mm sorbitol concentrations. Growth was recorded after a 3-day incubation at 30 °C. For growth curves, equal cell inocula were added into liquid SC medium buffered for Ca2+ at 100 μm with or without 400 mm NaCl. In some experiments, medium was also supplemented with amiloride (500 μm). Cells were incubated on an incubator shaker at 30 °C. Change in A600 was monitored over a period of 2 days. Alternatively, sensitivity curves were generated using log phase cells with or without preincubation with inhibitors such as 5 μm bafilomycin, N-(p-amylcinnamoyl) anthranilic acid (ACA) (30 and 40 μm), KN62 (20, 50, and 100 μm), and SB 203580 (10 and 200 μm) prior to administrating the NaCl insult. A600 was determined at the 18th hour of salt stress. Estimation of Total Cellular Na+ and Ca2+%Yeast strains were grown to an A600 of 0.6 in SC medium buffered at 100 μm Ca2+. Cells were either taken as such or stressed with 400 mm NaCl, and 2-ml aliquots were collected over a range of time points extending up to 18 h. Cells were collected by centrifugation, washed four times in ice-cold Buffer B (10 mm Tris-HCl, pH 6.0, 2 mm MgCl2, 1% glucose, 0.6 m sorbitol), and dried at 50 °C for 4 days. Dried cells were acid-digested in a 4:1 diacid mixture of perchlorate, and nitrate and Na+ levels were estimated by flame photometry using a Systronics Flame Photometer 128 (Ahmedabad, India) (41Anil V.S. Krishnamurthy P. Kuruvilla S. Sucharitha K. Thomas G. Mathew M.K. Physiol. Plant. 2005; 124: 451-464Crossref Scopus (63) Google Scholar). In a few experiments, growth was carried out at Ca2+ concentrations of 100 μm and 5 mm. At an A600 of 0.6, a set of cells grown in 100 μm Ca2+ was washed three times with SC medium and adapted to 20 nm Ca2+ for 1 h before processing these cells, as well as those at 100 μm and 5 mm for total Ca2+ estimation using a Zeeman atomic absorption spectrophotometer (model Z-6100; Hitachi). Permeabilization of Plasma Membranes with Cu2+ IonsYeast strains were grown in 300 ml of SC medium, to A600 of 0.6 and stressed for 3 h with 400 mm NaCl. Cells were harvested by centrifugation, washed twice in ice-cold Buffer B, and resuspended in the same buffer at room temperature at a density of 1 × 108 cells/ml. After withdrawing 2-ml aliquots for total Na+ estimation, the remaining cell suspension was subjected to plasma membrane permeabilization with Cu2+ as described by Anraku and co-workers (42Ohsumi Y. Kitamoto K. Anraku Y. J. Bacteriol. 1988; 170: 2676-2682Crossref PubMed Google Scholar). CuSO4 was added to a final concentration of 500 μm and incubated at 23 °C. At the indicated times, aliquots were withdrawn and washed twice with buffer B, and cells were dried for acid digestion and flame photometry. Aequorin Luminescence Measurements and [Ca2+]cyt QuantificationAequorin luminescence was determined using a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA) following procedures used to quantify [Ca2+]cyt in yeast (19Anil V.S. Rao K.S. J. Plant Physiol. 2001; 158: 1237-1256Crossref Scopus (49) Google Scholar). Cells were transformed with the 2μ plasmid pKC147/AEQ (provided by Dr. K. Cunningham) containing the APOAEQUORIN gene. Transformants, grown to an A600 of ∼0.4-0.6, were harvested by centrifugation, resuspended in fresh medium (SC-Ura + 1 mm EGTA) at an A600 of 10, and loaded with 25 μg/ml coelenterazine (Molecular Probes, Inc., Eugene, OR) for 3 h in the dark at room temperature. Cells were then collected by centrifugation and resuspended in the same volume of fresh medium buffered at 100 μm Ca2+. After a preincubation of 1 h, the cells were diluted with the same medium to an A600 of 0.5, and a cellular luminescence base line was determined by 2-3 min of recordings at 10-s intervals. Hypertonic/ionic shock was administered to yeast cells by the addition of a range of NaCl (100-1200 mm), 300 mm KCl, 50 mm CaCl2, or 750 mm sorbitol. When required, loaded cells were preincubated for 20 min with 5 μm bafilomycin A1 or 30 μm ACA dissolved in Me2SO or 10 mm LaCl3 dissolved in water prior to the acquisition of the basal luminescence reading and the subsequent osmotic/ionic shock. Luminescence from aequorin that remained in cells at the end of an experiment was determined by diluting the cell suspension 1:1 with a solution containing 10% Triton X-100 and 4 m CaCl2. [Ca2+]cyt was calculated using Equation 1, [Ca2+]((LLmax)13+(118(LLmax)13)−1)(7×106−(7×106(LLmax)13))(Eq. 1) where L represents the luminescence intensity at any time point, and Lmax is the integrated luminescence intensity (34Denis V. Cyert M.S. J. Cell Biol. 2002; 156: 29-34Crossref PubMed Scopus (228) Google Scholar). L values did not exceed ∼1% of the maximal light emission capacity in all three strains. Figures are representative of a minimum of three experiments in each case. Complementation by Full-length ACA2K616 transformed with pYX112-ACA2 was selected by Ura autotrophy. K616 is a mutant for the endogenous Ca2+ pumps PMC1 and PMR1, a potentially lethal combination but for a third knock-out mutation of CNB1. In the absence of CNB1, the Ca2+/H+-antiporter (VCX1) located in the vacuolar membrane is active and suffices to populate intracellular calcium stores, provided K616 is grown in adequately high Ca2+. Ura-selected colonies expressing ACA2 exhibited growth on complementation plates comprising SC medium depleted of calcium with 10 mm EGTA, unlike K616 and K616 transformed with the vector alone (Fig. 1A). The ACA2 transformant strain will be referred to as K616-ACA2 henceforth for simplicity, whereas the strain transformed with vector alone will be referred to as K616-V. K616, in turn, is derived from the strain K601, which will be treated as wild type for this study. Saline and Osmotic Sensitivity of Yeast StrainsAt very low Ca2+ of 20 nm, K616-V grew very poorly on SC medium (Fig. 1B) and not at all under 400 mm NaCl stress. Interestingly, at a moderate free Ca2+ level of 100 μm, K616-V showed effective growth under unstressed conditions but failed to grow under salt stress (Fig. 1B). With 5 mm Ca2+ in the medium, however, K616-V was able to grow effectively under unstressed conditions, as well as mount a reasonable salt tolerance response when stressed with NaCl (Fig. 1B). Thus, K616-V requires two levels of media Ca2+: a lower concentration (∼100 μm) for growth and millimolar calcium for reasonable salt tolerance. K616 and K616-V behaved identically in Ca2+-depleted medium (Fig. 1A) and under hyperosmotic stress (supplemental Fig. 1D). The expression of ACA2 in K616 eliminated both requirements, transformants growing well and exhibiting salt tolerance even at 20 nm Ca2+ (Fig. 1B). NaCl titration carried out at 100 μm medium Ca2+ showed that K616 did not grow in 300 mm NaCl (Fig. 1C). Sensitivity to NaCl was detected even at 100 mm, where a significant lag was observed in the growth curve (Fig. 1C). K601 and K616-ACA2, on the other hand, grew well in medium containing up to 1.2 m NaCl (growth curves not shown). To evaluate whether other hyperosmotic stresses affect these strains similarly, they were subjected to saline and osmotic stress comprising 300 mm KCl or 400/800 mm NaCl or 750 mm sorbitol in SC medium containing 100 μm free Ca2+. Although both K601 and K616-ACA2 grew well on plates containing 400 or 800 mm NaCl, K616-V did not grow on these plates (Fig. 1D). However, all three strains grew reasonably well on plates containing either 750 mm sorbitol or 300 mm KCl, indicating that purely osmotic stresses and ionic stresses up to 300 mm were tolerated by all of them (Fig. 1D). Curing the K616-ACA2 strain of pYX112-ACA2 generated colonies that did not grow on calcium-depleted media and were as sensitive to NaCl stress as was K616 (supplemental Fig. 1, A-D). ATP-induced Ca2+ pumping and vanadate-sensitive-ATPase activities were detected in ER-enriched vesicles prepared from K616-ACA2 but not in those from K616, indicating the expression of a functional Ca2+-ATPase in K616-ACA2 (supplemental Fig. 1, E-G). An N-terminal, autoinhibitory domain in ACA2 (43Hwang I. Harper J.F. Liang F. Sze H. Plant. Physiol. 2000; 122: 157-168Crossref PubMed Scopus (59) Google Scholar) does not appear to prevent activity of the full-length construct used here. ACA2 Changes Ca2+ Homeostasis in K616To test the degree to which internal calcium stores get filled, cellular Ca2+ of yeast preincubated with external Ca2+ of 20 nm, 100 μm, or 5 mm was estimated by atomic absorption spectrophotometry. Total cellular calcium is tightly controlled in wild type, K601, and K616-ACA2, remaining below 0.2 mg/g, dry weight, under all of the Ca2+ concentrations tested (Fig. 2A). K616 showed >8-fold higher total calcium than K601 when subjected to 20 nm external Ca2+ (Fig. 2A). However, at both 100 μm and 5 mm calcium, cellular Ca2+ levels were comparable among all of the yeast strains tested, suggesting that internal stores of Ca2+ are adequately filled in all. We focused on 100 μm Ca2+, since it is the concentration that is adequate for growth of K616 but insufficient for mounting a salt tolerance response. At 100 μm Ca2+, the sensitivity of K616 to hypersodic media could be a consequence of altered signaling due to an aberrant [Ca2+]cyt transient in response to saline stress. To test this hypothesis, we used aequorin to monitor [Ca2+]cyt. The addition of 400 mm NaCl to the yeast suspension resulted in rapid increases in aequorin luminescence in all three strains (Fig. 2B). In the case of K601 and K616-ACA2, the rise in [Ca2+]cyt was limited to a peak value between 0.6 and 0.8 μm, from which maximum it decayed to basal levels in 2-3 min (Fig. 2B). In K616, on the other hand, [Ca2+]cyt rose to over 1.2 μm over the course of 1 min. Thereafter, it declined very slowly, not reaching basal values even after 20 min (Fig. 2B). Effect of Bafilomycin A1 on the NaCl-induced" @default.
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• W2000615534 title "A Plant Ca2+ Pump, ACA2, Relieves Salt Hypersensitivity in Yeast" @default.
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