The Golgi Apparatus Plays a Significant Role in the Maintenance of Ca2+ Homeostasis in the vps33Delta Vacuolar Biogenesis Mutant of Saccharomyces cerevisiae*

Attila MisetaDagger , Lianwu Fu, Richard Kellermayer, Jessica Buckley, and David M. Bedwell§

From the Department of Microbiology, the University of Alabama at Birmingham, Birmingham, Alabama 35294

    ABSTRACT
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Abstract
Introduction
References

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 vps33Delta vacuolar biogenesis mutant. We found that growth of the vps33Delta 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, the vps33Delta 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 a vps33Delta /pmr1Delta 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.

    INTRODUCTION
Top
Abstract
Introduction
References

Like all eukaryotes, the yeast Saccharomyces cerevisiae normally maintains a resting cytosolic Ca2+ concentration of 50-200 nM (1-3). 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 (4). 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+ (5, 6). Accordingly, many different vacuolar mutations result in an inability to grow in the presence of high concentrations of extracellular Ca2+ (7-13).

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 PMC1 gene, 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+ (7). The second protein known to be involved in vacuolar Ca2+ transport is the H+/Ca2+ exchanger encoded by the VCX1 (HUM1) gene (14, 15). Although mutants that do not express Vcx1p show little or no decrease in Ca2+ tolerance, the combination of pmc1Delta and vcx1Delta mutations 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)1 and FK506 (16, 17). 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 (14, 15).

Several other genes encoding potential Ca2+ ATPases have been identified within the yeast genome (18); 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 (19-22). 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 pmc1Delta strain is 2-3-fold lower than normal, but the total cellular Ca2+ level in the pmr1Delta 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 (23). 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 vps33Delta mutation lack a morphologically distinguishable vacuole but instead accumulate small vesicular and Golgi-like structures (24-26). 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 (27). A vps33Delta 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 alpha -mannosidase to the cell surface (24).

In this study we asked how the severe defects in vacuolar biogenesis associated with the vps33Delta mutation affect cellular Ca2+ homeostasis. We found that the vps33Delta strain 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 vps33Delta 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 vps33Delta strain. We found that PMR1 expression is elevated in the vps33Delta strain. We also found that a vps33Delta /pmr1Delta 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.

    MATERIALS AND METHODS

Strains Used-- Strains used in this study are listed in Table I. The PMC1 and VCX1 genes were disrupted using the one-step gene replacement method (28). 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 the PMC1 gene was then removed by digestion with AflIII and EcoRI and replaced by the TRP1 gene taken from pJJ280 plasmid (29). A KpnI/NotI fragment containing the disrupted pmc1Delta ::TRP1 fragment was then used to transform yeast. Trp+ colonies were selected, and the correct gene replacement was confirmed by PCR.

                              
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Table I
Yeast strains used in this study

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 with HincII and HindIII endonucleases and replaced with a fragment containing the URA3 gene obtained from pJJ244 (29). A KpnI/BamHI fragment containing the disrupted vcx1Delta ::URA3 fragment from this plasmid was used to transform yeast. The replacement of wild type VCX1 was confirmed by PCR analysis. Other genetic manipulations were carried out by standard methods (30).

Culture Media-- Bacterial strains were grown on standard media (31). 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 (30). Growth media were routinely buffered with 40 mM MES-Tris, pH 5.5.

Determination of Ca2+ Concentration in Media-- 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 CaCl2 stock 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.

Measurements of Total Cellular Ca2+, Mg2+, Na+, K+, and Phosphate Levels-- 50-100 A600 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. 1 M 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 (32).

Measurement of Cytosolic Free Ca2+ Concentration-- A pEVP11-based plasmid containing a functional apoaequorin gene (pAEQ) was transformed into yeast using the LEU2 gene as selectable marker (1). 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 A600 units of cells were resuspended in 0.2 ml of aequorin test medium, which consists of SMD medium (which contains 1 mM Ca2+) 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 µM coelenterazine (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 (33). The total amount of reconstituted aequorin was determined routinely in a crude extract of each strain by measuring the maximum light emission (Lmax) value in the presence of a saturating concentration of Ca2+. To prepare the crude extract, 2 A600 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 mM MgCl2; 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 Lmax of this sample was induced by injecting 25 µl of a 50 mM CaCl2 solution. The Lmax 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 the Lmax 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 (33). 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 the L observed in intact cells and the Lmax emission observed in a crude extract of the same cells were determined. The ratio between these values (L:Lmax) 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 Lmax value. Thus, the absolute amount of reconstituted aequorin was not limiting in any of these experiments.

45Ca2+ Uptake and Release-- To determine the rate of Ca2+ uptake by different mutant strains, cells were grown in SMD medium to approximately 1.0 A600/ml. Cells were harvested and resuspended in a buffer containing 40 mM MES-Tris, pH 6.5, and 20 mM D-glucose. An aliquot of 45Ca2+ (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 mM MgCl2, 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 A600. The medium was then supplemented with 45Ca2+, and the cells were grown to a cell density of 0.5-1 A600/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.

Northern Analysis-- RNA extraction and Northern analysis were carried out as described previously (34). 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 A600/ml. A 0.56-kb region of the PMR1 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 [alpha -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.

    RESULTS

Sensitivity of Yeast Vacuolar Mutants to Different Environmental Ca2+Concentrations-- We initially compared the Ca2+ tolerance of yeast strains containing knockouts of genes involved in vacuolar Ca2+ transport (pmc1Delta , vcx1Delta , or pmc1Delta /vcx1Delta ) vacuolar biogenesis (vps33Delta ) or a combination of both classes (vps33Delta /pmc1Delta /vcx1Delta ). 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, pmc1Delta , vcx1Delta , and pmc1Delta /vcx1Delta strains grew similarly on standard YPD plates (buffered to pH 5.5) containing 0.3 mM Ca2+ (Fig. 1A), whereas the colony size of the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta strains was slightly smaller. The wild type, pmc1Delta , and vcx1Delta strains also grew similarly on YPD medium supplemented with 100 mM CaCl2, whereas the growth rate of the pmc1Delta /vcx1Delta double mutant was reduced significantly on this medium (Fig. 1B). In contrast, neither the vps33Delta strain nor the vps33Delta /pmc1Delta /vcx1Delta strain was able to form visible colonies under these growth conditions during the 48-h incubation period.


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Fig. 1.   Effect of different concentrations of extracellular Ca2+ on the growth of vacuolar mutants. The growth of the indicated strains is shown on standard YPD (0.3 mM Ca2+, panel A), YPD plus 100 mM CaCl2 (panel B), YPD plus 200 mM CaCl2 (panel C), and YPD plus 10 mM EGTA (panel D). The plates were incubated at 30 °C for 48 h. WT, wild type.

When the YPD plates were supplemented with 200 mM CaCl2, both the wild type and vcx1Delta strains grew somewhat more slowly than on YPD plates supplemented with 100 mM CaCl2. The pmc1Delta /vcx1Delta double mutant was unable to grow under these conditions, whereas the pmc1Delta strain grew much more slowly than the wild type strain (Fig. 1C). A further doubling of the Ca2+ concentration in the YPD plate to 400 mM completely inhibited growth of the pmc1Delta strain but not the growth of the wild type and vcx1Delta 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 mM CaCl2 did not cause the growth sensitivity described above. We conclude that strains harboring the vps33Delta mutation show greater sensitivity to high extracellular Ca2+ than strains carrying the pmc1Delta mutation, the vcx1Delta mutation, or both mutations together. Overall, the rank order of Ca2+ sensitivity observed for these strains was: vps33Delta /pmc1Delta /vcx1Delta and vps33Delta strains > pmc1Delta /vcx1Delta strain > pmc1Delta strain > vcx1Delta and wild type strains.

We also examined whether the growth of these strains was sensitive to inhibition by the chelating agent EGTA. We found that pmc1Delta , vcx1Delta , and pmc1Delta /vcx1Delta strains grew similarly to the wild type strain on YPD plates buffered to pH 5.5 and supplemented with 10 mM EGTA (Fig. 1D). In contrast, the growth of the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta 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 vps33Delta 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 vps33Delta and vps33Delta /pmc1Delta /vcx1Delta strains are more sensitive to either high or low levels of environmental Ca2+ than the wild type, pmc1Delta , vcx1Delta , and pmc1Delta /vcx1Delta strains.

Measurement of Rapid Changes in the Cytosolic Ca2+ Concentration upon External Ca2+ Challenge-- Yeast cells, like mammalian cells, have been reported to maintain cytosolic free Ca2+ levels in the range of 50-200 nM (1-3). 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 (1). 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 (33). 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 the Lmax each sample was capable of discharging (Fig. 2A). 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").


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Fig. 2.   Measurement of rapid changes in cytosolic free Ca2+ levels after a Ca2+ shock. Panel A, standard curve correlating free Ca2+ concentration to aequorin-dependent light emission as measured in crude extracts. Panel B, changes in the cytosolic free Ca2+ concentration were measured in strains containing aequorin after the addition of 50 mM CaCl2 to the medium. Light emission was monitored over a 2-min period from wild type, pmc1Delta /vcx1Delta , vps33Delta , and vps33Delta /pmc1Delta /vcx1Delta strains. The Ca2+ shock was initiated by injecting 50 mM CaCl2 into the test medium after measuring the basal light emission for 10 s (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. 2B). 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 pmc1Delta /vcx1Delta 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 pmc1Delta /vcx1Delta 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 suggests that the loss of the Pmc1p and Vcx1p vacuolar Ca2+ transporters severely compromises the ability of this strain to return its cytosolic Ca2+ concentration to a low resting level after exposure to elevated extracellular Ca2+.

We next examined the response of strains carrying the vps33Delta mutation to Ca2+ shock. We found that the initial resting cytosolic Ca2+ concentration was ~165 nM, which was 2-fold higher than the wild type strain. The basal cytosolic Ca2+ level measured in the vps33Delta /pmc1Delta /vcx1Delta strain was ~210 nM, which was almost 3-fold higher than the wild type strain. When the vps33Delta strain was exposed to Ca2+ shock, the maximum light emission was nearly 100-fold higher than observed with the wild type strain and corresponded to a peak cytosolic Ca2+ concentration of ~1.75 µM (Fig. 2B). This level was 5-fold higher than the peak observed with the wild type strain. Like the pmc1Delta /vcx1Delta strain, the recovery of the vps33Delta strain from the peak cytosolic Ca2+ level was much weaker than the wild type control and reached a new steady-state level at ~470 nM (6-fold higher than the wild type strain). The vps33Delta /pmc1Delta /vcx1Delta strain exhibited a high peak of cytosolic Ca2+ which corresponded to ~1.5 µM, which was somewhat lower than was observed with the vps33Delta strain. However, the recovery of this strain from the peak level was even weaker than the vps33Delta strain and reached a new steady-state level of ~660 nM (more than 8-fold higher than the wild type strain). The weaker recovery of this strain may indicate that a low level of residual function of the Pmc1p and/or the Vcx1p transporters remains within the vesicles that accumulate in the vps33Delta strain. When taken together, these results indicate that strains carrying the vps33Delta mutation are severely compromised in their ability to regulate basal cytosolic Ca2+ levels and are unable to sequester efficiently the cytosolic Ca2+ that enters the cell after an acute Ca2+ shock.

Measurement of Total Cellular Ca2+, Mg2+, and Phosphate Levels in Yeast Vacuolar Mutants-- A large fraction of total cellular Ca2+, Mg2+, and polyphosphate normally resides within the vacuole (6, 8, 35, 36). To determine how these various vacuolar mutations affect the capacity to store these compounds within the vacuole, we measured their total cellular levels (Fig. 3). We did not detect a significant change in the level of Mg2+ in the pmc1Delta /vcx1Delta strain, although a small (22%) decrease in total cellular inorganic phosphate (orthophosphate and polyphosphate) was observed. In contrast, the total cellular Ca2+ level was reduced nearly 2-fold in the pmc1Delta /vcx1Delta strain.


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Fig. 3.   The vps33Delta mutation causes a large decrease in cellular Mg2+ and phosphate but does not reduce the amount of cellular Ca2+. Cultures of the indicated strains were grown in standard YPD medium, and the relative amounts of Mg2+ (panel A), inorganic phosphate (panel B), and Ca2+ (panel C) were determined as described under "Materials and Methods." WT, wild type.

The cellular levels of these three compounds were significantly different in strains carrying the vps33Delta mutation. We found that the total amount of cellular Mg2+ was 3-fold lower in both the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta strains. Similarly, the total inorganic phosphate level was reduced more than 4-fold in the vps33Delta strain and 6-fold in the vps33Delta /pmc1Delta /vcx1Delta / strain. Thus, strains carrying the vps33Delta mutation exhibited a severe reduction in the cellular content of Mg2+ and inorganic phosphate, consistent with a reduced capacity to store these ions within the vacuole of strains carrying the vps33Delta mutation.

Because >90% of total cellular Ca2+ is normally stored within the vacuole (5, 6), we expected the vps33Delta strain also to contain a much lower level of total Ca2+. However, we found that both the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta / strains contained 15-20% more cellular Ca2+ than the wild type strain. Thus, the vacuolar biogenesis defect associated with the vps33Delta mutation resulted in a net increase in total cellular Ca2+, and this phenotype was epistatic to the decrease in total cellular Ca2+ observed in the pmc1Delta /vcx1Delta mutant. When taken in conjunction with the observation that the vacuolar storage of Mg2+ and inorganic phosphate is compromised in strains carrying the vps33Delta mutation, these results suggest that another intracellular compartment is capable of compensating for the defects in Ca2+ storage and homeostasis in strains carrying the vps33Delta mutation.

Membrane Permeability of the Vacuolar Mutants-- The results described above indicate that the vps33Delta mutation has effects on Ca2+ homeostasis which differ significantly from the combined loss of the Pmc1p and Vcx1p vacuolar Ca2+ transporters. One possible explanation for the higher level of Ca2+ observed is that the rate of Ca2+ uptake in the vps33Delta strain is increased. To test this possibility, we measured the rate of 45Ca2+ uptake in each strain (Fig. 4). A CaCl2 solution containing the radionuclide was added to cells at a final concentration of 1 mM. Aliquots were then collected at intervals over a period of 90 s to determine the rate of Ca2+ uptake. All four strains (wild type, pmc1Delta /vcx1Delta , vps33Delta , and vps33Delta /pmc1Delta /vcx1Delta ) showed a similar rate of Ca2+ uptake, indicating that the vps33Delta mutation does not significantly alter the rate of Ca2+ uptake under the conditions examined (1 mM extracellular Ca2+). If the vps33Delta mutation altered the plasma membrane permeability in a more general, nonspecific way, it is likely that the concentration of other intracellular ions would also be altered. To examine this possibility, we measured the steady-state concentrations of the monovalent K+ and Na+ ions in each strain when grown in YPD medium (Fig. 5). We found that none of the strains had any significant differences in the total cellular concentrations of either cation. Taken together, these results indicate that the membrane permeabilities of Ca2+, K+, and Na+ are not altered significantly in strains carrying the vps33Delta mutation under the conditions examined.


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Fig. 4.   Measurement of the rate of 45Ca2+ uptake. The uptake of 45Ca2+ was measured in the indicated strains over a time interval of 90 s in cells growing in SMD medium. Squares, wild type; diamonds, pmc1Delta /vcx1Delta ; circles, vps33Delta ; triangles, vps33Delta /pmc1Delta /vcx1Delta .


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Fig. 5.   Measurement of total cellular Na+ and K+ levels. Cultures of the indicated strains were grown in standard YPD medium, and the relative amounts of K+(panel A) and Na+ (panel B) were determined as described under "Materials and Methods." WT, wild type.

45Ca2+ Exchange in the Vacuolar Mutants-- The total cellular Ca2+ found in yeast cells exists in two distinct forms, termed the exchangeable and nonexchangeable pools (6, 8). The exchangeable pool represents Ca2+ that can readily leave the cell, whereas the nonexchangeable pool is thought to represent a more stable pool of Ca2+ located primarily within the vacuole in a complex with polyphosphate. To determine the partitioning of cellular Ca2+ between the exchangeable and nonexchangeable pools in the vps33Delta strains, we measured 45Ca2+ efflux. Strains were grown in YPD medium containing 45Ca2+ for four generations. After washing and resuspending the cells in fresh YPD medium containing 50 mM CaCl2, the amount of 45Ca2+ that remained associated with cells from each strain was determined at various times (Fig. 6A). We found that the wild type and pmc1Delta /vcx1Delta strains quickly exchanged a small portion of the total cellular Ca2+ during the first 15 min and subsequently exchanged Ca2+ at a much slower rate. In contrast, both strains carrying the vps33Delta mutation exhibited a much longer period of Ca2+ exchange which extended for 90 min for the vps33Delta /pmc1Delta /vcx1Delta strain and 210 min for the vps33Delta strain. This indicates that most of the Ca2+ within strains carrying the vps33Delta mutation does not reside within a nonexchangeable pool. Although the larger size of the exchangeable Ca2+ pool may partially account for the increased period of time required to release the exchangeable Ca2+ pool in these strains, other factors may also be involved.


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Fig. 6.   Measurement of 45Ca2+ efflux. The indicated strains were grown for four generations in YPD medium supplemented with 45Ca2+. To initiate Ca2+ release, the strains were harvested, washed, and resuspended in YPD supplemented with 50 mM CaCl2. The amount of 45Ca2+ that remained cell-associated was determined at the indicated times and converted to total cellular Ca2+ as described under "Materials and Methods." Panel A, absolute amounts of cell-associated Ca2+. Squares, wild type; diamonds, pmc1Delta /vcx1Delta ; circles, vps33Delta ; and triangles, vps33Delta /pmc1Delta /vcx1Delta . Panel B, nonexchangeable Ca2+ pools. Panel C, exchangeable Ca2+ pools. WT, wild type.

Because the strains carrying the vps33Delta mutation exhibited a prolonged time of release of their exchangeable pools, we compared the nonexchangeable and exchangeable Ca2+ pools in each strain after Ca2+ efflux was allowed to proceed for 210 min (Fig. 6, B and C). Under these conditions, we found that the wild type strain contained 4.9 mmol of Ca2+/kg dry mass in its nonexchangeable pool. In contrast, the pmc1Delta /vcx1Delta strain retained only 0.5 mmol of Ca2+/kg of dry mass after 210 min of efflux. Similarly, the vps33Delta strain held 1.0 mmol of Ca2+/kg dry mass, and the vps33Delta /pmc1Delta /vcx1Delta strain held 0.6 mmol of Ca2+/kg dry mass in their nonexchangeable pools. Thus, the nonexchangeable pool in each of these mutant strains is 5-10-fold smaller than in the wild type strain, indicating that all three mutant strains are severely compromised in their ability to store Ca2+ within the vacuolar nonexchangeable pool.

When we calculated the amount of Ca2+ that was readily mobilized during 210 min of efflux, we found that the exchangeable pool in the wild type strain contained 1.2 mmol of Ca2+/kg dry mass. This pool held 6.9 mmol of Ca2+/kg dry mass in the vps33Delta strain and 8.1 mmol of Ca2+/kg dry mass in the vps33Delta /pmc1Delta /vcx1Delta strain. Thus, the absolute amount of Ca2+ in the exchangeable pool in these strains was 6-7-fold larger than in the wild type strain. In contrast, the amount of Ca2+ in the exchangeable pool in the pmc1Delta /vcx1Delta strain was 1.8 mmol of Ca2+/kg dry mass, which was only 1.5-fold higher than the wild type strain. These results indicate that although the nonexchangeable Ca2+ pools within the three vacuolar mutant strains are similar, the exchangeable pools found in the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta strains are roughly 4-fold larger than those found in the pmc1Delta /vcx1Delta strain.

Sensitivity of Vacuolar Mutants to Cyclosporin A-- Several studies have found that the loss of calcineurin function leads to a significant increase in the steady-state level of cellular Ca2+ (7, 14, 37-39). To determine how the vps33Delta strain responds to such an increase in intracellular Ca2+, we compared the growth of these strains on YPD plates (pH 5.5) with and without 20 µg/ml CsA (Fig. 7). In the absence of CsA, the vps33Delta and the vps33Delta /pmc1Delta /vcx1Delta strains again had a slightly slower growth rate than the other strains. However, when CsA was added to the plates the growth of the vps33Delta /pmc1Delta /vcx1Delta strain was completely blocked, whereas the vps33Delta strain showed a severe growth defect compared with plates lacking CsA. In contrast, growth of the wild type and pmc1Delta /vcx1Delta strains was unaffected by the presence of CsA. These results are consistent with the possibility that the vps33Delta mutation reduces the ability of these strains to sequester adequately the increased intracellular Ca2+ that accumulates upon the inhibition of calcineurin function.


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Fig. 7.   Sensitivity of vacuolar mutants to CsA. The indicated strains were streaked on YPD (panel A) or YPD supplemented with 20 µg/ml CsA (panel B). WT, wild type.

The Golgi Ca2+ ATPase Pmr1p Participates in the Maintenance of Cellular Ca2+ Homeostasis during Ca2+ Stress-- The results presented above clearly demonstrate that the vps33Delta mutation severely disrupts intracellular Ca2+ homeostasis. Despite the severe defects in vacuolar structure and Ca2+ sequestration which result from this mutation, they remain viable and accumulate a normal amount of total cellular Ca2+. This raised the possibility that other intracellular organelles may compensate for the loss of vacuolar Ca2+ storage in these strains. Besides the vacuole, two compartments within the secretory pathway have also been implicated in Ca2+ storage in yeast. The PMR1 gene encodes a Ca2+ ATPase that has been localized to the Golgi apparatus (19, 21, 22) and was also recently reported to influence the rate of degradation of proteins within the endoplasmic reticulum (40). Given this well defined role of Pmr1p as a Ca2+ ATPase within a non-vacuolar compartment, we next tested whether Pmr1p may be involved in maintaining Ca2+ homeostasis in strains defective in vacuolar biogenesis.

First, we examined PMR1 mRNA levels to determine whether its expression changes in response to either the concentration of environmental Ca2+ or mutations that effect Ca2+ homeostasis. To provide the broadest range of environmental Ca2+ concentrations during this experiment, strains were grown in YPD containing 1 mM EGTA (calculated to reduce the free Ca2+ concentration to approximately 0.01 mM) or in YPD supplemented with 50 mM CaCl2. RNA was extracted from each strain, and the level of PMR1 mRNA was determined (relative to an ACT1 control). In the wild type strain, we found that the relative level of PMR1 mRNA increased 1.4-fold as extracellular Ca2+ increased (Fig. 8). In the pmr1Delta /vcx1Delta strain, we found that the PMR1 mRNA level was slightly elevated in the low Ca2+ medium and was increased to 1.6-fold above the wild type control when the environmental Ca2+ was increased. Finally, the PMR1 mRNA level in the vps33Delta strain was 1.6-fold higher than the wild type strain when grown in the presence of low Ca2+ and was increased to 2.2-fold higher than the wild type control when grown in the presence of 50 mM Ca2+. These results indicate that PMR1 gene expression increases moderately as a function of the Ca2+ stress on a wild type strain or as a consequence of mutations that effect the maintenance of intracellular Ca2+ homeostasis.


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Fig. 8.   Northern analysis of PMR1 mRNA isolated from strains grown in YPD containing 1 mM EGTA (calculated to yield 0.01 mM free Ca2+) or YPD containing 50 mM CaCl2. The abundance of PMR1 mRNA was corrected for the recovery of ACT1 mRNA in each strain and normalized to the PMR1 mRNA level measured in the wild type (WT) strain grown under low Ca2+ conditions.

To address further the role of Pmr1p in maintaining Ca2+ homeostasis in strains carrying the vps33Delta mutation, we examined the progeny of a cross between a pmr1Delta strain and a vps33Delta /pmc1Delta /vcx1Delta strain. A total of 36 tetrads was dissected, and the genotype of the 107 viable spores was determined. We found that all but three possible combinations of mutations were obtained. The nonviable combinations, which all contained the pmr1Delta mutation, were: pmr1Delta /pmc1Delta /vcx1Delta , pmr1Delta /vps33Delta /pmc1Delta , and pmr1Delta /vps33Delta /pmc1Delta /vcx1Delta . This indicates that the loss of both Ca2+ transporters located in the vacuole (pmc1Delta and vcx1Delta ) in conjunction with the Golgi apparatus Ca2+ transporter (pmr1Delta ) is lethal. Although strains lacking both the vacuolar Ca2+ ATPase (Pmc1p) and Golgi apparatus Ca2+ ATPase (Pmr1p) were viable, the introduction of mutations that further compromised Ca2+ homeostasis (either the vps33Delta or the vcx1Delta mutation) apparently resulted in a lethal imbalance in Ca2+ homeostasis. These results indicate that specific combinations of both vacuolar and Golgi mutations lead to insurmountable defects in Ca2+ homeostasis.

Previous studies reported that disruption of the PMR1 gene does not confer sensitivity to elevated levels of environmental Ca2+ (19-21). This led to the conclusion that the Golgi apparatus does not play a significant role in maintaining cellular Ca2+ homeostasis under conditions of Ca2+ stress. To determine whether the Golgi apparatus plays a more significant role in this process when vacuolar Ca2+ storage is compromised, we next examined the ability of the pmr1Delta /vps33Delta strain to grow in the presence of elevated environmental Ca2+ (Fig. 9). This strain grew somewhat slower than the vps33Delta strain on standard YPD medium. Although the vps33Delta and vps33Delta /pmc1Delta /vcx1Delta strains were capable of growth on YPD plates containing 50 mM CaCl2, growth of the pmr1Delta /vps33Delta strain was completely inhibited. These results indicate that the Golgi apparatus acts to compensate for the defective Ca2+ homeostasis associated with the vps33Delta strain.


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Fig. 9.   The pmr1Delta mutation increases Ca2+ sensitivity in conjunction with the vps33Delta mutation. WT, wild type.

Finally, we examined whether strains carrying the pmr1Delta mutation alone also exhibited a growth defect in the presence of high environmental Ca2+ (Fig. 10). We found that each of the four strains examined (wild type, pmr1Delta , pmc1Delta , and vcx1Delta ) grew with similar rates on plates containing 100 mM CaCl2. However, we found that the pmr1Delta and pmc1Delta strains were unable to grow on plates containing 500 mM CaCl2, whereas the wild type and vcx1Delta strains did grow under these conditions. These results indicate that in cells with intact vacuolar function, Pmr1p plays a more important role in maintaining Ca2+ homeostasis upon exposure to extreme Ca2+ stress than the vacuolar Vcx1p transporter.


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Fig. 10.   Pmr1p plays a role in Ca2+ homeostasis in strains with intact vacuolar function under conditions of extreme Ca2+ stress. WT, wild type.


    DISCUSSION

Wild type strains of S. cerevisiae are capable of maintaining intracellular Ca2+ levels within a narrow range when faced with extracellular Ca2+ concentrations ranging from <1 µM to >100 mM. Consistent with the fact that the yeast vacuole normally contains >90% of the total cellular Ca2+, mutations in Ca2+ transporters which limit vacuolar Ca2+ uptake have been shown to cause a 2-3-fold reduction in the total cellular Ca2+ levels (7, 14, 15). Similarly, we observed a 2-fold decrease in total cellular Ca2+ in the pmc1Delta /vcx1Delta strain. In contrast, we found that strains carrying the vps33Delta vacuolar biogenesis mutation have total cellular Ca2+ levels that are slightly higher than the wild type strain. This result was surprising based upon the severe defects in vacuolar biogenesis caused by mutations in this gene (24-26) in conjunction with our finding that the steady-state levels of two other substances normally stored primarily within the vacuole (Mg2+ and polyphosphate) were greatly reduced. Because the vps33Delta /pmc1Delta /vcx1Delta strain (which lacks both known vacuolar Ca2+ transporters) also had this high level of total cellular Ca2+, the increased accumulation of Ca2+ cannot be attributed to the residual function of these transporters in a prevacuolar compartment. Instead, our results suggest that the loss of most (or all) vacuolar Ca2+ storage in strains carrying the vps33Delta mutation leads to the redistribution of a significant portion of intracellular Ca2+ into the Golgi apparatus and possibly other intracellular compartments as well.

The Golgi apparatus contains the only non-vacuolar Ca2+ ATPase (Pmr1p) that has been characterized in yeast (19-22). Because the pmr1Delta strain was not previously found to be sensitive to elevated extracellular Ca2+, it was not thought to play a significant role in maintaining cellular Ca2+ homeostasis. However, we found that a pmr1Delta /vps33Delta strain is more sensitive to elevated extracellular Ca2+ than the vps33Delta strain alone, and PMR1 gene expression is elevated in the vps33Delta strain. In addition, we found that certain combinations of mutations affecting both vacuolar and Golgi Ca2+ transport (pmr1Delta /pmc1Delta /vcx1Delta , pmr1Delta /vps33Delta /pmc1Delta , and pmr1Delta /vps33Delta /pmc1Delta /vcx1Delta ) resulted in synthetic lethality. Taken together, these results indicate that the Golgi apparatus of yeast plays a significant role in cellular Ca2+ homeostasis through a Pmr1p-dependent mechanism when vacuolar Ca2+ storage is compromised. We also found that a pmr1Delta strain with normal vacuolar function is sensitive to high levels of Ca2+ in the growth medium. Given the fact that the Golgi has not previously been observed to play a role in Ca2+ homeostasis under other growth conditions, Golgi Ca2+ sequestration may only play a significant role in cellular Ca2+ homeostasis when the cytosolic Ca2+ load exceeds the capacity of the vacuolar Ca2+ storage system.

Although our study clearly implicates Pmr1p in the maintenance of Ca2+ homeostasis in vps33Delta strains, we observed only a 2-fold increase in PMR1 transcription. Although a larger increase might have been expected, it is possible that PMR1 expression is regulated primarily at a post-transcriptional level. In this way a significant increase in Pmr1p activity could occur without a concomitant increase in mRNA abundance (or protein abundance if the regulation is exerted at a post-translational level). Alternatively, Pmr1p may be present and active under all conditions, but the vacuolar Ca2+ uptake system may sequester cytosolic Ca2+ more efficiently than the Golgi apparatus under all but the most severe conditions. This could occur, for example, if the vacuolar transporters were activated at a lower cytosolic Ca2+ concentration than Pmr1p. By either mechanism, a high level of Golgi Ca2+ storage would not be observed under most growth conditions that did not subject the cells to high Ca2+ stress. Such an overlapping hierarchy of transporter activation to control Ca2+ homeostasis (either at the level of synthesis or function) would be consistent with the observations obtained in the current study. Such a mechanism would also explain why Pmr1p was not attributed a role in the maintenance of cellular Ca2+ homeostasis in previous studies.

Other results obtained in this study are also consistent with a hierarchical control of Ca2+ homeostasis. First, we found that strains carrying the vps33Delta mutation exhibit a 2-3-fold higher basal level of cytosolic Ca2+ when incubated in a medium containing only 10 µM Ca2+ (Fig. 2). This finding provides evidence that Pmr1p function may be activated at a higher cytosolic Ca2+ concentration than the vacuolar Ca2+ transporters. Because our results suggest that the secondary system utilizing Pmr1p plays a larger role in Ca2+ homeostasis in strains carrying the vps33Delta mutation, it would be expected that the basal cytosolic Ca2+ would be maintained near the concentration that activates this transporter. We also found that strains carrying the vps33Delta mutation exhibited a severe defect in the maintenance of cytosolic Ca2+ homeostasis when exposed to 50 mM extracellular CaCl2. Under these conditions, we found that the cytosolic Ca2+ concentration of the vps33Delta strain quickly rose to 1.75 µM, a level that was 6-fold higher than the wild type strain. Furthermore, the rate of recovery was slower, and the new steady-state level that was reached was also much higher than the control strain. Again, these results suggest that this secondary system of Ca2+ sequestration cannot remove excess Ca2+ from the cytosol as quickly as the vacuolar system. Despite these limitations, this system remains capable of maintaining intracellular Ca2+ homeostasis (at least to the extent required to maintain growth) in strains carrying the vps33Delta mutation when challenged by environmental concentrations as high as 50 mM Ca2+ (see Fig. 9).

Previous studies have shown that strains carrying vps33 mutations mislocalize the vacuolar membrane protein alkaline phosphatase to the cell surface (24-26). Unfortunately, neither the extent of the mislocalization of other vacuolar membrane proteins nor the composition of vesicles that accumulate in the vps33Delta strain has been characterized further. Nevertheless, it is possible that vacuolar Ca2+ transporters may also be mislocalized to the cell surface and thus could potentially contribute to the increased cytosolic Ca2+ levels observed upon exposure to high extracellular Ca2+. However, we found that the peak cytosolic Ca2+ level was still 5-fold higher than the wild type strain in the vps33Delta /pmc1Delta /vcx1Delta strain. This indicates that the mislocalization of the Pmc1p and Vcx1p transporters to the plasma membrane is not responsible for most of the elevated cytosolic Ca2+ observed in strains carrying the vps33Delta mutation. Two additional lines of evidence suggest that the vps33Delta mutation does not significantly alter the general permeability of the plasma membrane. First, the steady-state cellular concentrations of two other cations, K+ and Na+, were unaffected by the vps33Delta mutation. In addition, the rate of 45Ca2+ uptake measured in strains carrying the vps33Delta mutation was identical to that of the wild type strain. Taken together, these results suggest that the vps33Delta mutation does not significantly alter the permeability of the plasma membrane in the vps33Delta strain. As discussed above, it is more likely that the higher peak in cytosolic Ca2+ is caused by a reduced capacity to sequester the Ca2+ into other cellular compartments rapidly.

Several studies have reported that the loss of calcineurin function increases the total cellular Ca2+ level (7, 14, 37-39). Our finding that the vps33Delta strain shows an increased sensitivity to CsA on standard YPD medium is also consistent with the model that this secondary system of Ca2+ sequestration is not capable of transporting Ca2+ from the cytosol into intracellular compartments as efficiently as the wild type strain. In addition, it has been shown that the induction of PMR1 expression is prevented by the immunosuppressive drug FK506 (which functions to inhibit calcineurin activation in a manner analogous to CsA) (14). Thus, the combined effects of increased cellular Ca2+ uptake and lack of PMR1 induction could account for the increased CsA sensitivity that was observed in the vps33Delta strains.

The results of this study provide evidence that the Golgi apparatus plays a significant role in the maintenance of cellular Ca2+ homeostasis under conditions where the accumulation of cytosolic Ca2+ exceeds the capacity of the vacuole. This suggests that the vacuolar storage system that normally mediates the bulk of Ca2+ homeostasis in yeast may have been superimposed upon another system that is functionally related to the Ca2+ storage and signaling system found within the secretory pathway of mammalian cells. Further studies are required to determine whether other intracellular organelles of yeast (such as mitochondria) also participate in the maintenance of cellular Ca2+ homeostasis under conditions of extreme Ca2+ stress.

    ACKNOWLEDGEMENTS

We thank Scott Emr, Patrick Masson, and Ann Batiza for providing strains and plasmids. We also thank Kyle Cunningham for helpful discussions and Richard Marchase and Barclay Browne for providing critical comments on the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Visiting Scientist from the Department of Clinical Chemistry, University Medical School, Peçs, Hungary.

§ To whom correspondence should be addressed: Dept. of Microbiology, Bevill Biomedical Research Bldg., Rm. 432, University of Alabama, Birmingham, AL 35294-2170. Tel.: 205-934-6593; Fax: 205-975-5482; E-mail: dbedwell{at}uab.edu.

    ABBREVIATIONS

The abbreviations used are: CsA, cyclosporin A; kb, kilobase; MES, 4-morpholineethanesulfonic acid; L, light emission; Lmax, maximum light emission.

    REFERENCES
Top
Abstract
Introduction
References
  1. Batiza, A. F., Schulz, T., and Masson, P. H. (1996) J. Biol. Chem. 271, 23357-23362[Abstract/Free Full Text]
  2. Iida, H., Yagawa, Y., and Anraku, Y. (1990) J. Biol. Chem. 265, 13391-13399[Abstract/Free Full Text]
  3. Nakajima-Shimada, J., Iida, H., Tsuji, F. I., and Anraku, Y. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6878-6882[Abstract]
  4. Cunningham, K. W., and Fink, G. R. (1994) J. Exp. Biol. 196, 157-166[Abstract/Free Full Text]
  5. Ohsumi, Y., Kitamoto, K., and Anraku, Y. (1988) J. Bacteriol. 170, 2676-2682[Medline] [Order article via Infotrieve]
  6. Eilam, Y., Lavi, H., and Grossowicz, N. (1985) J. Gen. Microbiol. 131, 623-629
  7. Cunningham, K. W., and Fink, G. R. (1994) J. Cell Biol. 124, 351-363[Abstract]
  8. Dunn, T., Gable, K., and Beeler, T. (1994) J. Biol. Chem. 269, 7273-7278[Abstract/Free Full Text]
  9. Garrett-Engele, P., Moilanen, B., and Cyert, M. S. (1995) Mol. Cell. Biol. 15, 4103-4114[Abstract]
  10. Hemenway, C. S., Dolinski, K., Cardenas, M. E., Hiller, M. A., Jones, E. W., and Heitman, J. (1995) Genetics 141, 833-844[Abstract/Free Full Text]
  11. Kitamoto, K., Yoshizawa, K., Ohsumi, Y., and Anraku, Y. (1988) J. Bacteriol. 170, 2687-2691[Medline] [Order article via Infotrieve]
  12. Ohya, Y., Miyamoto, S., Ohsumi, Y., and Anraku, Y. (1986) J. Bacteriol. 165, 28-33[Medline] [Order article via Infotrieve]
  13. Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977[Abstract/Free Full Text]
  14. Cunningham, K. W., and Fink, G. R. (1996) Mol. Cell. Biol. 16, 2226-2237[Abstract]
  15. Pozos, T. C., Sekler, I., and Cyert, M. S. (1996) Mol. Cell. Biol. 16, 3730-3741[Abstract]
  16. Breuder, T., Hemenway, C. S., Movva, N. R., Cardenas, M. E., and Heitman, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5372-5376[Abstract]
  17. Foor, F., Parent, S. A., Morin, N., Dahl, A. M., Ramadan, N., Chrebet, G., Bostian, K. A., and Nielsen, J. B. (1992) Nature 360, 682-684[CrossRef][Medline] [Order article via Infotrieve]
  18. Catty, P., and Goffeau, A. (1996) Biosci. Rep. 16, 75-85[Medline] [Order article via Infotrieve]
  19. Antebi, A., and Fink, G. R. (1992) Mol. Biol. Cell 3, 633-654[Abstract]
  20. Halachmi, D., and Eilam, Y. (1996) FEBS Lett. 392, 194-200[CrossRef][Medline] [Order article via Infotrieve]
  21. Rudolph, H. K., Antebi, A., Fink, G. R., Buckley, C. M., Dorman, T. E., LeVitre, J. A., Davidow, L. S., Mao, J., and Moir, D. T. (1989) Cell 58, 133-145[Medline] [Order article via Infotrieve]
  22. Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895-9901[Abstract/Free Full Text]
  23. Bryant, N. J., and Stevens, T. H. (1998) Microbiol. Mol. Biol. Rev. 62, 230-247[Abstract/Free Full Text]
  24. Banta, L. M., Robinson, J. S., Klionsky, D. J., and Emr, S. D. (1988) J. Cell Biol. 107, 1369-1383[Abstract]
  25. Banta, L. M., Vida, T. A., Herman, P. K., and Emr, S. D. (1990) Mol. Cell. Biol. 10, 4638-4649[Medline] [Order article via Infotrieve]
  26. Robinson, J. S., Klionsky, D. J., Banta, L. M., and Emr, S. D. (1988) Mol. Cell. Biol. 8, 4936-4948[Medline] [Order article via Infotrieve]
  27. Rieder, S. E., and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327[Abstract/Free Full Text]
  28. Rothstein, R. J. (1983) Methods Enzymol. 101, 202-209[Medline] [Order article via Infotrieve]
  29. Jones, J. S., and Prakash, L. (1990) Yeast 6, 363-366[Medline] [Order article via Infotrieve]
  30. Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in Yeast Genetics: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  31. Miller, J. H. (1992) A Short Course in Bacterial Genetics., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  32. Stanton, M. G. (1968) Anal. Biochem. 22, 27-34[Medline] [Order article via Infotrieve]
  33. Allen, D. G., Blinks, J. R., and Prendergast, F. G. (1977) Science 195, 996-998[Medline] [Order article via Infotrieve]
  34. Bonetti, B., Fu, L., Moon, J., and Bedwell, D. M. (1995) J. Mol. Biol. 251, 334-345[CrossRef][Medline] [Order article via Infotrieve]
  35. Okorokov, L. A., Letrikevich, S. B., Lichko, L. P., and Mel'nikova, E. V. (1978) Biol. Bull. Acad. Sci. USSR 5, 638-640[Medline] [Order article via Infotrieve]
  36. Okorokov, L. A., Lichko, L. P., and Kulaev, I. S. (1980) J. Bacteriol. 144, 661-665[Medline] [Order article via Infotrieve]
  37. Tanida, I., Hasegawa, A., Iida, H., Ohya, Y., and Anraku, Y. (1995) J. Biol. Chem. 270, 10113-10119[Abstract/Free Full Text]
  38. Tanida, I., Takita, Y., Hasegawa, A., Ohya, Y., and Anraku, Y. (1996) FEBS Lett. 379, 38-42[CrossRef][Medline] [Order article via Infotrieve]
  39. Withee, J. L., Mulholland, J., Jeng, R., and Cyert, M. S. (1997) Mol. Biol. Cell 8, 263-277[Abstract]
  40. Durr, G., Strayle, J., Plemper, R., Elbs, S., Klee, S. K., Catty, P., Wolf, D. H., and Rudolph, H. K. (1998) Mol. Biol. Cell 9, 1149-1162[Abstract/Free Full Text]


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