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Originally published In Press as doi:10.1074/jbc.M400833200 on July 13, 2004

J. Biol. Chem., Vol. 279, Issue 37, 38495-38502, September 10, 2004
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The Ca2+ Homeostasis Defects in a pgm2{Delta} Strain of Saccharomyces cerevisiae Are Caused by Excessive Vacuolar Ca2+ Uptake Mediated by the Ca2+-ATPase Pmc1p*

David P. Aiello{ddagger}§, Lianwu Fu{ddagger}§, Attila Miseta¶, Katalin Sipos||, and David M. Bedwell{ddagger}**

From the {ddagger}Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, and Departments of Clinical Chemistry and ||Biochemistry, University Medical School, Peçs H-7624, Hungary

Received for publication, January 26, 2004 , and in revised form, June 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Loss of the major isoform of phosphoglucomutase (PGM) causes an accumulation of glucose 1-phosphate when yeast cells are grown with galactose as the carbon and energy source. Remarkably, the pgm2{Delta} strain also exhibits a severe imbalance in intracellular Ca2+ homeostasis when grown under these conditions. In the present study, we examined how the pgm2{Delta} mutation alters yeast Ca2+ homeostasis in greater detail. We found that a shift from glucose to galactose as the carbon source resulted in a 2-fold increase in the rate of cellular Ca2+ uptake in wild-type cells, whereas Ca2+ uptake increased 8-fold in the pgm2{Delta} mutant. Disruption of the PMC1 gene, which encodes the vacuolar Ca2+-ATPase Pmc1p, suppressed the Ca2+-related phenotypes observed in the pgm2{Delta} strain. This suggests that excessive vacuolar Ca2+ uptake is tightly coupled to these defects in Ca2+ homeostasis. An in vitro assay designed to measure Ca2+ sequestration into intracellular compartments confirmed that the pgm2{Delta} mutant contained a higher level of Pmc1p-dependent Ca2+ transport activity than the wild-type strain. We found that this increased rate of vacuolar Ca2+ uptake also coincided with a large induction of the unfolded protein response in the pgm2{Delta} mutant, suggesting that Ca2+ uptake into the endoplasmic reticulum compartment was reduced. These results indicate that the excessive Ca2+ uptake and accumulation previously shown to be associated with the pgm2{Delta} mutation are due to a severe imbalance in the distribution of cellular Ca2+ into different intracellular compartments.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The regulation of intracellular Ca2+ homeostasis in eukaryotic cells is a remarkably intricate process. Ca2+ transport across the plasma membrane and its intracellular sequestration is tightly regulated such that the resting cytosolic Ca2+ concentration is maintained in a range of 50–200 nM (1–4). Small variations in cytosolic Ca2+ that occur in response to a number of stimuli are sufficient to activate a variety of Ca2+-sensing proteins, such as calmodulin and calcineurin. This then leads to the induction of various downstream signal transduction pathways (5). Equally as important, the Ca2+ concentrations within the lumen of the endoplasmic reticulum (ER)1 and Golgi apparatus are carefully maintained to ensure the proper folding and processing of proteins transported through the secretory pathway (6).

In the budding yeast Saccharomyces cerevisiae, the vacuole is the major cellular Ca2+ storage compartment and contains >95% of total cellular Ca2+ (7). This large store of Ca2+ is maintained through the action of two transporters, the Ca2+-ATPase Pmc1p and the Ca2+/H+ exchanger Vcx1p (8, 9). Once thought to be relatively static by virtue of its association with inorganic polyphosphate (7), the vacuolar Ca2+ store has recently been suggested to be more dynamic in nature. The recently identified yeast transient receptor potential channel homologue, Yvc1p, was shown to localize to the vacuolar membrane and mediate Ca2+ efflux out of the vacuole (10). Additional reports have shown that vacuolar Ca2+ efflux by Yvc1p can be specifically induced by hypotonic shock, which may be mediated by a mechano-sensitive mechanism (11, 12).

In addition to the vacuole, the ER and Golgi apparatus are also important for maintaining proper intracellular Ca2+ homeostasis in yeast. The transporters responsible for maintaining proper Ca2+ levels in the secretory pathway include the Ca2+-ATPases Pmr1p (3, 13–16) and Cod1p/Spf1p (17–20). Pmr1p is localized primarily to the Golgi apparatus, where it plays an essential role in maintaining the lumenal Ca2+ concentration required for the proper glycosylation and processing of proteins in this compartment (13, 14). The loss of Pmr1p results in a number of alterations in Ca2+ homeostasis, including an increased rate of cellular Ca2+ uptake from the extracellular environment and a greater sensitivity to elevated extracellular Ca2+ levels (15). The elevated Ca2+ uptake observed in the pmr1{Delta} mutant is mediated by the MID1 and CCH1 gene products and is reminiscent of the mammalian capacitative Ca2+ entry (CCE) response (21). The depletion of secretory pathway Ca2+ stores caused by the pmr1{Delta} mutation also leads to improper folding and processing of proteins that transit through the ER and Golgi (14, 22). It was recently reported that this lumenal Ca2+ depletion induces the unfolded protein response (UPR) (23). The UPR is activated by the presence of unfolded proteins in the ER and results in the increased expression of molecular chaperones that aid in protein folding in this compartment (24). This increased expression is mediated by the transcription factor Hac1p/Ern4p (25–27).

Recent evidence suggests that some products of carbohydrate metabolism also influence intracellular Ca2+ homeostasis in yeast cells. In particular, the sugar phosphates Glc-6-P and Glc-1-P have been proposed to play a role in modulating intracellular Ca2+ homeostasis (28, 29). The enzyme phosphoglucomutase (PGM) interconverts Glc-1-P and Glc-6-P and is required for the metabolism of galactose. Yeast strains lacking the major isoform of this enzyme (pgm2{Delta}) accumulate a high level of intracellular Glc-1-P when galactose is utilized as the carbon source due to a metabolic bottleneck in the conversion of Glc-1-P to Glc-6-P. This strain also exhibits alterations in cellular Ca2+ homeostasis under these conditions, including dramatically increased Ca2+ uptake and accumulation, sensitivity to high extracellular Ca2+ concentrations, and increased sensitivity to the calcineurin inhibitor cyclosporin A (28). Most recently, it was demonstrated that the simultaneous overproduction of both Glc-1-P and Glc-6-P restored normal Ca2+ homeostasis, suggesting that the ratio of these glucose metabolites plays an important role in controlling this process (29). In the present study our results indicate that the pgm2{Delta} strain experiences elevated vacuolar Ca2+ uptake and reduced ER Ca2+ sequestration when grown with galactose as the carbon source. Furthermore, we show that these phenotypes are suppressed by the deletion of the MC1 gene. These results demonstrate that the relative levels of Glc-1-P and Glc-6-P play an important role in regulating the distribution of Ca2+ into different intracellular compartments.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Plasmids Used—Yeast strain YDB0316 is a wild-type strain described previously (28). The PGM2 gene in YDB0316 was disrupted by insertion of a previously described fragment containing the LEU2 gene (28) to generate YDB0473. To generate strains YDB0484 (pmc1{Delta}) and YDB0483 (pgm2{Delta}/pmc1{Delta}), the PMC1 gene was disrupted using standard methods (30) in YDB0316 (wt) and YDB0473 (pgm2{Delta}) using the S. cerevisiae HIS3 open reading frame that was PCR-amplified from pRS313 (31). The primers used to generate this product were DB1158 (5'-TAGAAAAGTG GTTCTAAAAA AAAAAAACTG TGTGCGTAAC AAAAAAAATA GCTTGGTGAG CGCTAGGAGT-3') and DB1159 (5'-CAATTTTGAA AATATAACTA TTACACACAT CTTTTCATTT GGTCACTTAC CTGTTCGTAT ACATACTTAC TGAC-3'). To generate strains YDB0475 (vcx1{Delta}) and YDB0474 (pgm2{Delta}/vcx1{Delta}), the VCX1 gene was disrupted using standard methods (30) in YDB0316 (wt) and YDB0473 (pgm2{Delta}) with a previously described knockout construct (3). For each strain, gene disruptions were confirmed by PCR, Southern blotting, and/or PGM enzymatic assays (28). Finally, strain BY4741 (wild type) and the isogenic yvc1{Delta} strain BY4741 (clone 1863) were purchased form Open Biosystems. A pgm2::LEU2 disruption construct (28) was used to disrupt the PGM2 gene in BY4741 to generate the isogenic pgm2{Delta} strain YDB0626 and in BY4741 (clone 1863) to generate the isogenic pgm2{Delta}/yvc1{Delta} strain YDB0627. The induction of UPR signaling was monitored using the reporter plasmid pMCZ-Y (a kind gift from Kazutoshi Mori) that contains lacZ under the control of the yeast CYC1 promoter containing an unfolded protein response element (26).

Culture Media—Bacterial strains used for cloning and plasmid maintenance were grown in standard media as described (32). Similarly, yeast media were prepared as described (30). Yeast extract/peptone (YP) medium and synthetic medium (SM) were supplemented with 2% glucose (dextrose) (YPD or SMD) or 2% galactose (YPGal or SMGal). YPD and YPGal media were routinely buffered to pH 5.5 with 40 mM MES-Tris. To prepare membranes for in vitro 45Ca2+ uptake assays, cells were grown in Yeast Mito medium containing 6.7 g of yeast nitrogen base (Difco) and 0.3% yeast extract (Difco)/liter in the presence of 2% glucose or 2% galactose (YMMG) (33). Liquid cultures were grown for a minimum of five generations to ≤1.0 A600 units/ml before harvesting.

Measurement of Whole Cell Ca2+ Uptake, Total Cellular Ca2+, and Exchangeable Ca2+ Pools—Whole cell Ca2+ uptake measurements were performed as described previously (15, 29). Measurement of total cell Ca2+ by flame photometry was also carried out as previously described (3, 29).

Cells for Ca2+ exchange experiments were grown in YPGal medium to a density of 0.05 A600. The medium was then supplemented with 45Ca2+, and growth was continued to a cell density of 0.5–1 A600/ml. The cells were then harvested by centrifugation at 4000 x g for 5 min, washed, and resuspended in fresh YPGal supplemented with 50 mM CaCl2. At the indicated times, aliquots were removed, filtered, washed, and processed for scintillation counting as previously described (3).

{beta}-Galactosidase Assays—Yeast strains transformed with plasmid pMCZ-Y were grown to mid-log phase in SMGal medium as indicated. Cells were then harvested by centrifugation and permeabilized by repeated freeze-thawing in liquid nitrogen (34). {beta}-Galactosidase activity was assayed using the colorimetric substrate 2-nitrophenyl-{beta}-D-galactopyranoside according to a previously described protocol (35). To examine the affect of extracellular Ca2+ and dithiothreitol (DTT), cells were treated with 20 mM CaCl2 or 5 mM DTT, respectively, for 4 h before harvest. Units of {beta}-galactosidase activity are defined as the absorbance at 420 nm x 103/min/A600 unit of cells.

Isolation and Subcellular Fractionation of Total Cell Membranes— Total cell membranes were isolated from yeast using a protocol based on previous publications (14, 16, 36). Yeast strains were grown at 30 °Cin the indicated media to 0.6–0.8 A600 units/ml. Before harvest, NaN3 was added to a final concentration of 10 mM, and the cells were rapidly chilled in ice water for 10 min. Cells were then harvested by centrifugation at 4 °C for 5 min at 6000 rpm (6000 x g) in a Sorvall SLA-3000 rotor. Cell pellets were resuspended in spheroplasting buffer (1.4 M sorbitol, 50 mM Tris-HCl, pH 7.5, 10 mM NaN3, 40 mM {beta}-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride (Sigma), and 0.3 mg/ml yeast lytic enzyme (ICN). Cells were converted to spheroplasts by incubation for 30–45 min at 37 °C. The spheroplasts were then harvested by centrifugation at 4 °C for 5 min at 2500 rpm (750 x g) in a Sorvall SS-34 rotor and gently resuspended in lysis buffer (0.3 M sorbitol, 20 mM triethanolamine acetate, pH 7.2, 1 mM EDTA, and protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml chymostatin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin; all from Sigma). The spheroplasts were then disrupted mechanically using 20 strokes of a Wheaton Type A (tight) Dounce homogenizer. The cell lysates were then cleared of unbroken cells by centrifugation twice at 4 °C for 5 min at 2000 rpm (450 x g) in a Sorvall SS-34 rotor. To fractionate membranes, 3 ml of lysate was loaded onto a 10-step (18–54% in 4% increments) sucrose gradient in 10 mM HEPES, pH 7.5, 1 mM MgCl2. Gradients were centrifuged at 4 °C for 2 h at 27,000 rpm in a Beckman SW-28 rotor. After centrifugation, gradient fractions were collected manually in 3-ml aliquots from top to bottom. Individual fractions from multiple gradients were then pooled and stored at –80 °C in 1-ml aliquots. Protein concentrations in each gradient fraction were determined by the method of Bradford using bovine serum albumin to generate a standard curve (37).

Ca2+ Uptake Assays in Isolated Membranes—To assay for Ca2+ transport activity in sucrose gradient fractions, 0.7 ml of O-Buffer (10 mM HEPES-NaOH, pH 6.7, 150 mM KCl, 5 mM MgCl2, 0.5 mM ATP (pre-buffered to pH 6.7), 5 mM NaN3, 0.5 µCi/ml 45CaCl2 (9.6 mCi/mg)) was added to 0.3 ml of gradient fraction and incubated for 12 min at 25 °C (36). The entire 1-ml sample was then collected by filtration through a pre-washed nitrocellulose filter (Millipore, HAWP02500) and washed twice with 5 ml of ice-cold wash buffer (10 mM HEPES-NaOH, pH 7.5, 150 mM KCl) (36). Membrane-sequestered 45Ca2+ was then determined by liquid scintillation counting. Where indicated, Ca2+ transport by Vcx1p was inhibited by the addition of 25 µM carbonyl cyanide m-chlorophenylhydrazone (Sigma). To test for the effect of sugar phosphates on Ca2+ uptake, pre-buffered (pH 6.7) glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, or mannose 6-phosphate was added to the O-Buffer mix at the indicated concentrations as indicated. To test for the effect on Ca2+ uptake by free phosphate, 5 mM sodium phosphate was added to the O-Buffer mix as indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A Sustained Increase in Cellular Ca2+ Uptake Occurs upon Carbon Source Shift—Previous studies show that the pgm2{Delta} mutation causes increased cellular Ca2+ uptake and accumulation when galactose is utilized as the carbon source (28, 29, 38). This increase in Ca2+ uptake coincides with a large increase in the level of intracellular Glc-1-P and a resulting alteration in the Glc-1-P/Glc-6-P ratio. It has been proposed that the altered ratio of these glucose metabolites induces a mechanism that normally couples their relative abundance to intracellular Ca2+ homeostasis (28, 29, 38). This model predicts that wild-type cells shifted from the metabolism of glucose to galactose should also experience a significant increase in Ca2+ uptake. To test this hypothesis, we measured 45Ca2+ uptake in the wild-type and pgm2{Delta} strains after a carbon source shift from glucose to galactose (Fig. 1). The rates of 45Ca2+ uptake in the wild-type and pgm2{Delta} strains were similar before the shift. We found that the rate of 45Ca2+ uptake in the wild-type strain increased 2-fold within 6 h of re-suspending the cells in medium containing galactose as the carbon source and remained constant thereafter. In contrast, the pgm2{Delta} mutant exhibited a 4-fold increase in 45Ca2+ uptake 6 h after the shift, and uptake increased to 8-fold higher than the pre-shift level after 12 h. These results support the hypothesis that a component of the normal adaptive response of yeast cells to the utilization of galactose as the carbon source is an increase in Ca2+ transport across the plasma membrane. Furthermore, these findings suggest that the defect in Ca2+ homeostasis observed in the pgm2{Delta} strain may result from an inability to properly regulate this normal physiological response due to the overproduction of Glc-1-P in this strain.



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  		FIG. 1.
Yeast strains exhibit an increase in Ca2+ uptake and accumulation when shifted to growth media containing galactose as the carbon source. 45Ca2+ uptake was measured in wild-type and pgm2{Delta} yeast strains after a carbon source shift from YPD to YPGal medium. Data represent the 45Ca2+ taken up over 10 min as described under "Experimental Procedures." Cells grown in YPD medium were harvested in mid-log phase and resuspended to the same cell density in YPGal medium. The YPGal cultures were maintained in mid-log phase by monitoring the cell density and dilution of the culture as needed. After the indicated incubation period, equal numbers of cells were harvested for 45Ca2+ uptake assays. Data are represented as the mean ± S.D. Similar data were obtained in four independent experiments, each performed in triplicate.

 
Disruption of the PMC1 Gene Partially Suppresses Phenotypes Associated With the pgm2{Delta} Mutation—The vacuole serves as the major Ca2+ storage compartment in yeast (7, 39). Given the high rate of cellular Ca2+ uptake observed when the pgm2{Delta} strain utilizes galactose as carbon source, we reasoned that efficient vacuolar Ca2+ sequestration might be critical for the viability of this strain. To determine the consequences of reducing the level of vacuolar Ca2+ sequestration in the pgm2{Delta} mutant, we disrupted the genes encoding the vacuolar Ca2+ ATPase Pmc1p and the vacuolar Ca2+/H+ exchanger Vcx1p both independently and together in the pgm2{Delta} strain. The loss of Vcx1p activity had no effect on the growth of the pgm2{Delta} mutant (data not shown). Surprisingly, the pmc1{Delta} mutation partially suppressed the slow growth and Ca2+ sensitivity phenotypes of the pgm2{Delta} mutant on both standard YPGal plates and on YPGal plates supplemented with 50 mM CaCl2 (Fig. 2, A and B). Combining both the pmc1{Delta} and vcx1{Delta} mutations together in the pgm2{Delta} mutant provided no greater suppression than that observed for the pmc1{Delta} mutation alone (data not shown). These results suggest that vacuolar Ca2+ sequestration mediated by Pmc1p may be detrimental to growth of the pgm2{Delta} strain.



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  		FIG. 2.
The pmc1{Delta} mutation partially suppresses the slow growth phenotype associated with the pgm2{Delta} strain on YPGal plates. 50 mM CaCl2 was added to YPGal medium as indicated. Plates were incubated for 6 days at 30 °C.

 
To determine whether this partial suppression of the pgm2{Delta} growth defect by the pmc1{Delta} mutation correlated with changes in cellular Ca2+ homeostasis, we next measured the level of total cellular Ca2+ accumulation in strains grown with galactose as the carbon source (Fig. 3). As reported previously, the pgm2{Delta} mutant exhibited a 4-fold higher level of total cell Ca2+ than the wild-type strain. Consistent with the observed growth phenotypes, the pgm2{Delta}/vcx1{Delta} double mutant had a level of total cell Ca2+ that was similar to the level measured in the pgm2{Delta} mutant. In contrast, the pgm2{Delta}/pmc1{Delta} strain had a level of total cell Ca2+ that was only 1.5-fold higher than that found in the wild-type strain. These results demonstrate that the introduction of the pmc1{Delta} mutation (and presumably a reduction in vacuolar Ca2+ sequestration) coincides with the suppression of the Ca2+ homeostasis phenotypes observed in the pgm2{Delta} strain.



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  		FIG. 3.
The pmc1{Delta} mutation suppresses the high Ca2+ accumulation phenotype of the pgm2{Delta} strain grown in YPGal medium. The indicated strains were grown in YPGal medium to mid-log phase and harvested for total cell Ca2+ determination by flame photometry as described under "Experimental Procedures." Data are represented as the mean ± S.D.

 
Intracellular Ca2+ in yeast has been shown to exist in two distinct states termed the exchangeable and non-exchangeable pools (7). The exchangeable pool was so named because it was rapidly released from yeast cells when they were introduced into medium containing a limiting level of extracellular Ca2+. In contrast, the non-exchangeable pool of cellular Ca2+ was released from the cell under these conditions at a much slower rate. Until recently, the non-exchangeable pool of cellular Ca2+ was thought to be largely synonymous with the vacuole pool, where it is thought to exist in complex with polyphosphate (7). In contrast, the exchangeable pool was thought to represent Ca2+ located primarily within the ER and Golgi compartments. Thus, our finding that the exchangeable Ca2+ pool in the pgm2{Delta} strain was not reduced when grown with galactose as the carbon source suggested that the Ca2+ level was not depleted in the compartments of the secretory pathway. However, Cyert and Denis (11) recently found that a fraction of the vacuolar Ca2+ store is more dynamic than previously thought and can be rapidly released from the vacuole into the cytoplasm under certain conditions. They also showed that Yvc1p, a Ca2+ channel in the vacuolar membrane, was responsible for mediating this release of vacuolar Ca2+. Significantly, these findings indicated that a significant portion of the exchangeable Ca2+ pool is localized to the vacuole.

To test whether the exchangeable Ca2+ pool in the vacuole is altered by the pgm2{Delta} mutation, we next compared the effect of the yvc1{Delta} mutation on Ca2+ partitioning in the wild-type and pgm2{Delta} strains. To do this we measured the relative size of the exchangeable and non-exchangeable pools in wild-type, yvc1{Delta}, pgm2{Delta}, and pgm2{Delta}/yvc1{Delta} strains. We found that the size of the non-exchangeable pools ranged from 79.6 to 86.5% of total cellular Ca2+ in these strains (Fig. 4A), and the presence of the yvc1{Delta} mutation led to a small increase in the size of the non-exchangeable pool in either the wild-type or pgm2{Delta} strains. These differences in Ca2+ partitioning were more striking when the exchangeable pools were examined (Fig. 4B). The presence of the yvc1{Delta} mutation reduced the exchangeable Ca2+ pool from 17.2 to 13.5% in the wild-type background (a decrease of 21.5%), whereas the yvc1{Delta} mutation reduced the fraction of total cellular Ca2+ in the exchangeable pool of the pgm2{Delta} strain from 20.4 to 13.9% (a decrease of 31.9%). These results confirm that the vacuole contains a significant portion of the total exchangeable fraction. Furthermore, the 50% increase in the size of the exchangeable pool within the vacuole (based on the 31.9% decrease in the exchangeable vacuolar Ca2+ pool in the pgm2{Delta} strain versus the corresponding 21.5% decrease observed in the wild-type strain) suggests that the size of the exchangeable Ca2+ pool in the ER or Golgi may undergo a corresponding decrease in its contribution to the total exchangeable pool in the pgm2{Delta} strain.



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  		FIG. 4.
Loss of the vacuolar Ca2+ channel Yvc1p reduces the size of the exchangeable Ca2+ pool in wild-type and pgm2{Delta} strains. Quantitation of the fraction of total cellular Ca2+ present in (the non-exchangeable pool (A) and the exchangeable pool (B). The distribution of total cellular Ca2+ in each pool was determined as described under "Experimental Procedures."

 
The Unfolded Protein Response Is Activated in the pgm2{Delta} Strain and Reversed by the pmc1{Delta} Mutation—We previously used two assays to ask whether the Ca2+ level in the ER or Golgi was reduced. First, we found that the rate of ER degradation of a mutant form of carboxypeptidase Y (CPY*) was normal, suggesting that the level of divalent cations in the ER is normal. Similarly, we found that the glycosylation of invertase (more specifically, outer chain addition) was normal, suggesting that the level of divalent cations in the Golgi was also normal. Both of these processes are dependent upon the presence of Ca2+ or Mn2+. Complicating the use of these assays to estimate compartmental Ca2+ levels is the fact that Mn2+ can effectively replace the requirement for Ca2+ to promote the growth of yeast cells (40), and Mn2+ was found to suppress the defects in invertase glycosylation caused by the pmr1 mutation more effectively than Ca2+ (22). Thus, these results did not provide conclusive evidence that the Ca2+ level was normal in either compartment. In addition, our previous finding that a yeast strain carrying both the pgm2{Delta} and pmr1{Delta} mutations is unable to grow on media containing galactose as the carbon source (28) suggested that the pgm2{Delta} mutation may further reduce the depleted level of divalent cations in the secretory pathway caused by the pmr1{Delta} mutation. This led us to reexamine the level of divalent cations in the ER of the pgm2{Delta} strain.

The efficient sequestration of divalent cations is required for the proper folding and processing of proteins in the secretory pathway. Consequently, mutations that prevent the uptake of divalent cations into the ER, such as the pmr1{Delta} and cod1{Delta} mutations, result in an elevated UPR (23). To further examine whether the level of divalent cations in the ER are affected in the pgm2{Delta} mutant, we assayed the level of UPR induction in different strains metabolizing galactose as the carbon source. This analysis was carried out using a reporter plasmid that contained UPR elements upstream of the {beta}-galactosidase gene (26). Remarkably, we found that expression of the UPR reporter protein was 40-fold higher in the pgm2{Delta} mutant than in the wild-type strain (Fig. 5A). This finding provides strong evidence that the level of divalent cations in the ER is reduced. Significantly, the pgm2{Delta}/pmc1{Delta} double mutant exhibited a level of UPR induction that was only 3-fold higher than the level observed in the wild type strain when grown in a medium with galactose as carbon source. This indicates that the loss of Pmc1p function can largely suppress the UPR induction associated with the pgm2{Delta} mutation. Given the well characterized role of Pmc1p as a vacuolar Ca2+ transporter (3, 16, 39), these results suggest that this suppression of the UPR results from a specific reduction in Ca2+ sequestration into the vacuole.



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  		FIG. 5.
The pgm2{Delta} mutant exhibits an elevated UPR. The indicated strains were grown in SMGal medium (A) to mid-log phase and harvested for {beta}-galactosidase assays as described under "Experimental Procedures." The magnitude of the UPR was also determined in strains grown in SMGal medium supplemented with 20 mM CaCl2 (B) or 5 mM DTT (C). Data are represented as the mean ± S.D. {beta}-Gal, {beta}-galactosidase.

 
We next sought to characterize in greater detail the UPR induction observed in the pgm2{Delta} strain. A recent report by Bonilla et al. examining the induction of UPR in the pmr1{Delta} mutant found that the addition of Ca2+ to the growth medium reduces the magnitude of UPR induction (23). Consistent with this finding, we found that the addition of 20 mM Ca2+ to the growth medium reduced the UPR response observed in the pgm2{Delta} mutant by ~35% (compare the magnitude of responses in Fig. 5, A and B). In addition, the 3-fold greater level of UPR induction observed with the pgm2{Delta}/pmc1{Delta} double mutant was completely eliminated in the presence of 20 mM extracellular Ca2+ (Fig. 5B). Several reports have also shown that the addition of DTT to the growth medium can cause an accumulation of unfolded proteins in the ER and activation of the UPR response (23, 41, 42). We found that the addition of 5 mM DTT to the growth medium led to a 7-fold increase in {beta}-galactosidase activity in the wild-type strain and a 4.5-fold increase in the pmc1{Delta} mutant (Fig. 5C). Both of these values are much lower than the 40-fold induction that was observed when the pgm2{Delta} strain was grown with galactose as the carbon source. Interestingly, the pgm2{Delta} mutant showed no further UPR induction upon exposure to DTT, suggesting that a maximal level of induction had already been reached. Overall, the massive induction of UPR in the pgm2{Delta} mutant suggests that Ca2+ stores in the ER are significantly reduced when this strain is grown with galactose as the carbon source. The suppression of this UPR response by the introduction of the pmc1{Delta} mutation indicates that this depletion of ER Ca2+ becomes much less severe when vacuolar Ca2+ uptake is reduced.

Ca2+ Transport Activity in Cellular Membranes Is Increased in the pgm2{Delta} Strain—Our results indicate that yeast cells normally increase Ca2+ uptake and accumulation when shifted to a growth medium containing galactose as the carbon source. Furthermore, Ca2+ uptake is increased much more in the pgm2{Delta} strain under these conditions. To gain further insights into where this additional Ca2+ is sequestered inside the cell, we next assayed Ca2+ transport into isolated intracellular membrane vesicles after their fractionation on a sucrose step gradient as previously described (9, 14, 16, 36). We first examined ATP-dependent Ca2+ transport into membranes isolated from wild-type cells grown with galactose as the carbon source. As reported previously, we found 45Ca2+ transport capacity to be greatest in the higher density membrane fractions, with a peak in activity occurring in Fraction 7 (Fig. 6). Furthermore, we found that the majority of the 45Ca2+ transport observed was dependent on the presence of Pmc1p. A pmc1{Delta} strain exhibited a large decrease in total ATP-dependent 45Ca2+ transport activity (and a 6.8-fold reduction in the activity centered on Fraction 7) as compared with the wild-type strain. These results indicate that the vacuolar transporter Pmc1p is primarily responsible for the ATP-dependent Ca2+ transport activity in yeast membranes.



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  		FIG. 6.
Ca2+ sequestration into membrane vesicles isolated from wild-type cells correlates with Pmc1p activity. 45Ca2+ sequestration into membrane fractions isolated from wild-type (squares) or pmc1{Delta} (diamonds) cells grown in SMGal medium. ATP-dependent 45Ca2+ uptake assays were carried out in the presence of 25 µM carbonyl cyanide m-chlorophenylhydrazone. The data are presented as mean ± S.D. See "Experimental Procedures" for further details.

 
We next compared 45Ca2+ transport into membrane fractions harvested from the wild-type and pgm2{Delta} strains grown with galactose as the carbon source. We previously found that the pgm2{Delta} strain grows very slowly in synthetic media containing galactose as the carbon source. To normalize the growth rates between the wild-type and pgm2{Delta} strains as much as possible, the synthetic medium used to grow both strains was supplemented with 0.3% yeast extract. As shown above, membranes from both the wild-type and pgm2{Delta} strains again showed peak 45Ca2+ transport activity in Fraction 7 (Fig. 7). However, peak 45Ca2+ transport activity was 3.2-fold higher in this peak fraction of membranes harvested from the pgm2{Delta} mutant. These results are consistent with the hypothesis that vacuolar Ca2+ sequestration is significantly increased in the pgm2{Delta} mutant.



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  		FIG. 7.
Increased Ca2+ sequestration occurs in membrane vesicles harvested from the pgm2{Delta} strain metabolizing galactose as the carbon source. In vitro 45Ca2+ uptake assay on membrane fractions isolated from wild-type (squares) or pgm2{Delta} (diamonds) cells grown in YMMG medium. The data are presented as the mean ± S.D. See "Experimental Procedures" for further details.

 
Ca2+ Transport into Membranes Isolated from Wild-type and pgm2{Delta} Strains Is Not Stimulated Directly by Glc-6-P or Glc-1-P—A previous report demonstrated that the altered Ca2+ homeostasis in the pgm2{Delta} mutant correlates with an increase in the intracellular ratio of Glc-1-P relative to Glc-6-P (29). To determine whether this effect is due to a direct stimulation of Ca2+ transport, we used the in vitro 45Ca2+ transport assay to test whether Glc-1-P or Glc-6-P can directly stimulate Ca2+ transport into membranes harvested from the wild-type or pgm2{Delta} strains. For both strains membranes were again prepared from cells grown with galactose as the carbon source, and the fractions showing the peak 45Ca2+ transport activity in preliminary experiments were examined further. For the wild-type strain, we found that neither Glc-6-P (Fig. 8A) nor Glc-1-P (Fig. 8B) stimulated 45Ca2+ transport into these membranes. Instead they decreased 45Ca2+ transport in a dose-dependent manner. In the presence of 10 mM Glc-6-P, 45Ca2+ accumulation decreased 33%. Similarly, the presence of 10 mM Glc-1-P decreased 45Ca2+ accumulation 45%. To determine whether these decreases were specific for Glc-6-P and Glc-1-P, we measured 45Ca2+ transport activity in the presence of 5 mM fructose 6-phosphate or mannose 6-phosphate. Interestingly, although no significant change was observed on 45Ca2+ transport in the presence 5 mM fructose 6-phosphate, 5 mM mannose 6-phosphate led to a ~20% increase in 45Ca2+ uptake that was reproducible in multiple experiments (Fig. 8C). Because previous reports predicted that Ca2+ is retained in the yeast vacuole by an interaction with polyphosphate (7), we also assayed 45Ca2+ transport in the presence 5 mM sodium phosphate. Under these conditions, membranes from the wild-type strain reproducibly exhibited an increase in 45Ca2+ accumulation of greater than 50% (Fig. 8C).



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  		FIG. 8.
Neither Glc-6-P nor Glc-1-P can directly stimulate Ca2+ uptake into membrane vesicles from the wild-type or pgm2{Delta} strains. Fractions showing maximal 45Ca2+ uptake activity for each strain were assayed for 45Ca2+ uptake in the presence of the indicated sugar phosphate at the indicated concentration. A–C, 45Ca2+ uptake into membranes harvested from the wild-type strain. D–F, 45Ca2+ uptake into membranes harvested from the pgm2{Delta} strain. The data are presented as the mean ± S.D. G6P, glucose 6-phosphate; G1P, glucose 1-phosphate; F6P, fructose 6-phosphate; M6P, mannose 6-phosphate. ctrl., control.

 
We also examined the effects of sugar phosphates on 45Ca2+ transport using membranes harvested from the pgm2{Delta} mutant grown with galactose as carbon source. As described above for membranes harvested from the wild-type strain, we found that both Glc-6-P and Glc-1-P decreased 45Ca2+ accumulation into membranes from the pgm2{Delta} mutant in a dose-dependent manner. The addition of 10 mM Glc-6-P decreased 45Ca2+ transport 37% (Fig. 8D), whereas the addition of 10 mM Glc-1-P decreased 45Ca2+ transport nearly 50% (Fig. 8E). As observed with the wild-type strain, the addition of 5 mM fructose 6-phosphate had no significant effect on 45Ca2+ transport, whereas the addition of 5 mM mannose 6-phosphate led to an increase of ~ 20% (Fig. 8F). The addition of 5 mM sodium phosphate increased 45Ca2+ transport in vitro in membranes harvested from the pgm2{Delta} mutant by 75% (Fig. 8F). When taken together, these results indicate that the sugar phosphates Glc-6-P and Glc-1-P are unable to directly stimulate Ca2+ transport into the vacuolar compartment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study we found that cellular Ca2+ uptake in the pgm2{Delta} strain increased 8-fold after a shift from glucose to galactose as the carbon source. In addition, several observations indicate that increased vacuolar Ca2+ uptake by Pmc1p plays a key role in the manifestation of the Ca2+ homeostasis defects observed in the pgm2{Delta} mutant. First, we found that the introduction of the pmc1{Delta} mutation reduced total cellular Ca2+ in the pgm2{Delta} strain to a level similar to that observed in the wild-type strain. The observation that the massive 40-fold induction of UPR observed in the pgm2{Delta} strain is greatly reduced in the pgm2{Delta}/pmc1{Delta} strain closely parallels the levels of Ca2+ uptake and accumulation observed in these strains. Further evidence that these Ca2+ homeostasis defects are largely attributable to Pmc1p function is provided by experiments in which the genes encoding other intracellular Ca2+ transporters are disrupted. Disruption of the vacuolar Ca2+/H+ exchanger Vcx1p did not significantly alter the Ca2+ homeostasis phenotype observed in the pgm2{Delta} mutant. Similarly, disruption of the gene encoding the ER Ca2+-ATPase Cod1p was unable to rescue the pgm2{Delta}-related Ca2+ defects (data not shown), whereas the pmr1{Delta} mutation was found to exacerbate those defects (28, 29). When taken together, our data suggest that the pgm2{Delta} mutation simultaneously leads to elevated vacuolar Ca2+ uptake and reduced ER/Golgi Ca2+ accumulation in yeast cells grown with galactose as the carbon source. Given our previous finding that a Glc-1-P/Glc-6-P ratio is responsible for the imbalance in Ca2+ homeostasis in the pgm2{Delta} strain (29), this suggests that the relative levels of these key glucose metabolites play a key role in determining the distribution of intracellular Ca2+ into different intracellular compartments.

The results of in vitro Ca2+ transport assays carried out with membranes harvested from wild-type and pgm2{Delta} strains also suggest that the bulk of Ca2+ transport activity in intracellular membranes is attributable to the vacuolar Ca2+-ATPase Pmc1p. This finding is consistent with the previous conclusion that the vacuole contains >95% cellular Ca2+ in yeast (7). Other studies have also shown that pmc1{Delta} strains exhibit a reduced tolerance to high levels of extracellular Ca2+ and contain reduced amounts of total cellular Ca2+ (Refs. 3 and 39 and this study). However, a recent study using a similar Ca2+ uptake assay with isolated yeast membranes concluded that the Golgi Ca2+-ATPase Pmr1p rather than the vacuolar Pmc1p is the major intracellular Ca2+ transporter under normal growth conditions (16). The reason for this discrepancy remains to be determined.

The UPR can be induced by a defect in protein glycosylation or a reduction in the level of ER Ca2+ (23). Because the pgm2{Delta} mutation alters the relative cellular levels of Glc-1-P and Glc-6-P, it is possible that this imbalance in glucose metabolites inhibits the core glycosylation of proteins after their translocation into the ER. However, our finding that both the Ca2+ homeostasis defects and UPR induction can be suppressed by the pmc1{Delta} mutation strongly suggests that the primary defect associated with the pgm2{Delta} mutation is a defect in Ca2+ homeostasis rather than a defect in protein glycosylation. Based on this reasoning we propose the following model to explain how the pgm2{Delta} mutation alters Ca2+ homeostasis in yeast (Fig. 9). First, growth of the pgm2{Delta} strain in media containing galactose as the carbon source causes Glc-1-P to accumulate due to the metabolic bottleneck in the conversion of Glc-1-P to Glc-6-P (28). This results in an altered cellular ratio of Glc-1-P to Glc-6-P (29) that leads to an increase in Pmc1p activity (this study). The increased rate of vacuolar Ca2+ uptake reduces the level of cytosolic Ca2+, which in turn causes a depletion of free Ca2+ in the ER. This decrease in the level of free Ca2+ in the lumen of the ER has two consequences. First, it has an adverse effect on protein folding in the ER, which leads to an induction of the UPR. In addition, it leads to an increase in Ca2+ uptake across the plasma membrane by a CCE-like mechanism. Evidence that excessive vacuolar Ca2+ uptake can deplete ER Ca2+ stores was previously provided by Cunningham and co-workers (21), who found that increased vacuolar Ca2+ sequestration could effectively out-compete the secretory pathway Ca2+ transporters for free Ca2+ in the cytosol. Notably, this led to an induction of a CCE-like response due to the depletion of ER Ca2+ stores (21). According to our model, the suppression of these Ca2+ homeostasis defects by the pmc1{Delta} mutation occurs by moderating the excessive vacuolar Ca2+ sequestration, thus allowing the Ca2+ levels in the cytoplasm and ER lumen to rise to concentrations that are closer to normal levels.



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  		FIG. 9.
Model; excessive vacuolar Ca2+ uptake in the pgm2{Delta} mutant leads to the depletion of ER Ca2+ and induction of the UPR and CCE responses. Step 1, increased ratio of Glc-1-P/Glc-6-P stimulates vacuolar Ca2+ sequestration by Pmc1p. Step 2, increased vacuolar Ca2+ sequestration results in a decrease in the cytosolic Ca2+ level, which leads to Ca2+ store depletion in the ER. Step 3, the low cytosolic Ca2+ level and decreased lumenal Ca2+ level in the secretory pathway induces the UPR and activates a signal for Ca2+ uptake across the plasma membrane. See "Discussion" for further details. G1P, glucose 1-phosphate; G6P, glucose 6-phosphate.

 
It was previously shown that yeast possess both high affinity and low affinity Ca2+ uptake mechanisms (21). The high affinity pathway is mediated by the MID1 and CCH1 gene products, which are thought to oligomerize to form a single high affinity Ca2+ channel in the plasma membrane. In contrast, the low affinity pathway has not been characterized in detail, and the gene products that encode components of this channel have not been identified. The high affinity pathway was considered to be a good candidate to mediate the CCE-like response proposed for the pgm2{Delta} strain, since the MID1/CCH1 gene products were previously shown to be required for a CCE-like mechanism in a pmr1{Delta} strain (21). However, we found that the introduction of a mid1{Delta} mutation into the pgm2{Delta} strain was unable to suppress the Ca2+ homeostasis defects associated with the pgm2{Delta} mutation.2 Because both the rate of cellular Ca2+ uptake and UPR induction are much larger in the pgm2{Delta} mutant than was previously shown to be associated with the pmr1{Delta} mutation (21, 23), it is possible that the more robust CCE response observed in this strain is mediated by either the low affinity Ca2+ uptake pathway or a combination of both.

Although we found that cellular Ca2+ uptake in the pgm2{Delta} strain increased 8-fold after a shift from glucose to galactose as the carbon source, we also observed that wild-type yeast cells undergo a 2-fold increase in Ca2+ uptake. This finding is consistent with our hypothesis that an increased Glc-1-P/Glc-6-P ratio stimulates cellular Ca2+ uptake via an activation of Pmc1p, since the steady-state Glc-1-P level increases in wild-type cells when galactose is utilized as carbon source. Thus, the increased level of Ca2+ uptake appears to be part of a normal adaptive response to this carbon source shift. In other experiments, we found that this 2-fold elevation in Ca2+ uptake correlated with a ~1.5-fold increase in PMC1 mRNA levels in both wild-type and pgm2{Delta} strains (data not shown). The much larger increase in Ca2+ uptake observed in the pgm2{Delta} strain suggests that a post-transcriptional mechanism may complement this modest increase in PMC1 transcription and is again consistent with the hypothesis that Glc-6-P and/or Glc-1-P acts as a signaling molecule whose level provides a sensitive metabolic readout of carbon source that is subsequently amplified via Ca2+ signaling mechanisms. Our finding that neither Glc-6-P nor Glc-1-P can enhance Ca2+ transport into intracellular membranes in vitro suggests that these metabolites do not directly stimulate the activity of a Ca2+ transporter such as Pmc1p in vivo. However, it remains a formal possibility that a metabolite derived from one of these compounds in vivo could function as the true physiological activator of this process. It is unlikely that a derivative of UDP-glucose (which is derived from Glc-1-P and UTP via the enzyme UDP-Glc pyrophosphorylase) plays this role, since it was previously shown that the overproduction of UDP-glucose in vivo did not result in changes in Ca2+ homeostasis like those observed in the pgm2{Delta} strain (28, 29).

If Glc-1-P and Glc-6-P do not stimulate intracellular Ca2+ sequestration by a direct mechanism, how does an imbalance in these sugar phosphates mediate this affect? A large body of evidence suggests that Glc-6-P normally functions as an intracellular signaling molecule. Recent studies have suggested that Snf3p and Rgt2p activate glucose signaling pathways by binding Glc-6-P on the cytosolic side of the cell membrane. This conclusion stems from the observation that the expression of only the C-terminal cytosolic domain of Snf3p can restore relatively normal glucose signaling in snf3{Delta} cells (43, 44). Consistent with a hypothesized signaling role for Glc-6-P, another study found that the transient elevation of cytosolic Ca2+ response shown to occur upon the re-addition of glucose to cells starved for carbon source was dependent on the ability of the cell to phosphorylate glucose to Glc-6-P (38). Our previous reports that the altered Ca2+ homeostasis phenotypes in the pgm2{Delta} mutant are due to an altered cellular ratio of Glc-6-P to Glc-1-P are also consistent with a signaling role for these sugar metabolites (28, 29).

Several proteins, including Hxk2p (45), Snf3p (44), Rgt2p (46), and Gpr1p/Gpa2p (47) have also been proposed to respond to the level of cytosolic Glc-6-P. As such, we sought to determine whether the disruption of the genes encoding any of these putative sugar phosphate sensors could suppress the Ca2+ homeostasis abnormalities observed in the pgm2{Delta} strain. Unfortunately, none of the mutations tested (or various combinations thereof) could suppress phenotypes associated with the pgm2{Delta} mutation (data not shown). These results indicate that signaling changes mediated by an altered ratio of Glc-6-P to Glc-1-P in the pgm2{Delta} strain either occur via a complex interplay of these signaling proteins or is mediated by another mechanism. Although a substantial body of experimental evidence indicates that increased cellular concentrations of Glc-6-P and/or Glc-1-P affect cellular Ca2+ homeostasis, further studies will be required to characterize the nature by which this metabolic signal is recognized and transduced to downstream components of the signaling pathway.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant T32 HL07553 (to D. P. A.), American Heart Association Grant 0255121B (to D. M. B.), and Hungarian National Science Foundation Grant T-038144 (to A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work and should be considered co-first authors. Back

** To whom correspondence should be addressed: Dept. of Microbiology, BBRB 432/Box 8, 1530 Third Ave., South, The University of Alabama at Birmingham, Birmingham, AL 35294-2170. Tel.: 205-934-6593; Fax: 205-975-5482; E-mail: dbedwell{at}uab.edu.

1 The abbreviations used are: ER, endoplasmic reticulum; CCE, capacitative Ca2+ entry; PGM, phosphoglucomutase; wt, wild-type strain; UPR, unfolded protein response; Glc-6-P, glucose 6-phosphate; Glc-1-P, glucose 1-phosphate; YP, yeast extract/peptone; SM, synthetic medium; MES, 4-morpholineethanesulfonic acid; DTT, dithiothreitol. Back

2 D. Aiello and D. Bedwell, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Patricia Kane, Todd Graham, Kyle Cunningham, Randy Hampton, and Kazutoshi Mori for generously providing strains and reagents.



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