Intracellular glucose 1-phosphate and glucose 6-phosphate levels modulate Ca2+ homeostasis in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase plays a key role in cellular metabolism by virtue of its ability to interconvert Glc-1-P and Glc-6-P. It was recently shown that a yeast strain lacking the major isoform of phosphoglucomutase (pgm2Delta) accumulates a high level of Glc-1-P and exhibits several phenotypes related to altered Ca(2+) homeostasis when d-galactose is utilized as the carbon source (Fu, L., Miseta, A., Hunton, D., Marchase, R. B., and Bedwell, D. M. (2000) J. Biol. Chem. 275, 5431-5440). These phenotypes include increased Ca(2+) uptake and accumulation and sensitivity to high environmental Ca(2+) levels. In the present study, we overproduced the enzyme UDP-Glc pyrophosphorylase to test whether the overproduction of a downstream metabolite produced from Glc-1-P can also mediate changes in Ca(2+) homeostasis. We found that overproduction of UDP-Glc did not cause any alterations in Ca(2+) uptake or accumulation. We also examined whether Glc-6-P can influence cellular Ca(2+) homeostasis. A yeast strain lacking the beta-subunit of phosphofructokinase (pfk2Delta) accumulates a high level of Glc-6-P (Huang, D., Wilson, W. A., and Roach, P. J. (1997) J. Biol. Chem. 272, 22495-22501). We found that this increase in Glc-6-P led to a 1.5-2-fold increase in total cellular Ca(2+). We also found that the pgm2Delta/pfk2Delta strain, which accumulated high levels of both Glc-6-P and Glc-1-P, no longer exhibited the Ca(2+)-related phenotypes associated with high Glc-1-P levels in the pgm2Delta mutant. These results provide strong evidence that cellular Ca(2+) homeostasis is coupled to the relative levels of Glc-6-P and Glc-1-P in yeast.

Ca 2ϩ homeostasis in eukaryotic cells is a complex process involving the regulated internalization and sequestration of Ca 2ϩ ions into a variety of intracellular compartments. The basic mechanisms that mammalian and yeast cells employ to carry out this process are similar, such that the cytosolic Ca 2ϩ concentration in both cell types is normally maintained in the range of 50 -200 nM (1)(2)(3)(4).
In mammalian cells, the endoplasmic reticulum (ER) 1 serves as a storage compartment that provides a readily mobilizable source of Ca 2ϩ for use in Ca 2ϩ signaling. Upon receiving an appropriate stimulus, ER Ca 2ϩ stores can be released to generate a transient increase in the cytosolic Ca 2ϩ concentration. This elevated Ca 2ϩ level can then activate various signaling pathways in a tightly regulated manner (5). In a variety of non-excitable cell types, the release of ER Ca 2ϩ can lead to the generation of a store depletion signal that results in an influx of Ca 2ϩ ions across the plasma membrane in a process termed capacitative Ca 2ϩ entry (CCE). CCE amplifies the transient increase in cytosolic Ca 2ϩ initiated by the release of ER Ca 2ϩ and ultimately serves as a source of cytosolic Ca 2ϩ for refilling the depleted ER Ca 2ϩ store (6). Mammalian ER Ca 2ϩ stores are maintained by the activity of the sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase pumps (7,8). In addition, past work suggests that glucose metabolites also play a role in sequestering mammalian ER Ca 2ϩ stores. Several studies have shown that both the rate and yield of ATP-dependent Ca 2ϩ transport in microsomes isolated from a variety of tissues are stimulated by Glc-6-P (9 -13). This effect appears to be mediated by a dedicated Glc-6-P transporter located in the ER membrane. The ER-localized Glc-6-P transporter has been cloned and is expressed as two distinct mRNA species (14 -16). The longer isoform is highly enriched in heart, brain, and skeletal muscle, whereas the shorter isoform shows a more ubiquitous tissue distribution.
The vacuole, which is the major Ca 2ϩ storage compartment in yeast cells, contains Ͼ95% of the total cellular Ca 2ϩ (17). Until recently, this large store of vacuolar Ca 2ϩ was thought to be relatively inert due to its association with polyphosphate. However, a recent study found that vacuolar Ca 2ϩ can be released in a regulated manner through the action of Yvc1p, a TRP channel homolog (18). Ca 2ϩ release through this channel was shown to be induced by hypotonic shock and may be responsive to the HOG/mitogen-activated protein kinase signaling cascade (19).
The Golgi apparatus has also been shown to play an important role in cellular Ca 2ϩ storage in yeast through the action of the Golgi-localized Ca 2ϩ -ATPase Pmr1p (3, 20 -24). It has been shown that pmr1⌬ mutants display a constitutively elevated cellular Ca 2ϩ uptake phenotype that may be related to the CCE response of mammalian cells (25). This yeast version of the CCE response is mediated through the action of a high affinity Ca 2ϩ channel in the plasma membrane containing at least the MID1 and CCH1 gene products as subunits (26). The increased Ca 2ϩ accumulation that occurs in the pmr1⌬ strain can stimulate the expression of a large number of genes by the transcription factor Tcn1p/Crz1p following its activation by the calmodulin-calcineurin signaling pathway (27)(28)(29)(30). These results suggest that the yeast Golgi apparatus may normally play a role that is functionally similar to the that of mammalian ER because it has the potential to mediate a transient rise in cytosolic Ca 2ϩ that can subsequently be amplified by a CCElike mechanism.
The yeast ER appears to play a lesser role in cellular Ca 2ϩ storage because the free Ca 2ϩ concentration of this compartment has been reported to be only ϳ10 M (31). It was recently shown that the COD1/SPF1 gene product is an ER-localized P-type Ca 2ϩ -ATPase that sequesters cytosolic Ca 2ϩ into the ER (32)(33)(34). A number of studies have also suggested that Pmr1p (20 -23, 34) and the vacuolar Ca 2ϩ -ATPase Pmc1p (23, 34) also play a role in maintaining ER Ca 2ϩ stores under certain conditions. In contrast to mammalian cells, a Glc-6-P transporter capable of stimulating Ca 2ϩ sequestration into the ER (or possibly the Golgi apparatus) has not been identified in yeast cells. However, it has been shown that Glc-1-P appears to play a role in yeast Ca 2ϩ homeostasis (35).
Saccharomyces cerevisiae contains two genes that encode phosphoglucomutase (PGM), a key metabolic enzyme that interconverts Glc-6-P and Glc-1-P (see Fig. 1). The PGM2 gene encodes the major isoform that accounts for ϳ90% of the total PGM activity in galactose-grown cells, whereas the PGM1 gene product accounts for the remaining 10% (36). When glucose is utilized as the carbon source, a low level of PGM activity is required to maintain enough Glc-1-P for use as substrate in the synthesis of sugar nucleotides such as UDP-Glc. UDP-Glc is needed for anabolic reactions such as glycogen synthesis and cell wall biosynthesis. However, a much higher level of PGM activity is required during growth in medium containing galactose as the carbon source to provide the Glc-6-P used in both the glycolytic and pentose phosphate pathways. Consistent with this need for a greater metabolic flux from Glc-1-P to Glc-6-P, a pgm2⌬ mutant accumulates a high level of intracellular Glc-1-P when grown in medium containing galactose as the sole carbon source (35). In addition, it was found that this high level of Glc-1-P is accompanied by a high rate of Ca 2ϩ uptake and accumulation, suggesting a possible link between glucose metabolites and Ca 2ϩ homeostasis in yeast.
The goal of this study was to further examine the hypothesis that the glucose metabolites Glc-1-P and/or Glc-6-P play a role in Ca 2ϩ homeostasis in yeast. We show that Ca 2ϩ homeostasis defects in the pgm2⌬ mutant are specific to accumulated Glc-1-P using three strategies. First, we show that Escherichia coli PGM expression can rescue galactose-specific growth defects in pgm2⌬ yeast strains. These data support the hypothesis that the loss of Pgm2p enzymatic activity is directly responsible for the observed alterations in cellular Ca 2ϩ homeostasis. Second, we show that increased synthesis of UDP-Glc, a metabolite synthesized directly from Glc-1-P and UTP by the enzyme UDP-Glc pyrophosphorylase, has no effect on Ca 2ϩ homeostasis. This result demonstrates that the increased synthesis of Glc-1-P, rather than sugar nucleotides derived from Glc-1-P, influences cellular Ca 2ϩ homeostasis. Third, we show that the accumulation of Glc-6-P also has an effect on Ca 2ϩ homeostasis that is distinct from Glc-1-P accumulation. (MATa ade2-101 his3-⌬200 leu2,3-112 ura3-52 pgm2⌬::URA3 pfk2⌬::HIS3). Strain Sc252 was kindly provided by J. E. Hopper. The pfk2⌬ strain YDB0397 was generated by disruption of the PFK2 gene in YDB0355 (MATa ade2-101 his3-⌬200 leu2,3-112 lys2-⌬201 ura3-52) by insertion of HIS3. To do this, a 2.9-kb fragment containing the PFK2 gene was amplified by PCR using yeast genomic DNA as template. The forward primer was DB732 (5Ј-ACG CGT CGA CCA TAC GCA ATG ACT GTT AC), which contains a SalI restriction endonuclease site (underlined). The reverse primer was DB733 (5Ј-GCT CTA GAC CAA ATG GTC AGC AAT GAG), which contains an XbaI site (underlined). The PCR product was digested with SalI and XbaI and subcloned into the same sites within plasmid pBluescript II KS(ϩ) (Stratagene) to generate pDB0657. The 1.3-kb BglII/EcoRI fragment in PFK2 was removed from pDB0657 and replaced with an EcoRI/XhoI fragment from pJJ215 (37) to generate pDB0658. The SalI/XbaI fragment containing the disrupted PFK2 gene was then used to transform strain YDB0355 by standard methods (38). The pgm2⌬/pfk2⌬ strain YDB0395 was constructed by transforming the same SalI/XbaI fragment from pDB0658 into YDB0313 (MATa ade2-101 his3-⌬200 leu2,3-112 ura3-52 pgm2⌬::URA3). In each strain constructed above, gene knockouts were confirmed by Southern blotting and enzyme assay for either PGM (35) or phosphofructokinase (39) activity.
Culture Media-Bacterial strains used for cloning and plasmid maintenance were grown in standard medium as described (40). Similarly, yeast media were prepared as described (38). Yeast extract/peptone (YP) medium and synthetic medium were supplemented with 2% glucose (dextrose) (YPD or SMD) or 2% galactose (YPGal). YPD and YPGal media were routinely buffered to pH 5.5 with 40 mM MES/Tris. Liquid cultures were grown for a minimum of six to seven generations to Յ0.8 A 600 units/ml prior to harvesting unless otherwise indicated.
Plasmids Used-The centromeric plasmid pDB0608 was used to express E. coli PGM from the PGM2 promoter in yeast strains SJ21R and YDB0171. Plasmid pDB0608 was constructed as follows. pDB0197, which contains a 3.3-kb BamHI/XhoI fragment with the yeast PGM2 locus in pBluescript II KS(ϩ), was digested with HpaI/EcoRI to remove the entire PGM2 coding sequence. This sequence was replaced with an HpaI/EcoRI fragment from pNK3476 containing the 3Ј-coding sequence of E. coli PGM (41). The 5Ј-coding sequence was amplified by PCR using DB431 (5Ј-AAA GTT AAC ATA ACA TGG CAA TCC ACA ATC G) and DB432 (5Ј-CTG ATC GTT AAC GAT AGT CAG G), both of which contain an HpaI cut site (underlined). The resultant PCR product was cut and ligated into the HpaI site in the plasmid described above to yield full-length E. coli PGM under the control of the yeast PGM2 promoter in pDB0540. A 3.6-kb EcoRI/BamHI fragment containing the entire E. coli PGM gene under the control of the yeast PGM2 promoter was removed from pDB0540 and ligated into the same sites of pSEYC58 (42) to give pDB0608. Finally, the multicopy plasmid pYJM1 (a kind gift from Jean François) was used to overexpress the UGP1 gene. This plasmid contains the UGP1 gene on a 4.7-kb SphI/BamHI fragment inserted into the same site of plasmid YEp351 (43).
Measurement of Metabolite Levels-Glc-6-P and Glc-1-P levels were measured as described previously (35). Glc-6-P was measured in acidsoluble cell extracts using a coupled enzyme assay with Glc-6-P dehydrogenase (Sigma) (44). Glc-1-P was measured similarly with the addition of PGM (Sigma). UDP-Glc was measured in acid-soluble extracts of mid-log phase cells grown in YPD medium using a coupled enzyme assay with UDP-Glc dehydrogenase (45). Measurement of Ca 2ϩ Uptake and Total Cellular Ca 2ϩ -Ca 2ϩ uptake measurements were carried out as described previously (22,35). Cells were harvested at a cell density of Ͻ0.8 A 600 units/ml, washed, and resuspended at a cell density of 1 A 600 unit/ml in uptake buffer (40 mM MES/Tris (pH 5.5) and 20 mM D-glucose). Cells were incubated in uptake buffer for 10 min at 30°C, and 45 Ca 2ϩ uptake was initiated by the addition of 45 Ca 2ϩ to a final concentration of 1 Ci/ml. At the indicated times, 1-ml aliquots of cells were filtered through a 0.45-m Gelman GN-6 Metricel filter prewashed with buffer containing 20 mM MgCl 2 and 0.2 mM LaCl 3 . The filters were washed three additional times with the same buffer, and the amount of cell-associated 45 Ca 2ϩ was determined by liquid scintillation counting. Total cellular Ca 2ϩ in unlabeled cells was determined by flame photometry as previously described (3). Briefly, 50 -100 A 600 units of yeast grown in YPD or YPGal medium as indicated were harvested and washed with YP medium. The cell pellets were lyophilized and then resuspended in 1 M HCl. Total Ca 2ϩ in this acid-soluble extract was measured by flame photometry and is expressed in mmol of Ca 2ϩ /kg of cells (dry mass).

Expression of E. coli PGM in the pgm2⌬ Strain Suppresses
Galactose-specific Growth Defects-Previous work has shown that disruption of the PGM2 gene in S. cerevisiae leads to high Ca 2ϩ uptake and accumulation when the pgm2⌬ strain is grown in medium containing galactose as the carbon source (35). This phenotype may be related to an inability to efficiently convert Glc-1-P to Glc-6-P, a step required for cells to utilize galactose as the carbon source (Fig. 1). However, it is also possible that Pgm2p has another cellular function that leads to alterations in Ca 2ϩ homeostasis when the gene encoding this protein is deleted. This alternative function could be related to the previous finding that Pgm2p undergoes a post-translational modification in the form of a glucosyl residue attached by a phosphodiester linkage to one or more O-linked mannose residues (46). The extent of the post-translational modification of Pgm2p is regulated by both the carbon source and heat shock; however, it is unclear what role this modification plays in Pgm2p function (47,48). Because cytosolic proteins are not glycosylated in bacteria and because E. coli PGM shares only limited homology (31% sequence identity) with yeast Pgm2p, we asked whether the expression of E. coli PGM can suppress the Ca 2ϩ homeostasis defects associated with the pgm2⌬ mutation. As shown in Fig. 2, the pgm2⌬ strain exhibited several phenotypes, including slow growth in YPGal medium, sensitivity to high extracellular Ca 2ϩ levels, and sensitivity to cyclosporin A (an inhibitor of calcineurin function). However, the pgm2⌬ strain expressing E. coli PGM grew normally under each of these conditions. These results suggest that the Ca 2ϩ homeostasis defects observed when the pgm2⌬ mutant was grown in YPGal medium are directly related to the loss of PGM enzymatic activity, and not other putative functions of Pgm2p.
An Elevated Level of UDP-Glc Is Not Responsible for Altered Ca 2ϩ Homeostasis in the pgm2⌬ Mutant-The results described above support the hypothesis that a loss of Pgm2p enzymatic activity and the resultant accumulation of cellular Glc-1-P are responsible for the observed alterations in cellular Ca 2ϩ homeostasis. We next asked whether these effects are attributable specifically to the accumulation of Glc-1-P or to a metabolic derivative of this molecule. Other than the formation of Glc-6-P by PGM, the major way to consume Glc-1-P is through the action of the enzyme UDP-Glc pyrophosphorylase, which uses Glc-1-P and UTP to catalyze the formation of UDP-Glc (Fig. 1). If the cellular level of either UDP-Glc or a downstream metabolite was increased in the pgm2⌬ mutant in medium containing galactose as the carbon source, it could be responsible for the observed Ca 2ϩ homeostasis defects in the pgm2⌬ null mutant, rather than the high level of Glc-1-P. To explore this possibility, we overexpressed UDP-Glc pyrophosphorylase to determine whether an elevated level of UDP-Glc can affect Ca 2ϩ homeostasis in yeast.
UDP-Glc pyrophosphorylase is encoded by the UGP1 gene. It was previously reported that introduction of UGP1 into a multicopy plasmid leads to an increase in both UDP-Glc pyrophosphorylase activity and the cellular UDP-Glc concentration in glucose-grown cells (43). Consistent with that report, we observed a 10-fold increase in the cellular UDP-Glc concentration in wild-type cells carrying a multicopy plasmid expressing UGP1 when this strain was grown in YPD medium (Fig. 3A). However, this strain exhibited normal 45 Ca 2ϩ uptake when grown in YPD medium (Fig. 3B) or YPGal medium (data not shown). These results demonstrate that an increased level of UDP-Glc is not responsible for the observed alterations in Ca 2ϩ homeostasis when the pgm2⌬ mutant is grown with galactose as the carbon source. Disruption of PFK2 Increases Cellular Glc-6-P in both Glucose-and Galactose-grown Cells-When grown in medium containing galactose as the carbon source, the pgm2⌬ mutant accumulates an elevated level of Glc-1-P and exhibits a reduced growth rate, as well as sensitivity to cyclosporin A and high levels of extracellular Ca 2ϩ (35). Because the related metabolite Glc-6-P has been shown to influence Ca 2ϩ sequestration into the ER of mammalian cells (9 -13), we next asked whether an elevated level of Glc-6-P could mediate similar phenotypes in yeast. Previous studies have shown that strains containing a disruption of the PFK2 gene, which encodes the ␤-subunit of phosphofructokinase, accumulate a high level of Glc-6-P (49). To determine whether Glc-6-P also plays a role in yeast Ca 2ϩ homeostasis, we compared the Ca 2ϩ -related phenotypes of the wild-type, pgm2⌬, pfk2⌬, and pgm2⌬/pfk2⌬ strains.
We first determined the cellular concentrations of Glc-6-P and Glc-1-P in strains carrying these mutations. As previously reported (35), the levels of these two metabolites were similar in the wild-type and pgm2⌬ strains when grown in YPD medium (Fig. 4A). In contrast, the cellular level of Glc-6-P was increased by 5-6-fold in the pfk2⌬ and pgm2⌬/pfk2⌬ strains grown in YPD medium. When grown in YPGal medium, the pgm2⌬ mutant accumulated 10 -15-fold more Glc-1-P than the wild-type strain (Fig. 4B), as previously reported (35). The pfk2⌬ strain accumulated 3-fold more Glc-6-P than the wildtype strain when grown under these conditions, whereas the pgm2⌬/pfk2⌬ strain accumulated high levels of both Glc-6-P and Glc-1-P when grown in YPGal medium. Thus, the double mutant accumulated Glc-1-P only when utilizing galactose as the carbon source, but accumulated Glc-6-P when utilizing either glucose or galactose.

FIG. 3. Increased levels of UDP-Glc do not cause changes in cellular Ca 2؉ homeostasis.
A, overexpression of UDP-Glc pyrophosphorylase (UGPase) leads to a 10-fold increase in the cellular UDP-Glc concentration. Cultures were initially grown to mid-log phase in SMD medium (minus uracil) and then diluted back to 0.05 A 600 units/ml in YPD medium and harvested at 0.6 -0.8 A 600 units/ml. Cell lysates were made and assayed for UDP-Glc as described under "Experimental Procedures." B, cells overexpressing UDP-Glc pyrophosphorylase (छ) show 45 Ca 2ϩ uptake rates similar to those of wild-type yeast cells (Ⅺ). Cultures were grown as described for A, and cells were harvested and assayed for 45 Ca 2ϩ uptake as described under "Experimental Procedures." FIG. 4. The pgm2⌬ and pfk2⌬ mutations increase Glc-1-P and Glc-6-P levels, respectively. Cells were grown in YPD (A) or YPGal (B) medium to mid-log phase and then harvested for Glc-6-P (G6P) and Glc-1-P (G1P) measurements as described under "Experimental Procedures." wt, wild-type strain.

Loss of PFK2 Suppresses Galactose-specific Growth Defects
Observed in the pgm2⌬ Strain-We next compared the growth phenotypes of the pgm2⌬, pfk2⌬, and pgm2⌬/pfk2⌬ mutants. We found that the pfk2⌬ mutant exhibited only a slightly reduced growth rate compared with the wild-type strain on both YPD medium (data not shown) and YPGal medium (Fig.  5A). In addition, this strain was able to tolerate high extracellular Ca 2ϩ levels (Fig. 5B) or cyclosporin A (Fig. 5C) under these conditions. These results indicate that an increased level of Glc-6-P does not cause the same growth defects seen with the pgm2⌬ mutant on YPGal plates.
Like the pfk2⌬ single mutant, we found that the pgm2⌬/ pfk2⌬ strain also showed a slightly reduced growth rate in both YPD and YPGal media. However, it grew better than the pgm2⌬ single mutant on YPGal plates (Fig. 5A). Surprisingly, combining the pfk2⌬ and pgm2⌬ mutations also suppressed the galactose-specific growth defects associated with the pgm2⌬ mutation in medium with high extracellular Ca 2ϩ levels ( Fig.  5B) or cyclosporin A (Fig. 5C). These results indicate that the elevated level of Glc-6-P in the pgm2⌬/pfk2⌬ strain can suppress the growth defects associated with a high level of Glc-1-P.

Loss of PFK2 Suppresses the High Ca 2ϩ Uptake and Accumulation Phenotypes Observed in the pgm2⌬ Mutant Grown in
Galactose-containing Medium-The data presented above indicate that an elevated level of Glc-6-P can suppress the growth defects observed when a strain carrying the pgm2⌬ mutation is grown in YPGal medium. To determine whether this is due to an alleviation of the Ca 2ϩ homeostasis defects associated with the pgm2⌬ mutation, we measured 45 Ca 2ϩ uptake and total cellular Ca 2ϩ accumulation in strains carrying various combinations of the pgm2⌬ and pfk2⌬ mutations. In glucose-grown cells, 45 Ca 2ϩ uptake measured in the pfk2⌬, pgm2⌬, and pgm2⌬/pfk2⌬ strains was comparable to that in the wild-type strain (Fig. 6A). When the strains were grown in YPGal medium, the pgm2⌬ strain exhibited a 6-fold higher rate of 45 Ca 2ϩ FIG. 5. Deletion of PFK2 suppresses galactose-specific growth defects in the pgm2⌬ mutant. 300 mM CaCl 2 or 10 g/ml cyclosporin A was added to YPGal medium as indicated. Plates were incubated for 6 days at 30°C. wt, wild-type strain.
FIG. 6. The pfk2⌬ mutation suppresses the high Ca 2؉ uptake phenotype associated with the pgm2⌬ mutant. The wild-type (Ⅺ), pgm2⌬ (छ), pfk2⌬ (E), pgm2⌬/pfk2⌬ (‚) strains were grown to mid-log phase in YPD (A) or YPGal (B) medium and harvested for 45 Ca 2ϩ uptake as described under "Experimental Procedures." uptake ( Fig. 6B), as previously reported (35). In contrast, the pfk2⌬ single mutant displayed a rate of 45 Ca 2ϩ uptake that was comparable to that of the wild-type strain. Finally, in close agreement with the growth phenotypes shown in Fig. 5, the introduction of the pfk2⌬ mutation into the pgm2⌬ background completely suppressed the high 45 Ca 2ϩ uptake phenotype seen in galactose-grown cells.
We next determined total cellular Ca 2ϩ levels by flame photometry of extracts from both glucose-and galactose-grown cells. In multiple experiments, we found that the pfk2⌬ strain accumulated 1.5-2-fold more total cellular Ca 2ϩ than the wildtype strain when grown in YPD medium (Fig. 7A). In contrast, the pgm2⌬ and pgm2⌬/pfk2⌬ mutants exhibited a slight decrease in total cellular Ca 2ϩ when grown under these conditions. When these strains were grown in YPGal medium, we found that the pgm2⌬ mutant exhibited a 9 -10-fold increase in total cellular Ca 2ϩ (Fig. 7B), as previously reported (35). The pfk2⌬ mutant again exhibited a smaller 1.5-2-fold increase in total cellular Ca 2ϩ . In agreement with its reduced rate of 45 Ca 2ϩ uptake, the excess Ca 2ϩ accumulation associated with the pgm2⌬ mutation was eliminated when the pgm2⌬/pfk2⌬ strain was grown in YPGal medium. In fact, the level of total cellular Ca 2ϩ in the pgm2⌬/pfk2⌬ strain was slightly lower than that observed in the wild-type strain. These results dem-onstrate that an increased level of Glc-6-P does not cause the same large increase in Ca 2ϩ uptake and accumulation that is observed when the pgm2⌬ strain is grown in galactose-containing medium. However, a small increase in Ca 2ϩ accumulation was observed that was independent of the carbon source. Finally, we found that the accumulation of Glc-6-P associated with the pfk2⌬ mutation completely suppressed the Ca 2ϩ homeostasis defects associated with the accumulation of Glc-1-P in the pgm2⌬ strain. DISCUSSION In this study, we found that cellular Ca 2ϩ homeostasis in yeast is responsive to the relative levels of the glucose metabolites Glc-1-P and Glc-6-P. Our results indicate that both the slow growth phenotype and the large increase in Ca 2ϩ uptake and accumulation observed when the pgm2⌬ strain is grown in galactose-containing medium are specific to the increased level of Glc-1-P in the cell, rather than metabolism of the Glc-1-P to UDP-Glc or another downstream metabolite. In addition, the slow growth phenotype of the pgm2⌬ mutant is not due to ATP limitation in the cell. The most sensitive way to measure the availability of high energy phosphate bonds available for use in ATP-dependent reactions is to determine the cellular energy charge (defined as the sum of the ATP concentration plus one-half of the ADP concentration divided by the sum of the concentrations of ATP, ADP, and AMP in the cell). A previous study found that the cellular energy charge is not reduced in the pgm2⌬ mutant grown in YPGal medium (35). Consistent with this result, we found that the cellular energy charge was not significantly different from that in the wild-type strain in either the pfk2⌬ or pgm2⌬/pfk2⌬ strain when grown in YPGal medium (data not shown). We also observed that the slow growth phenotype of the pgm2⌬ strain was partially suppressed in the pgm2⌬/pfk2⌬ strain. Because the combination of these mutations was also shown to normalize Ca 2ϩ homeostasis, we conclude that the slow growth of the pgm2⌬ strain in galactose-containing medium is partially attributable to Ca 2ϩ stress, rather than a limitation of metabolic flux from Glc-1-P to Glc-6-P.
The intracellular concentration of Glc-6-P was more than an order of magnitude higher than the Glc-1-P concentration when wild-type cells were grown in either YPD or YPGal medium. The ratio of these two metabolites was unchanged when the pgm2⌬ strain was grown in YPD medium, and Ca 2ϩ homeostasis remained indistinguishable from that in the wild-type strain. However, the ratio of these two metabolites was significantly altered when the pgm2⌬ mutant was grown in YPGal medium, such that Glc-1-P became ϳ2-fold more abundant than Glc-6-P. This large change in the relative levels of these metabolites coincided with a drastic change in cellular Ca 2ϩ homeostasis. These results suggest that either the increase in the absolute level of Glc-1-P or the accompanying large change in the ratio of Glc-6-P to Glc-1-P is responsible for the observed changes in Ca 2ϩ uptake and accumulation. The hypothesis that the altered ratio of Glc-6-P to Glc-1-P is responsible for the changes in Ca 2ϩ homeostasis observed in the pgm2⌬ mutant is strongly supported by our results with the pgm2⌬/pfk2⌬ strain. This strain, like the pgm2⌬ strain, contained a high level of Glc-1-P when grown in medium containing galactose as the carbon source. However, the increased level of Glc-6-P in the pgm2⌬/pfk2⌬ strain again made it the more abundant of these two metabolites. Remarkably, this "normalization" of the relative levels of Glc-6-P and Glc-1-P in the pgm2⌬/pfk2⌬ strain was accompanied by a return to nearly normal levels of Ca 2ϩ uptake and accumulation in galactose-containing medium. This strain also regained the ability to grow in the presence of high extracellular Ca 2ϩ levels or cyclosporin A under these FIG. 7. The pfk2⌬ mutation suppresses the high Ca 2؉ accumulation phenotype associated with the pgm2⌬ mutation. The strains were grown in YPD (A) or YPGal (B) medium to mid-log phase and harvested for total cellular Ca 2ϩ determination by flame photometry as described under "Experimental Procedures." wt, wild-type strain. growth conditions. Taken together, these results suggest that the high Ca 2ϩ uptake and accumulation observed in the pgm2⌬ strain are directly attributable to the change in the relative levels of Glc-6-P and Glc-1-P.
It was previously shown that the increased level of Glc-1-P observed when the pgm2⌬ strain is grown in medium containing galactose as the carbon source correlates with a large increase in Ca 2ϩ uptake and accumulation (35). In contrast, the increased level of Glc-6-P observed in the pfk2⌬ strain was accompanied by a much more subtle 1.5-2-fold increase in the level of total cellular Ca 2ϩ . Furthermore, unlike the pgm2⌬ strain, this change in the cellular Ca 2ϩ level was observed when the pfk2⌬ mutant was grown in medium containing either glucose or galactose as the carbon source. These findings suggest that Glc-6-P also plays a role in yeast Ca 2ϩ homeostasis that has not been reported previously. A number of studies have shown that Glc-6-P can stimulate the ATP-dependent sequestration of Ca 2ϩ into the ER of several mammalian cell types (9 -12). Consistent with these findings, an isoform of the ER-localized Glc-6-P transporter is ubiquitously expressed in mammalian cells. To date, a dedicated Glc-6-P transporter has not been reported in yeast. However, a BLAST search of the GenBank TM /EBI Data Bank using the sequence of the cloned mammalian Glc-6-P transporter indicated that several yeast open reading frames share limited sequence homology with this mammalian protein. 2 Some of these potential candidates are members of the hexose transporter gene family and have yet to have definitive subcellular locations assigned. Future work should be directed at determining whether any of these proteins possess the ability to transport Glc-6-P into an intracellular compartment in a manner that influences cellular Ca 2ϩ homeostasis.
Currently, we do not know the mechanism by which Glc-6-P and Glc-1-P influence Ca 2ϩ homeostasis in yeast. However, the results obtained to date are consistent with at least two models. The first model (Fig. 8A) is based on previous studies showing that Glc-6-P can stimulate Ca 2ϩ uptake into mammalian microsomes (9 -13). By analogy with this mechanism, the excess intracellular Glc-1-P that accumulates in the pgm2⌬ mutant may act as a competitive inhibitor of Glc-6-P transport, resulting in a reduction of Ca 2ϩ sequestration into an intracellular compartment (depicted for illustrative purposes as the ER/ Golgi apparatus). If this inhibition of Ca 2ϩ transport ultimately leads to Ca 2ϩ depletion within the affected intracellular compartment, it may result in an increased rate of cellular Ca 2ϩ uptake and accumulation by a CCE-like mechanism. There is evidence that a CCE-like mechanism exists in yeast because it was recently reported that cellular Ca 2ϩ uptake and accumulation are increased when Ca 2ϩ sequestration into the ER/Golgi apparatus is blocked by a pmr1⌬ mutation (26). This model could also account for the suppression of Ca 2ϩ homeostasis phenotypes in the pgm2⌬ mutant by the pfk2⌬ mutation because the elevated level of Glc-6-P caused by the pfk2⌬ mutation would largely restore the normal Glc-6-P/Glc-1-P ratio and thus relieve the Glc-1-P-mediated inhibition of Glc-6-P transport.
The second model (Fig. 8B) proposes that the high level of Glc-1-P that accumulates in the pgm2⌬ strain acts to stimulate the transport of Ca 2ϩ out of the cytosol. Again using the analogy of Glc-6-P-stimulated Ca 2ϩ uptake into mammalian microsomes (9 -13), Glc-1-P may stimulate the sequestration of Ca 2ϩ into an intracellular compartment such as the vacuole, ER, or Golgi apparatus in yeast. The imbalance in the Glc-6-P/Glc-1-P ratio in the pgm2⌬ mutant may enhance this sequestration to the point that the free cytosolic Ca 2ϩ level drops below a critical threshold level, thus leading to the induction of a signal to take up more Ca 2ϩ from the external environment. Inherent in this model is a mechanism by which the Glc-6-P/Glc-1-P ratio is monitored by an intracellular sensor that couples the relative abundance of these metabolites to Ca 2ϩ sequestration. Under normal conditions, an external stimulus such as a change in availability of a carbon source may alter the Glc-6-P/Glc-1-P ratio, causing this sensor to activate a signaling pathway that alters either the activity or expression of intracellular Ca 2ϩ transporters. In this scenario, the high Glc-1-P levels present 2 D. P. Aiello, L. Fu, A. Miseta, and D. M. Bedwell, unpublished data.
FIG. 8. Two models explaining how the accumulation of Glc-1-P may disrupt cellular Ca 2؉ homeostasis in yeast. A, a Glc-1-Pmediated block in the sequestration of Ca 2ϩ into an intracellular compartment induces Ca 2ϩ uptake and accumulation.
Step 3, store depletion leads to the generation of a CCE-like uptake signal.
Step 4, Ca 2ϩ channels in the plasma membrane are opened to allow the influx of Ca 2ϩ in an attempt to refill the depleted internal stores. B, an elevated level of Glc-1-P increases the sequestration of cytosolic Ca 2ϩ into an intracellular compartment, leading to induction of Ca 2ϩ uptake and accumulation.
Step 3, reduced cytosolic Ca 2ϩ levels induce a signal for Ca 2ϩ uptake through the plasma membrane.
Step 4, Ca 2ϩ channels in the plasma membrane open to allow the influx of Ca 2ϩ to restore a normal cytosolic Ca 2ϩ level. See "Discussion" for further details.
in the pgm2⌬ mutant would inappropriately (and constitutively) activate this Glc-6-P/Glc-1-P sensor. This would lead to a much greater increase in cytosolic Ca 2ϩ sequestration than normally occurs, leading to the induction of Ca 2ϩ uptake from the extracellular environment. Such a model is supported by the observation that the altered Glc-6-P/Glc-1-P ratio is the key factor controlling Ca 2ϩ homeostasis in the pgm2⌬ mutant, and not the high absolute level of Glc-1-P.
Several mechanisms could be used to monitor the Glc-6-P/ Glc-1-P ratio in the second model. Snf3p is known to be a high affinity glucose receptor located at the plasma membrane and has been shown to regulate the expression of a variety of downstream target genes via Mth1p and Std1p (50 -52). Snf3p has a large C-terminal domain that is thought to transduce a signal indicating the presence of extracellular glucose, but the detailed mechanism by which this occurs is not clear (53,54). It was recently reported that Snf3p can signal via the C-terminal domain in the absence of extracellular glucose, and it was hypothesized that Snf3p may be responsive to intracellular glucose metabolites such as Glc-6-P (55). Inappropriate signaling via Snf3p, which might occur when excess Glc-1-P is present, could lead to the increased sequestration of cytosolic Ca 2ϩ . It is not currently known whether Mth1p or Std1p regulates any intracellular Ca 2ϩ transporters. However, the global changes in gene expression in response to glucose derepression make this an intriguing possibility. A second related possibility is inappropriate signaling via the Ras-cAMP pathway. Import and phosphorylation of glucose to Glc-6-P can induce the Ras-cAMP signaling cascade (56). A clear role for Gpr1p and Gpa2p has been established in both sensing extracellular glucose and transmitting this signal downstream via the Ras-cAMP pathway (56 -58). Furthermore, Gpr1p and Gpa2p have been reported to be required for the Ca 2ϩ influx that occurs in response to glucose addition to nutrient-starved cells (2,59,60). The altered level of Glc-1-P in the pgm2⌬ mutant could upset the normal regulatory mechanisms that control this pathway and thus lead to the inadvertent increased activity or expression of transporters that sequester intracellular Ca 2ϩ .
The models presented above present several intriguing avenues for further study. Determining an intracellular compartment that has altered Ca 2ϩ levels and sequestration properties in the pgm2⌬ mutant will represent an important step in elucidating the mechanisms that couple Ca 2ϩ homeostasis and glucose metabolism. Ultimately, gaining a better understanding of these processes could lead to advances in the treatment of various diseases.