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J. Biol. Chem., Vol. 275, Issue 49, 38245-38253, December 8, 2000

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Cytosolic Ca²⁺ Homeostasis Is a Constitutive Function of the V-ATPase in Saccharomyces cerevisiae^*

Carola Förster and Patricia M. Kane^§

From the Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York 13210

Received for publication, July 25, 2000, and in revised form, September 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION 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 via a Ca²⁺ pump (Pmc1p) and a H⁺/Ca²⁺ antiporter (Vcx1p) driven by the vacuolar H⁺-ATPase (V-ATPase). We examined the function of the V-ATPase in cytosolic Ca²⁺ homeostasis by comparing responses to a brief Ca²⁺ challenge of a V-ATPase mutant (vma2) and wild-type cells treated with the V-ATPase inhibitor concanamycin A. The kinetics of the Ca²⁺ response were determined using transgenic aequorin as an in vivo cytosolic Ca²⁺ reporter system. In wild-type cells, the V-ATPase-driven Vcx1p was chiefly responsible for restoring cytosolic Ca²⁺ concentrations after a brief pulse. In cells lacking V-ATPase activity, brief exposure to elevated Ca²⁺ compromised viability, even when there was little change in the final cytosolic Ca²⁺ concentration. vma2 cells were more efficient at restoring cytosolic [Ca²⁺] after a pulse than concanamycin-treated wild-type cells, suggesting long term loss of V-ATPase triggers compensatory mechanisms. This compensation was dependent on calcineurin, and was mediated primarily by Pmc1p.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The vacuolar H⁺-ATPase (V-ATPase)¹ is a universal component of eukaryotic organisms. V-ATPases play both constitutive roles in internal organelles of all eukaryotic cells and specialized roles distinct to specific cell types (1, 2).V-ATPase activity generates and maintains an acidic pH inside the organelles of the central vacuolar system, including lysosomes, endosomes, the Golgi apparatus, secretory vesicles, and clathrin-coated vesicles (1, 2). Their action provides the driving force for solute transport across endomembranes and contributes to Ca²⁺ and pH homeostasis of the cytosol (1, 3).

The yeast V-ATPase has emerged as an excellent model for the constitutive V-ATPase found in all eukaryotic cells because of the strong similarity between the yeast enzyme and other eukaryotic V-ATPases and the number of methodological approaches established in this organism. Fungi appear to be able to tolerate loss of V-ATPase function, but exhibit a variety of growth and morphological phenotypes (4-6). Disruption of genes encoding for any V-ATPase subunit in Saccharomyces cerevisiae, with the exception of the functionally redundant STV1 and VPH1 genes (7), is conditionally lethal and generates a characteristic Vma phenotype. Yeast cells lacking a V-ATPase subunit can grow in medium buffered to pH 5, but fail to grow in medium buffered to pH 7.5, medium containing elevated calcium concentrations, or medium containing standard concentrations of non-fermentable carbon sources (1, 2). Although this set of Vma phenotypes has been used both to identify new VMA genes and to characterize subunit structure and function of the yeast V-ATPase (8-11), these phenotypes are still poorly understood. Furthermore, a temperature-conditional vma mutant, in which loss of V-ATPase function can be induced by a shift to 37 °C, exhibited an exaggerated set of morphological phenotypes, including defects in cell morphology, actin distribution, and cytokinesis (12). These phenotypes were more severe than those observed in a corresponding vma deletion strain, and indicate that an "acute" loss of V-ATPase function in the temperature conditional mutant can have different consequences, at least in the short term, from a "chronic" loss of V-ATPase function in vma deletion strains. This difference may be attributed to activation of compensatory mechanisms that allow cells to adapt to loss of V-ATPase function.

Tight control of cytosolic Ca²⁺ concentrations in yeast (50-200 nM range) is a prerequisite for proper function of Ca²⁺-dependent signal transduction pathways (13, 14) and may also be critical for maintaining the solubility of phosphorylated metabolites in the cell (15). Previous studies of Ca²⁺ homeostasis of S. cerevisiae have characterized a feedback mechanism for cytosolic Ca²⁺ control involving calmodulin, calcineurin, and three intracellular Ca²⁺ transporters: Vcx1p, a vacuolar H⁺/Ca²⁺ exchanger, Pmc1p, a vacuolar Ca²⁺ pump, and Pmr1p, a Golgi Ca²⁺ pump with homology to Pmc1p (16). Under typical growth conditions, the vacuole appears to be the major Ca²⁺ store in yeast (14). Recent results suggest that the two vacuolar transporters play complementary roles in Ca²⁺ homeostasis (17). Vcx1p is predominantly responsible for restoring cytosolic Ca²⁺ concentrations after a brief challenge with high extracellular Ca²⁺ concentrations; Pmc1p plays a minimal role under these conditions (17). In contrast, Pmc1p appears to be critical for long term Ca²⁺ tolerance, because vcx1 mutants are relatively tolerant of extended exposure to elevated extracellular [Ca²⁺] but pmc1 mutants are Ca²⁺-sensitive (16, 18, 19).

How loss of Ca²⁺ homeostasis contributes to the conditional lethality of vma mutants is only partially understood. V-ATPase activity drives H⁺/Ca²⁺ exchange by Vcx1p in vitro (19-21), and the elevated resting cytoplasmic [Ca²⁺] reported for vma mutants (8, 23) has been attributed to loss of activity of this exchanger. There are additional complexities in the role of the V-ATPase in Ca²⁺ homeostasis, however. The calcium sensitivity of vma mutants is much more severe than that of vcx1 mutants (8, 16, 19). vma mutants also exhibit synthetic lethality with calcineurin mutants (24, 25), and treatment with calcineurin inhibitors aggravates both the pH and Ca²⁺ sensitivity of vma mutants (25). Either elevated Ca²⁺ or elevated pH could induce morphological defects in vma4-1^ts mutants at the non-permissive temperature, suggesting that control of cytoplasmic pH and Ca²⁺ might be essential for a common set of processes (12). From this and other results, it is clear that the Ca²⁺ and pH sensitivity of vma mutants are linked, and this may reflect important links between cytosolic pH and Ca²⁺ homeostasis. In this work we have addressed the calcium-related aspects of the Vma phenotype by comparing how cellular response to a brief Ca²⁺ challenge is affected by an acute loss of V-ATPase activity (in a temperature-sensitive vma mutant or in wild-type cells treated with concanamycin A) or a chronic loss of V-ATPase activity (in a vma deletion mutant). We have investigated how extracellular pH modifies calcium responses of vma mutants and assessed the significance of alternative mechanisms of vacuolar acidification in supporting Ca²⁺ homeostasis. Finally, we have begun to address how other transport systems may help compensate for loss of the V-ATPase (17).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Zymolyase 100T was purchased from ICN. Native coelenterazine was purchased from Molecular Probes. All other reagents were purchased from Sigma.

Strains, Transformation, and Culture Conditions-- Yeast strains used in this study and their genotypes are listed in Table I. The congenic pmc1vma2 and vma2 strains were prepared by transforming strains YDB0224 and SEY6210 with XhoI- and HindIII-digested pCY40 (26) in a single-step gene disruption (27). Disruption of VMA2 was confirmed by Western blotting with monoclonal antibody 13D11 against Vma2p.

Yeast media were prepared as described by Sherman et al. (28). Buffered medium was prepared as described by Yamashiro et al. (26), except that 50 mM MES and 20 mM MPOS was used to buffer YEPD (yeast extract-peptone-2% dextrose) plates to pH 7.5 when the medium contained 60 mM CaCl₂. Bacterial strains were grown on standard media (29).

Measurement of Cytosolic Free Ionized [Ca²⁺]-- A pEVP11-based plasmid containing a functional apoaequorin gene (pAEQ; a gift from Patrick Masson, University of Wisconsin-Madison) was transformed into yeast using the LEU2 gene as a selectable marker (30). The measurement of cytosolic free [Ca²⁺] was carried out with modifications as described previously (17). Briefly, cells containing the pAEQ plasmid were grown overnight at 28 °C on SD-leucine medium (fully supplemented minimal medium lacking leucine) and were harvested in the exponential phase. Cells were than diluted to an A₆₀₀ of 0.15 into YEPD, pH 5.0, and allowed to double twice. Temperature-sensitive mutants were incubated at both the restrictive (37 °C) and permissive (28 °C) temperatures. 5 A₆₀₀ units of cells were resuspended in 0.1 ml of aequorin test medium, which consisted of SD medium containing 2 mM EGTA and 50 mM sodium succinate/50 mM sodium phosphate buffer, pH 5.8. To convert the apoaequorin to aequorin, 5 µl of 590 µM coelenterazine (dissolved in methanol) was added, and the cells were incubated for 20 min at room temperature on a rocker. They were then centrifuged briefly in a microcentrifuge, and the supernatant containing the excess coelenterazine was removed. The cells were washed in 0.5 ml of aequorin test medium and then resuspended in 0.5 ml of test medium and incubated at room temperature for another 20 min on a rocker before initiating the experiment. Aequorin-dependent light emission was measured for 10 s to determine the basal cytosolic Ca²⁺ levels. CaCl₂ was then added in a pulse to a final concentration of 50 mM. This calcium addition was calculated to give free ionized [Ca²⁺] concentrations of 35.5 mM at pH 5.0, 30.8 mM at pH 5.8, and 20.4 mM at pH 7.5 in the aequorin test buffer, using the Bound and Determined program (31). Changes in cytosolic Ca²⁺ levels were recorded for an additional 2-10 min. A Berthold 9050 Lumat luminometer was used to collect data at 1-s intervals. To determine the concentration of cytosolic Ca²⁺ using the aequorin reporter system, the total amount of reconstituted aequorin available for light emission and the relationship between Ca²⁺ concentration and light emission (32) were determined. The total amount of reconstituted aequorin was determined routinely in a crude extract of each strain by measuring the maximum light emission (L_max) value in the presence of a saturating Ca²⁺ pulse. To prepare the crude extract, 2 A₆₀₀ units of cells in aequorin standard buffer (50 mM sodium succinate/50 mM sodium phosphate buffer, 2 mM EDTA pH 5.8, 2 mM phenylmethylsulfonyl fluoride) was lysed by agitation in the presence of 4 mg/ml Zymolyase 100 T at room temperature. After 25 min, the lysate was spun at 14,000 rpm in a microcentrifuge for 3 min. The supernatant served as the extract for the luminescence assay. The L_max value was generally between 1.5 and 2.0 × 10⁶ relative light units/s.

To determine the relationship between the free [Ca²⁺] and aequorin-dependent light emission, a standard curve was prepared using a cell lysate as described previously (32). Briefly, increasing concentrations of CaCl₂ were added to a crude extract of wild-type cells (SF838-5A and SEY6210) prepared in aequorin standard buffer. To determine the cytosolic [Ca²⁺] within intact cells, both the L observed in intact cells and the L_max emission observed in a crude extract of the same cells were determined. The ratio between these values (L:L_max) was then used to estimate the cytosolic free [Ca²⁺] from the standard curves. In no case was the L value in an experiment greater than 2-3% of the L_max value. Thus, the absolute amount of reconstituted aequorin was not limiting in the experiments.

Inhibition of the V-ATPase with Concanamycin A-- Cells containing the pAEQ plasmid were grown overnight at 25 °C on SD-leucine medium and harvested in exponential phase. Cells were then diluted to A₆₀₀ 0.15 into the same medium at 28 °C and allowed to double twice. Apoaequorin was converted to aequorin. Cells were incubated and washed as described above then resuspended in test medium containing 300 nM or 1 µM concanamycin A in 1% (v/v) dimethyl sulfoxide and incubated at room temperature for another 20 min on a rocker before initiating the experiment. As a control for membrane lesion effects, an additional sample was incubated in test medium containing 1% (v/v) dimethyl sulfoxide.

Inhibition of Calcineurin-- Cells were diluted into YEPD, pH 5.0, as described and preincubated for 30 min at 28 °C. The culture was then supplemented with 50 µg/ml cyclosporin A, and cells were allowed to double twice. Cells were harvested by centrifugation, and the Ca²⁺ reporter assay was performed as described.

Viability Test-- Cells growing exponentially in YEPD, pH 5.0, medium (A₆₀₀ = 0.6) were shifted to aequorin test buffer at pH 5.0 or 7.5 (see above) and pulsed with 50 mM CaCl₂. The culture was then diluted 100-fold into aequorin test buffer at pH 5.0 or 7.5, respectively, and 100 µl of the cell suspension was spread on YEPD, pH 5.0, plates. After an incubation for 20 h at 30 °C, colonies were counted. All measurements were performed in duplicate and averaged.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sensitivity of Yeast vma Mutants to a Brief Ca²⁺ Pulse-- We initially examined the viability of vma mutants after exposure to a brief Ca²⁺ challenge at different extracellular pH. To minimize adaptation of cells to the loss of V-ATPase activity, we determined viability and Ca²⁺ response in a temperature-sensitive strain (vma4-1^ts) in which the Vma phenotype can be induced by a simple temperature shift from 28 to 37 °C. Exponentially growing cells were transferred to SD-leucine medium supplemented with 2 mM EGTA (aequorin test buffer) at pH 5.0 or 7.5, subjected to a brief CaCl₂ pulse (50 mM), then spread on YEPD plates buffered to pH 5.0 and incubated at 30 °C for 20 h. Without a Ca²⁺ shock, growth was comparable for the vma4-1^ts mutant grown at 28 °C at either pH 5.0 or 7.5 (Fig. 1A). After being exposed to a brief Ca²⁺ shock, no growth inhibition was observed for the vma4-1^ts mutant grown at 28 °C at pH 5.0. However, viability of the vma4-1^ts mutant grown at 28 °C and shocked at pH 7.5 was reduced by 60% after the Ca²⁺ pulse. Without a Ca²⁺ shock, incubation at 37 °C reduced viability of the cells by 66% at pH 5.0 and by 87% at pH 7.5, respectively. After exposure to a brief increase in extracellular Ca²⁺, viability of the mutants shocked at pH 5.0 was further compromised, whereas growth of the cells pulsed in a medium buffered to pH 7.5 was completely inhibited (Fig. 1A).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Sensitivity of temperature conditional vma mutants to a brief calcium pulse. A, viability following a calcium pulse: The vma4-1ts strain was incubated for 4 h at 28 °C and 37 °C, then subjected to a 50 mM CaCl2 pulse (filled bars) or a buffer pulse (open bars) at the indicated pH. Immediately after the pulse, cells were spread on YEPD, pH 5, plates, then incubated at 30 °C for 24 h and colonies counted. B, basal and post-shock cytosolic Ca2+ levels: Basal and post-shock cytosolic Ca2+ levels were determined using an aequorin Ca2+ reporter assay. Ca2+-dependent light emission was recorded for 10 s before and 2 min after a brief 50 mM Ca2+ pulse in cells treated as described in A. Open bars, buffer pulse; closed bars, 50 mM CaCl2 pulse. The data for both A and B represent the average and standard deviation of six independent experiments.

To better understand how high Ca²⁺ levels inhibited growth of the acute vma mutants and how Ca²⁺-dependent growth inhibition was related to extracellular pH, we monitored the cytoplasmic Ca²⁺ levels in vma4-1^ts cells before and after a Ca²⁺ challenge at pH 5.0 or 7.5 (Fig. 1B). Cytosolic free [Ca²⁺] was directly monitored using transgenic aequorin as an in vivo Ca²⁺ reporter system (33). Cultures were grown in YEPD medium buffered to pH 5.0, harvested, and resuspended in aequorin test buffer (17). Ca²⁺-dependent bioluminescence was measured for 5 s to determine basal cytosolic [Ca²⁺]. The resting cytosolic[Ca²⁺] of the vma4-1^ts mutant at 28 °C was in the 80-85 nM range at either pH 5.0 or 7.5. To examine the response to a Ca²⁺ challenge, 50 mM CaCl₂ was injected into the cell suspension (Fig. 1B), and the restored post-shock cytosolic [Ca²⁺] were determined after 2 min. The vma4-1^ts cells grown at 28 °C quickly recovered after the Ca²⁺ shock at either pH reaching a new steady-state level of 85-90 nM. In contrast, basal cytosolic Ca²⁺ levels in the vma4-1^ts cells incubated at 37 °C were significantly higher than in the cells grown at permissive temperature, ranging from 110 to 120 nM (Fig. 1B). The post-shock level of 125 nM Ca²⁺ in the mutant shocked at pH 5.0 was only slightly elevated, indicating that the Vma strain at low pH was still able to regulate its cytosolic Ca²⁺ levels fairly efficiently. In contrast, post-shock cytosolic [Ca²⁺] in the Vma cells at pH 7.5 decreased to a level of only 140 nM (Fig. 1B). These results indicate that, at elevated pH, the ability to compensate for loss of V-ATPase activity is more strongly compromised than at low pH. The elevated basal levels of cytosolic Ca²⁺ may partially account for loss of viability in vma mutants, but a calcium pulse can further compromise viability even under conditions where there is minimal effect on the post-shock cytosolic [Ca²⁺].

To further analyze the function of the V-ATPase in response to a brief Ca²⁺ shock and its relationship to the external pH, we monitored the kinetics of the cytosolic Ca²⁺ response to an extracellular Ca²⁺ pulse in wild-type cells and a vma2 mutant. The wild-type cells routinely exhibited a resting free cytosolic [Ca²⁺] of 90-95 nM whether the pH of the aequorin test buffer was pH 5.0 or 7.5 (Fig. 2A). However, the recovery profile after the Ca²⁺ pulse differed at the two pH values. For wild-type cells at low external pH, cytosolic [Ca²⁺] increased rapidly after injection of 50 mM CaCl₂ and reached a peak level corresponding to ~230 nM cytosolic [Ca²⁺] within 5 s (Fig. 2A). The [Ca²⁺] decreased rapidly thereafter and returned to a new steady-state free cytosolic [Ca²⁺] of ~95 nM within 60 s. At pH 7.5, however, the response of wild-type cells to the Ca²⁺ shock showed a biphasic profile. Light emission increased rapidly and reached a peak level corresponding to ~230 nM cytosolic Ca²⁺ within 5 s (Fig. 2A). Recovery occurred in two steps, suggesting a very fast removal of the cytosolic Ca²⁺ within the first 10 s after the pulse to ~140 nM and a much more gradual removal of excess cytosolic Ca²⁺ to a new steady state of 100 nM within 70 s after the pulse. Because of differential effects of buffering ions in the medium, a 50 mM CaCl₂ pulse delivers 35 mM free ionized Ca²⁺ at pH 5 and 20 mM at pH 7.5. To test whether the different Ca²⁺ profiles were an effect of the different free external [Ca²⁺] at pH 5.0 and pH 7.5 or a genuine pH effect, we administered a 20 mM Ca²⁺ pulse, delivering a free ionized [Ca²⁺] of 12.2 mM, at pH 5.0. A similar recovery profile was obtained at pH 5.0 for a 20 mM and 50 mM CaCl₂ pulse, distinct from the profile at pH 7.5 (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Rapid changes in cytosolic free Ca2+ levels and viability after a Ca2+ shock at different external pH. A, aequorin measurement of cytosolic Ca2+ levels in wild-type cells. Ca2+-dependent light emission was monitored over a 2-min period in strains containing aequorin, and light emission was converted to Ca2+ concentration as described under "Experimental Procedures." The Ca2+ shock was initiated by injecting 50 mM into the test medium after measuring the basal light emission for 10 s. The data represent the average of three independent kinetic measurements. Medium was buffered to pH 5.0 or 7.5 as indicated. , pH 5; , pH 7.5. B, aequorin measurement of cytosolic Ca2+ levels in vma2 cells. Measurements were performed as described in A. , pH 5; , pH 7.5. C, growth of wild-type and vma2 cells following a Ca2+ shock. Cells were plated on rich medium (YEPD, pH 5) following a Ca2+ shock administered at pH 5 or 7.5 as described for A and B (closed bars) or following administration of a buffer control (open bars). Colonies formed were counted after 20 h at 30 °C.

We next examined the response of strains carrying the vma2 mutation to Ca²⁺ shock to further define the role of the V-ATPase in short term Ca²⁺ recovery. In the experiment shown in Fig. 2B, the initial resting cytosolic Ca²⁺ was ~120 and ~130 nM at pH 5.0 and 7.5, respectively, 1.4-fold higher than the wild-type strain. When the vma2 strain was exposed to Ca²⁺ shock at low pH, the maximum cytosolic [Ca²⁺] was lower than observed in the wild-type strain and corresponded to a peak cytosolic [Ca²⁺] of 180 nM, reached within 20 s after the pulse. In other experiments (for example, Fig. 4B) peak cytosolic Ca²⁺ levels comparable to the wild-type were observed. Recovery was always much more gradual than observed for the wild-type cells, reaching a new steady-state level of cytosolic Ca²⁺ of 145 nM after 2 min (Fig. 2B). At elevated environmental pH (pH 7.5), injection of 50 mM CaCl₂ generated a very steep spike of cytosolic [Ca²⁺], corresponding to a maximum concentration of ~280 nM within 5 s (Fig. 2A). As in wild-type cells, recovery occurred in 2 steps, a very fast removal of the peak [Ca²⁺] within the first 10 s after the pulse to ~ 180 nM and a slow incomplete removal of further cytosolic Ca²⁺ to a new steady state of 150 nM within 100 s after the pulse (Fig. 2B). Similar results were obtained in a vma3 strain (data not shown). In general, the absolute peak values for cytosolic [Ca²⁺] after a pulse were more variable between experiments for the vma mutants than for the wild-type cells. The shapes of the recovery curves were very consistent, however.

Wild-type and vma2 strains were spread on YEPD, pH 5.0, plates to test the viability of the cells after the Ca²⁺ insult (Fig. 2C). Neither environmental pH nor a Ca²⁺ pulse affected viability of the wild-type cells. Comparison to the results with the vma4-1^ts mutant (Fig. 1A) suggests that the mutant is more sensitive to Ca²⁺ at pH 7.5 than wild-type cells, even at the permissive temperature. (Possible reasons for this difference are described under "Discussion.") Viability of the vma2 mutants was strongly compromised by the Ca²⁺ challenge even at pH 5 (Fig. 2C), however, as observed for the vma4-1^ts mutants (Fig. 1B). Taken together, the results indicate that strains carrying the vma2 mutation are defective in regulation of basal cytosolic Ca²⁺ levels after a brief Ca²⁺ insult and that V-ATPase activity is essential for proper Ca²⁺ homeostasis even at low pH.

Effect of Concanamycin A on Cytosolic Ca²⁺ Homeostasis-- As outlined above, loss of V-ATPase activity in vma2 mutants and even in the vma4-1^ts mutant might promote compensatory mechanisms. To test this possibility, we examined the Ca²⁺ response in wild-type cells in the presence of varied concentrations of concanamycin A, a rapid and specific inhibitor of the V-ATPase (34). Wild-type cells were incubated for 20 min with the inhibitor. CaCl₂ was then injected to a concentration of 50 mM, delivering a free ionized [Ca²⁺] of 30.8 mM at pH 5.8. After the Ca²⁺ challenge, bioluminescence was recorded for 10 min (Fig. 3A), and cell viability was assessed in parallel (Fig. 3B). We found that treatment of cells with either 300 nM or 1 µM concanamycin A caused the peak cytosolic Ca²⁺ level in the wild-type strain to increase from ~260 to ~350 nM. Recovery and viability differed between the 300 nM and 1 µM concanamycin A treatments (Fig. 3A). Although cells treated with 300 nM concanamycin A showed a slow, but still complete, recovery within 360 s after the pulse, the level of cytosolic Ca²⁺ in cells treated with 1 µM concanamycin A reached only ~190 nM (Fig. 3A). Viability of the cells after exposure to 300 nM concanamycin A was reduced by 45% without calcium addition and by 65% after a Ca²⁺ challenge (Fig. 3B). Treatment with 1 µM concanamycin compromised viability more strongly; viability was reduced by 60% in the absence and by more than 85% in the presence of a Ca²⁺ challenge (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effect of concanamycin A on viability and response of cytosolic Ca2+ levels to elevated extracellular Ca2+. A, aequorin measurement of cytosolic Ca2+ levels. Wild-type (SF838-5A) cells were pretreated for 20 min with 300 nM or 1 µM concanamycin (in Me2SO) or Me2SO alone. Ca2+-dependent light emission was then recorded for 10 s to determine the basal cytosolic [Ca2+]. A 50 mM CaCl2 pulse was administered, and light emission was recorded for an additional 110 s and converted to [Ca2+] as described under "Experimental Procedures." The data represent the average of three independent kinetic measurements. , control (Me2SO only); , 300 nM concanamycin A; , 1 µM concanamycin A. B, growth of concanamycin-treated cells following a Ca2+ shock. Cells treated with concanamycin A as described in A were plated on rich medium (YEPD, pH 5) following a Ca2+ shock (closed bars) or following administration of a buffer control (open bars). Colonies formed were counted after 20 h at 30 °C.

In comparison to concanamycin A-treated cells, vma mutants (vma2, vma4-1^ts) are better able to control cytosolic Ca²⁺ in the absence of V-ATPase activity. If a proton gradient across the vacuolar membrane generated by other mechanisms (4) were available to drive vacuolar Ca²⁺ sequestration at pH 5, then we would expect inhibition of V-ATPase activity by concanamycin A to have little effect. Because concanamycin A severely impairs calcium recovery even at low pH, it appears that the V-ATPase is necessary for Ca²⁺ sequestration at both low and high pH.

Calcineurin Compensates for Loss of V-ATPase Function in Ca²⁺ Homeostasis-- To elucidate how cells compensated for loss of V-ATPase activity to achieve control of cytosolic Ca²⁺, we compared the response of wild-type and vma2 strains to a brief Ca²⁺ shock after inactivation of calcineurin by addition of 50 µg/ml cyclosporin A to the growth medium (Fig. 4). Inactivation of calcineurin did not affect the ability of wild-type cells to recover quickly and completely from a Ca²⁺ challenge (Fig. 4A). In contrast, inactivation of calcineurin in vma mutants affects the ability of the cells to regulate their cytosolic [Ca²⁺] (Fig. 4B). Peak [Ca²⁺] was increased from ~250 to ~285 nM, and recovery was both slower and less complete in the presence of cyclosporin. Cells reached a new steady-state Ca²⁺ level of only ~190 nM cytosolic Ca²⁺ instead of ~145 nM without the inhibitor (Fig. 4B). The response of the vma2 cells in the presence of calcineurin strongly resembles the Ca²⁺ response of the wild-type cells in the presence of 1 µM concanamycin A (Fig. 3A).

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Ca2+ response of wild-type and vma mutant cells after inactivation of calcineurin. A and B, effect of calcineurin inhibition on cytosolic Ca2+ response to a Ca2+ shock. Exponentially growing cells were inoculated into YEPD, pH 5.0, medium with 50 µg/ml cyclosporin A in methanol or with methanol alone, allowed to double twice, harvested, and employed in the aequorin Ca2+ reporter assay. Ca2+-dependent light emission was recorded for 10 s in low Ca2+ medium to determine the basal cytosolic [Ca2+]. A 50 mM CaCl2 pulse was administered, and light emission was recorded for an additional 110 s and converted to [Ca2+] as described under "Experimental Procedures." The data represent the average of three independent kinetic measurements. A, wild-type (SF838-5A): , control (methanol only); , 50 µg/ml cyclosporin A. B, vma2 (SF838-5A vma2): , control (methanol only); , 50 µg/ml cyclosporin. C, synthetic effects of pmc1 and vma2 mutations: The growth of wild-type (SEY6210), vma2 (SEY6210vma2), and vma2pmc1 (YDB0224vma2) strains after 2 days of growth on the indicated medium were compared.

Previous results have indicated that transcription of the vacuolar Ca²⁺ pump Pmc1p is strongly induced by calcineurin (16). When V-ATPase activity is lost, Pmc1p may be the major system used for vacuolar Ca²⁺ sequestration. To test this possibility, we constructed a pmc1vma2 double mutant strain. If Pmc1p is important in the calcineurin-mediated compensation for loss of V-ATPase activity, then we would anticipate that the double mutant might be even less tolerant of elevated extracellular calcium than either single mutant. This proved to be true, as shown in Fig. 4C. Both the vma2 and pmc1 single mutants are able to tolerate an extracellular [Ca²⁺] of 60 mM in medium buffered to pH 5, but the double mutant was unable to grow under these conditions. The double mutant even grows somewhat more slowly than either single mutant in the absence of added CaCl₂ (Fig. 4C).

Vcx1 Is Chiefly Responsible for Restoring Cytosolic Ca²⁺ Concentrations after a Brief Pulse-- To further examine the targets of calcineurin activation, the response of vcx1 and pmc1 mutants to a brief Ca²⁺ pulse in the presence and absence of cyclosporin was monitored by the aequorin assay system and compared with the response of wild-type cells (Fig. 5, A and B).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Ca2+ response of vacuolar transporter mutants after inactivation of calcineurin. A and B, exponentially growing cells were inoculated into YEPD, pH 5.0, medium with 50 µg/ml cyclosporin or with an equivalent volume of the methanol solvent (control). The cells were allowed to double twice, harvested, and employed in the aequorin Ca2+ reporter assay as described. The data represent the average of three independent kinetic measurements. A, vcx1 cells (YDB0225): , control; , 50 µg/ml cyclosporin. B, pmc1 cells (YDB0224): , control; , 50 µg/ml cyclosporin.

Loss of Vcx1p activity severely compromises Ca²⁺ restoration following a Ca²⁺ pulse (Fig. 5A). In contrast to wild-type cells (~95 nM Ca²⁺), basal cytosolic [Ca²⁺] before the pulse was 135-140 nM in vcx1 mutants in the absence and presence of cyclosporin. After a Ca²⁺ challenge, peak [Ca²⁺] in vcx1 mutants reached values of ~350 nM, and cytosolic [Ca²⁺] fell to a new steady-state level of ~150 nM after the shock. Inactivation of calcineurin resulted in an even stronger phenotype (Fig. 5A). Recovery was even weaker, with post-shock cytosolic [Ca²⁺] reaching ~180 nM, comparable to the levels reported in wild-type cells treated with 1 µM concanamycin A (Fig. 3A).

The Ca²⁺ response monitored for the pmc1 strain was very similar to that obtained for the wild-type (Fig. 5B). Calcineurin has previously been reported to exert an inhibitory effect on Vcx1p activity at the post-translational level (16, 19). Consistent with this, a more rapid restoration of cytosolic Ca²⁺ levels was observed in pmc1 mutants after inactivation of calcineurin with cyclosporin. Basal cytosolic [Ca²⁺] was slightly lower than observed for wild-type and pmc1 mutants in the absence of cyclosporin (~85 nM), and cells recovered from a 50 mM Ca²⁺ pulse within 30 s to a new steady-state [Ca²⁺] of ~90 nM. Thus, the V-ATPase-driven Vcx1p seems to be chiefly responsible for restoring cytosolic Ca²⁺ levels after a brief Ca²⁺ challenge as proposed by Miseta et al. (17), and this system may restore [Ca²⁺]even more efficiently when calcineurin is inactivated. Inactivation of calcineurin in strains carrying a vcx1 mutation severely perturbs the regulation of cytosolic Ca²⁺ levels following a Ca²⁺ pulse, to an extent comparable to cyclosporin-treated vma mutants or concanamycin A-treated wild-type cells.

Significance of Alternative Mechanisms of Vacuolar Acidification for Ca²⁺ Homeostasis in vma Mutants-- Several alternative pathways for generating partial vacuolar acidification in vma mutants at low extracellular pH have been envisaged (1, 4, 35). To determine if such proton gradients can drive Ca²⁺ sequestration by Vcx1p in vma mutants, we examined the response to a Ca²⁺ shock in a vcx1 mutant at pH 5.0 and 7.5 (Fig. 6). If Vcx1p is using a proton gradient generated by alternative mechanisms at pH 5 to help drive Ca²⁺ uptake, we would anticipate that vcx1 cells would be less efficient in controlling cytosolic [Ca²⁺] at pH 5 than vma mutants. In vcx1 mutant cells, however, the pH dependence of recovery was similar to that observed in an vma mutant (Fig. 6A, see Fig. 2B). At pH 5.0, cells show a gradual recovery from a peak cytosolic [Ca²⁺] of ~220 nM, reaching a new steady-state cytosolic [Ca²⁺] of ~155 nM (Fig. 6A). At pH 7.5, injection of 50 mM CaCl₂ generated a very steep spike of cytosolic [Ca²⁺] (Fig. 6A). The difference in Ca²⁺ restoration at pH 5.0 and 7.5 may therefore result from pH-dependent activity of a compensatory vacuolar Ca²⁺ sequestration system, such as Pmc1p. The high spike in cytosolic Ca²⁺ arising from a Ca²⁺ challenge at pH 7.5 may be ascribed to a failure of this system to efficiently sequester Ca²⁺ at this pH.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   pH dependence of Ca2+ response in a vcx1 mutant. A, aequorin measurement of cytosolic Ca2+ levels. Ca2+-dependent light emission was monitored over a 2-min period in vcx1 (YDB0225) mutants containing aequorin, and light emission was converted to Ca2+ concentration as described under "Experimental Procedures." The Ca2+ shock was initiated by injecting 50 mM CaCl2 into the test medium after measuring the basal light emission for 10 s. The data represent the average of three independent kinetic measurements. Medium was buffered to pH 5.0 () or pH 7.5 () during the aequorin measurement. B, growth of vcx1 and vma2 in the presence of elevated pH and calcium concentrations. vcx1 (YDB0225) and vma2 (SF838-5A vma2) cells were grown on YEPD, pH 7.5, containing 60 mM CaCl2 for 72 h.

Although vcx1 mutants have been shown to be relatively Ca²⁺-insensitive (16, 19), their growth at high pH has not been examined. We compared the growth of vma2 and vcx1 mutants on medium containing 60 mM CaCl₂ at pH 7.5 (Fig. 6B). vma2 cells do not grow at all under these conditions. In contrast, vcx1 cells can grow, arguing that the spike of high cytosolic Ca²⁺ seen at pH 7.5 in both vcx1 and vma2 does not directly cause vma2 mutants to lose viability.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The vacuole has been shown to be the major site of intracellular Ca²⁺ storage in yeast and functions to maintain cytosolic Ca²⁺ levels within a narrow physiological range via a Ca²⁺ pump (Pmc1p) and a H⁺/Ca²⁺ antiporter (Vcx1p) driven by the V-ATPase (14, 16, 18). Here we present evidence that a general loss of cytosolic calcium control exacerbates, or may even be responsible for, the pH conditional lethality of vma mutants (4). To characterize the calcium-related aspects of the Vma phenotype, we compared the responses of a temperature-conditional V-ATPase mutant, a non-conditional V-ATPase mutant, and wild-type cells treated with the V-ATPase inhibitor concanamycin A to a brief Ca²⁺ challenge.

A temperature conditional vma mutant (vma4-1^ts (12)) was initially used to examine the effect of a brief Ca²⁺ challenge. The goal was to observe the immediate consequences of loss of V-ATPase activity on control of cytosolic [Ca²⁺], before onset of compensatory mechanisms. The more pronounced morphological defects in the vma4-1^ts mutant had previously been attributed to the absence of compensatory mechanisms in the temperature conditional strain (12). Fig. 1 demonstrates that this mutant is clearly more Ca²⁺-sensitive than wild-type cells even at the permissive temperature, however, it also suggests that V-ATPase function is compromised and compensatory mechanisms may be active, even at low temperature. Interestingly, the effects of a brief Ca²⁺ pulse on viability are clearly aggravated when the pulse is administered at elevated pH, even at the permissive temperature (Fig. 1B). These data are in accord with previous studies on the Vma phenotype, which report synergistic effects of high pH and high calcium (25).

To further investigate how extracellular pH modifies calcium responses, we used the in vivo aequorin Ca²⁺ reporter system (17, 30, 33) to monitor the time course of cytoplasmic [Ca²⁺] changes following a Ca²⁺ challenge. The vma4-1^ts mutant exhibits a variety of morphological aberrations and a strong tendency for the cells to clump in suspension. As a result, we were able to use the aequorin system to measure resting [Ca²⁺] in this strain (Fig. 1) but were unable to measure rapid cytosolic [Ca²⁺] changes. The time course of cellular response to a Ca²⁺ challenge was therefore determined in wild-type cells with and without a brief concanamycin A treatment and in vma2 mutant cells. The concanamycin treatment generated an "acute" loss of V-ATPase activity, without the leakiness of the vma4-1^ts mutant, and the vma2 cells represented long term loss of V-ATPase activity, with the potential for activation of compensatory mechanisms for pH and Ca²⁺ control. It is clear from Fig. 3 that the kinetics of the Ca²⁺ response in the concanamycin-treated wild-type strain are quite different from that of the vma2 strain. In the concanamycin-treated cells, the basal cytosolic [Ca²⁺] was strongly elevated, and the recovery from the Ca²⁺ pulse was slow and weak, in accord with previous observations (17). Concanamycin A had no effect on the Ca²⁺ response of the vma2 strain (data not shown). These results suggest that the deletion mutant is showing some adaptation in its response to the Ca²⁺ pulse that is not seen immediately following loss of V-ATPase activity. This adaptation appears to be more efficient at low pH; vma2 cells show decreased viability when the [Ca²⁺] pulse is administered at pH 7.5 (Fig. 2C) as well as a large spike in cytosolic [Ca²⁺] at pH 7.5 that is not seen at pH 5 (Fig. 2B).

These results shed some light on the Ca²⁺ sensitivity associated with loss of V-ATPase activity, but the picture is still incomplete. As reported previously for vma deletion mutants (8, 23), both the vma2 mutant and the vma4-1^ts mutant at 37 °C showed an elevated cytosolic [Ca²⁺] relative to wild-type cells. The cytosolic [Ca²⁺] measured in the vma2 mutant was not as high as those reported previously using Ca²⁺-sensitive dyes (8, 23). This may be due both to contributions of fura-2 and indo-1 in locations other than the cytosol in the earlier experiments and to some depletion of intracellular Ca²⁺ in our experiment, where the aequorin test buffer contains 2 mM EGTA. It is most notable, however, that elevated cytosolic [Ca²⁺] does not appear to fully account for the loss of viability in vma mutants. In both the vma4-1^ts mutants subjected to a Ca²⁺ pulse at pH 7.5, 28 °C (Fig. 1) and in the wild-type cells treated with 300 nM concanamycin A (Fig. 3), there is a significant loss of viability following a brief exposure to high extracellular [Ca²⁺], even though cytosolic [Ca²⁺] is restored to normal or near-normal levels. It is possible that viability is compromised by the spike in cytosolic [Ca²⁺] observed in the vma mutants at pH 7.5, perhaps because some Ca²⁺ signal transduction pathway is activated. The presence of a very similar spike in the vcx1 mutant argues against this, however, because growth of the vcx1 mutant is relatively insensitive to high extracellular [Ca²⁺] (Fig. 6B).

Nelson and Nelson (4) suggested that vma mutants acidify their vacuoles at pH 5.0 by endocytosis, and other means have subsequently been suggested for achieving vacuolar acidification at pH 5, including activation of the plasma membrane H⁺-ATPase and passive proton permeability via weak bases present in yeast media (6, 35). These models would connect extracellular pH and cytosolic Ca²⁺ control by proposing that a proton gradient capable of driving H⁺/Ca²⁺ exchange at the vacuole can be generated at extracellular pH 5.0 but not at pH 7.5. Our data do not support the presence of a vacuolar pH gradient driving Ca²⁺ uptake at pH 5. A pH-dependent cytosolic Ca²⁺ response identical to that in the vma2 mutant is observed in the vcx1 mutant, which lacks the exchanger. In addition, concanamycin A treatment severely impairs recovery from a Ca²⁺ pulse even at low pH, suggesting that the V-ATPase is essential at both low and high pH. It could be argued that elevated pH and Ca²⁺ are not specifically connected but instead represent two insults to cells weakened by lack of the V-ATPase. This is hard to eliminate entirely, but it is notable that the effects on viability shown in Figs. 1A and 2C were observed when the cells were exposed to pH 7.5 medium only during the time of the calcium pulse (less than 10 min). The explanation for the pH dependence of Ca²⁺ responses that is most consistent with our data is that the cellular compensation triggered by long term loss of V-ATPase function operates more efficiently at low pH. Further biochemical studies will be necessary to verify this explanation.

The data presented here clearly implicate calcineurin activation in adaptation of cells to loss of V-ATPase activity. vma2 mutants incubated with cyclosporin A, a calcineurin inhibitor, respond to a calcium pulse very similarly to wild-type cells treated with 1 µM concanamycin A, suggesting that adaptation in the deletion mutant has been eliminated (Figs. 3A and 4B). This result is consistent with previous results demonstrating synthetic lethality between calcineurin and vma mutants (24, 25). Calcineurin has been shown to generate strong transcriptional activation of Pmc1p in response to elevated cytosolic [Ca²⁺] (16, 19). Our results (Fig. 5), like those of Miseta et al. (17) suggest that H⁺/Ca²⁺ exchange driven by the V-ATPase is the predominant mechanism for lowering cytosolic [Ca²⁺] in response to a brief Ca²⁺ pulse. Pmc1p is the major vacuolar target of calcineurin activation and long term adaptation to elevated [Ca²⁺] (16, 19). Taken together, these results indicate that activation of Pmc1p by calcineurin may be the main mechanism by which cells adapt to loss of V-ATPase activity. The increased calcium sensitivity of the pmc1vma2 double mutant (Fig. 4C) strongly supports this.

It is probable that Pmc1p activation is not the sole means of adaptation to loss of V-ATPase activity. Although vma and calcineurin double mutants were reported to be inviable (24), the pmc1vma2 mutant is viable in the presence of low pH and low extracellular [Ca²⁺]. In addition, Tanida et al. (25) observed a pronounced, but slightly less severe, effect of calcineurin inhibition in vph1 mutants, which specifically affect V-ATPase activity at the vacuole. Miseta et al. (36) have demonstrated that uptake into the exchangeable Ca²⁺ pool becomes important under conditions where vacuolar function is severely compromised, and the Golgi Ca²⁺ pump, Pmr1p, also shows a modest activation in response to calcineurin. We frequently observed some "blunting" of the Ca²⁺ response in the vma2 mutant, similar to that seen in Fig. 2B. This blunting could arise from a reduction in Ca²⁺ uptake by the plasma membrane Ca²⁺ channel Mid1/Cch1 (37) or from a very rapid removal of cytosolic Ca²⁺ by another mechanism (38), either of which could show some calcineurin dependence. Further experiments will be necessary to determine the relative importance of these adaptation mechanisms and their potential for pH sensitivity.

In conclusion, this work demonstrates that cytosolic Ca²⁺ homeostasis is a constitutive function of the V-ATPase in S. cerevisiae. In higher eukaryotic cells, cytosolic [Ca²⁺] fluctuates transiently to regulate such diverse processes as neurotransmitter release, muscle contraction, and T-cell activation (39-41). Recently, compelling evidence for synergistic action of V-ATPase and calcineurin in the modulation of macrophage effector function and in Ca²⁺ homeostasis of fibroblast cell lines and primary astrocytes has been reported (42). The work presented here provides interesting insights into the synergistic action of calcineurin and V-ATPase in intracellular Ca²⁺ homeostasis with implications for future studies in yeast and higher eukaryotes.

	ACKNOWLEDGEMENTS

We thank David Bedwell, Richard Kellermayer, Patrick Masson, and Ann Batiza for providing strains and plasmids. We thank Maureen Tarsio for excellent technical assistance. We also thank David Bedwell for helpful discussions and for providing critical comments on the manuscript.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant R01-GM50322 (to P. M. K.) and by Deutsche Forschungsgemeinschaft fellowship Fo315/1-1 (to C. F.).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.

Present address: Dept. of Medical Nutrition, Karolinska Institute, NOVUM, Huddinge S-14186, Sweden.

^§ An American Heart Association Established Investigator. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 750 East Adams St., State University of New York, Upstate Medical University, Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8736; E-mail: kanepm@mail.upstate.edu.

Published, JBC Papers in Press, September 15, 2000, DOI 10.1074/jbc.M006650200

	ABBREVIATIONS

The abbreviations used are: V-ATPase, vacuolar H⁺-ATPase; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; YEPD, yeast extract-peptone-2% dextrose.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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