©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cooperation of Calcineurin and Vacuolar H-ATPase in Intracellular CaHomeostasis of Yeast Cells (*)

Isei Tanida (1) (2), Akira Hasegawa (2), Hidetoshi Iida (3), Yoshikazu Ohya (1), Yasuhiro Anraku (1)(§)

From the (1) Department of Plant Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, (2) Tonen Corporation, Corporate Research and Development Laboratory, 1-3-1, Nishi-tsurugaoka, Iruma-gun, Ohi-machi, Saitama 356, Japan, and the (3) Division of Cell Proliferation, National Institute for Basic Biology, 38 Nishigonaka, Myodaijicho, Okazaki 444, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Saccharomyces cerevisiae VMA genes, encoding essential components for the expression of vacuolar membrane H-ATPase activity, are involved in intracellular ionic homeostasis and vacuolar biogenesis. We report here that the immunosuppressants FK506 and cyclosporin A cause general growth inhibition of the vma3 mutant. Upon addition of the drugs, the mutant grew neither in the presence of more than 5 mM Canor above pH 6.0. The action of the immunosuppressants is dependent on their binding proteins and ascribable to inhibition of calcineurin activity; a mutation of a calcineurin subunit ( cnb1) shows synthetic lethal interaction with the vma mutation. The addition of FK506 decreases the cytosolic free concentration of Cain the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of a nonexchangeable Capool. These results suggest that calcineurin controls calcium homeostasis by repression of Caflux into a cellular compartment(s) and that the vacuolar H-ATPase is essential for cell growth cooperating with calcineurin to regulate the cytosolic free concentration of Ca.


INTRODUCTION

Intracellular Caplays important roles in signal transduction and regulation of many cellular enzymes in eukaryotic cells (1, 2, 3) . During T-cell activation (4) , antigens bind to specific receptors of quiescent T cells, and Cachannels on the endoplasmic reticulum and the plasma membrane open (4) to induce elevation of cytosolic free Caconcentration ([Ca]).() This rapid elevation of [Ca] is necessary for expression of many genes including interleukin-2. Calcineurin, also known as Ca/calmodulin-dependent phosphoprotein phosphatase or phosphoprotein phosphatase 2B, is a key signaling enzyme in this process (5, 6) . Inhibition of calcineurin by immunosuppressant drugs FK506 and cyclosporin A, which have biological effects by binding to their cytosolic receptors FKBP-12 and Cyp-18, respectively, results in reduced levels of Ca-dependent interleukin-2 transcription and loss of T-cell activation (7, 8) .

In the yeast Saccharomyces cerevisiae, the mating pheromone pathway involves mechanisms similar to that of the T-cell activation. Influx followed by a rise of [Ca] is essential for the late stage of the mating pheromone pathway (9) . Several lines of evidence suggest that yeast calcineurin is involved in response to mating pheromone (10, 11) . Mutants lacking calcineurin activity (a cnb1 mutant or a cna1 cna2 double mutant) have a defect in recovery from -factor arrest. In addition, recovery from mating factor arrest is highly sensitive to FK506 and cyclosporin A, and this sensitivity requires the presence of its respective receptors, FKBP-12 (Fkb1p) and Cyp-18 (Cyp1p) (12) . These results suggest that the Ca/calcineurin signal transduction plays an essential role in the late stage of the mating pheromone pathway. In addition, calcineurin is essential for growth in the presence of high concentrations of some monovalent cations, since the mutants lacking calcineurin activity are sensitive to 1.2 M NaCl and 140 mM LiCl (13) . Although calcineurin appears to be a key component of Casignaling in the mating response and in the regulation of cation concentration, little is known about the function of calcineurin in cytosolic Cahomeostasis.

In eukaryotic cells, a very low basal level of [Ca] is maintained against a large gradient across the plasma membrane (1, 2, 14) . The resting [Ca] (100-200 nM) of yeast cells is maintained by active transport from the cytosol into organelles and by pumping out from the cell through the plasma membrane (9) . In yeast cells, the vacuole is a major cytosolic Capool, accumulating over 95% of the total calcium associated with cells (15) . In vitro studies have indicated that Cais transported into the vacuole by Ca/Hantiporter using the proton motive force created by the vacuolar H-ATPase (16) . Since the activity of the vacuolar H-ATPase is dramatically reduced in the vma ( vacuolar membrane ATPase deficient) mutants, isolated vacuolar membrane vesicles of the vma cells have no detectable ATP-dependent Cauptake (17, 18) . Measurement of [Ca] in individual cells showed that [Ca] in vma mutants ( vma1, vma2, vma3, vma11, vma12, and vma13) is much higher (900 ± 100 nM) than that in wild type (150 ± 80 nM) (17) . This is consistent with the observation that all of the vma mutants are unable to grow in the presence of 100 mM Cain the medium (17) . These results strongly suggest that the vacuolar membrane H-ATPase plays an indispensable role in intracellular Cahomeostasis (18, 19, 20) . Recently, a putative Ca-ATPase, the PMC1 gene product, was discovered on the vacuolar membrane (21) . The pmc1 null mutant does not grow in the presence of 200 mM CaCl. Another putative Ca-ATPase, the PMR1 gene product, is thought to transport Cainto the Golgi complex (22, 23) . The pmc1 pmr1 double mutant shows synthetic lethality, indicating that the function of PMC1 is required in the pmr1 mutants.

Cunningham and Fink (21) reported that inactivation of calcineurin by mutation or by addition of the immunosuppressant, FK506, suppresses the calcium sensitivity of the pmc1 mutant. Since the pmc1 mutant shows a Ca-sensitive phenotype similar to vma mutants, it is of interest to know whether inhibition of calcineurin activity has a similar effect on the phenotype of the vma mutants. In the present study, we found that inhibition of calcineurin activity results in the opposite effect, namely lethal effects on the vacuolar membrane H-ATPase mutants. In consideration of the results, we discuss a novel function for calcineurin in intracellular Cahomeostasis in yeast cells.


MATERIALS AND METHODS

Yeast Strains, Media, and Growth Conditions

YPD medium contained 1% (w/v) Bacto-yeast extract (Difco), 2% (w/v) Bacto-peptone, and 2% (w/v) glucose. YPD pH 5.0 medium was YPD medium buffered with 50 mM succinate/NaOH, pH 5.0. YPD pH 6.0, YPD pH 6.5, and YPD pH 7.0 media were YPD supplemented with 50 mM potassium phosphate, pH 6.0, 6.5, and 7.0, respectively. 0.01-1 µg/ml FK506, 5-50 µg/ml cyclosporin A, and 1-1000 mM CaCl, 1.2 M NaCl, or 5 mM EGTA were supplemented in the medium where indicated in the text. The SD media for auxotroph selection were described in Sherman et al. (24) . For plasmid-loss experiment, FOA plates were prepared as described by Sikorski and Boeke (25) .

Strains used in this study are listed in . All yeast strains used were derivatives of ANY21, YPH499, YPH500, or YPH501 (26, 27) and constructed by transformation using the lithium acetate procedure or by standard genetic crosses.

Recombinant DNA

All procedures for recombinant DNA were carried out with Escherichia coli strain XL1-blue (Stratagene) grown in Luria broth medium with appropriate antibiotics (28) . The polymerase chain reaction (Perkin-Elmer Corp.) was performed according to the manufacturer's direction with some modification. The pRS series of vectors was obtained from P. Hieter (27) , and the pJJ series was from Prakash (29) ; pBluescript II KS+ was bought from Stratagene, and the pGem-T vector was from Promega. The oligonucleotides were synthesized with a DNA synthesizer 870A (Milipore/Miligen), and DNA sequencing was performed with DNA sequencer 370A (ABI). The pVMA3-YO326 plasmid carrying VMA3 gene was prepared by R. Hirata (30) . The ApaI- SacI fragment of the VMA3 gene was subcloned into the ApaI- SacI site of pRS316 to make pVMA3-RS316.

CNB1 Gene Disruption

The CNB1 gene was cloned by PCR amplification of the S288C genomic DNA. The oligonucleotide CNB1-Fw primer (5`-ACTTGGTAACTCAATGGTG-3`, 19-mer, sense) and the CNB1-Rev primer (5`-CTTATTGTTTGTTACATATAC-3`, 21-mer, antisense) were synthesized according to Cyert and Thorner (11) . 30 cycles of amplification (denaturing at 94 °C for 1 min, annealing at 55 °C for 2 min, extension at 74 °C for 3 min) produced the 0.8-kb product. This product was cloned into pGem-T (Promega) to make a plasmid, pCNB1-GemT. Partial sequence of this fragment was identical to the YSCCNB1 sequence (GenBank Accession No. M87508, 1992).

A null allele of CNB1 ( cnb1::HIS3) was constructed after replacement of the 0.334-kb BsmI- BsmI fragment of the CNB1 gene with the 1.7-kb BamHI- BamHI fragment of the pJJ215 plasmid that contains HIS3. This disruption plasmid was linearized by digestion with ApaI and SacI (sites that flanked this segment in the polylinker of the pGem-T vector) and was introduced into the YPH500 strain. Histransformants were selected, and the CNB1 gene disruption was confirmed both by phenotypic analysis (13) and by PCR amplification of the genomic DNA with the CNB1-Fw primer and the CNB1-Rev primer.

PMR1 Gene Disruption

The NcoI- EcoRV fragment in the PMR1 gene was cloned by PCR amplification of the S288C genomic DNA. The oligonucleotide PMR1AFw primer (5`-CATCGCCATGGCTACTGCTATTTCGTCCACAGCTTAATAC-3`, 40-mer, sense) and the PMR1ARev primer (5`-AACGGTGTTTCTGATATCAGGCAT-3`, 24-mer, antisense) were synthesized according to Rudolph et al. (22) . 30 cycles of amplification (denaturing at 94 °C for 1 min, annealing at 50 °C for 2 min, extension at 74 °C for 3 min) produced the 2.0-kb product. The ends of the PCR products were blunted, and the fragment was cloned into a EcoRV site of pBluescript II KS+ (Stratagene). Partial sequence of this fragment was identical to the sequence of YSCPMR1 sequence (GenBank Accession No. M25488, 1989).

A null allele ( pmr1::HIS3) was constructed by replacement of the 1.153-kb BglII-to- BglII fragment in the PMR1 gene with the 1.7-kb fragment of HIS3. This allele was excised by digestion with ApaI and SacI (sites that flanked this segment in the polylinker of the pBluescript II KS+ vector) and was introduced into YPH499 strain. Histransformants were selected, and the PMR1 gene disruption was confirmed by PCR. This pmr1 mutant was sensitive to YPD medium supplemented with 5 mM EGTA, pH 8.0, as described by Rudolph et al. (22) .

FKB1, CYP1, and CYP2 Gene Disruption

The construction of the fkb1, cyp1, and cyp2/crg1 null mutants was described by Tanida et al. (31) .

Determination of Cell Viability

Viability of the cells incubated under various conditions was determined as described in Ohya et al. (17) .

Cell Growth Assay Using a Culture Dish with a 96-Flat-bottom Well

Cells were cultured in YPD pH 5.0 medium to grow in the early stationary phase (1-2 10cells/ml), suspended with 2 YPD medium (2% Bacto-yeast extract Difco, 4% Bacto-peptone, and 4% glucose) to give a final concentration of 1 10cells/ml, and pre-incubated for 30 min at 30 °C. Series of solution A were supplemented with appropriate concentrations of immunosuppressant and CaCl, which were 2-fold higher than those indicated. An equal volume of the cell suspension was mixed with the solution A, and immediately 100 µl of the mixture was poured into a well on a culture dish (NUNC). The culture dish was incubated at 30 °C for 24 h with shaking. Awas measured with MTP120 microplate reader (Corona Electric). Measurement of [Ca]c in Individual Yeast Cells-[Ca] were measured in fura-2 loaded cells as described by Iida et al. (9) with some modifications. Cells cultured in YPD pH 5.0 medium (1-3 10cells/ml) were washed three times with distilled water by filtration. When indicated in the text, 2 µg/ml FK506 was added, and the cells were incubated before filtration. The cells were resuspended in distilled water containing 80 µM fura-2 (Molecular Probes) (32, 33) with the concentration of 2 10cells/ml, and the cell suspensions were subjected to electroporation under the same condition as described in Ref. 17. This condition was much milder than that for transformation, and 80-90% of the cells were still viable for 2 h after electroporation. Other details are given in Ref. 9. Pseudo color images were printed by using a color video printer (GZ-P11; Sharp).

Measurement of Exchangeable and Nonexchangeable CaPools

Intracellular exchangeable and nonexchangeable Capools were measured as described by Cunningham and Fink (21) with some modification. Yeast cultures growing exponentially (1-1.5 10cells/ml) in YPD pH 5.0 medium were shifted to YPD pH 5.0 medium supplemented with Ca (about 550 cpm/µl; DuPont NEN) and grown at 30 °C for 6.5 h. The total cell-associated Cawas calculated by measuring the radioactivity recovered from 0.1-ml culture aliquots, which were diluted into 5 ml of ice-cold buffer A1 (5 mM MES-Tris, pH 6.5, 10 mM CaCl), filtered rapidly onto a 25-mm nitrocellulose membrane filter (pore size, 0.45 µm; Milipore), washed three times with ice-cold buffer A, dried in vacuo, and processed for liquid scintillation counting. The nonexchangeable Capools were determined by the same procedure except that each culture was first diluted 10-fold into YPD pH 5.0 medium supplemented with 20 mM CaCland incubated an additional 20 min before filtration. The radioactivity released from the cells by this equilibration procedure was the exchangeable Capool, which was calculated as the difference between the total cell-associated Capool and the nonexchangeable Capool. All measurements were performed in duplicate and averaged.


RESULTS

Immunosuppressants Enhance the Calcium and pH Sensitivities of Cells Lacking Vacuolar Membrane H-ATPase

To study the effects of FK506 on growth of cells lacking the vacuolar membrane H-ATPase, the vma3 mutant was employed. We found that the mutant did not grow on YPD plates containing 1.0 µg/ml FK506 (A). Since the vma strain is incapable of growing in several conditions, we then tested whether the growth inhibition by FK506 is due to enhancement of any Vma phenotypes.

As previously reported (17) , the vma3 mutant is unable to grow in YPD medium buffered at pH 7.0, showing a neutral pH-sensitive phenotype. While the vma strain grew well on YPD pH 6.0 medium, the mutant did not grow on the same medium supplemented with 1.0 µg/ml FK506 (A). At pH 5.0, the vma strain grew irrespective to the presence of FK506 (A).

The effect of FK506 on the calcium sensitivities of the vma3 mutant was tested. The mutant grew on YPD pH 5.0 medium containing up to 50 mM CaCl(B1). In the presence of 0.1 and 1 µg/ml FK506, the mutant did not grow on YPD pH 5.0 medium containing more than 25 and 5 mM CaCl, respectively (B1). These results implied that FK506 increases both neutral pH and Casensitivities of the vma mutant in a dose-dependent manner. Cyclosporin A had similar effects on the vma3 mutant as FK506 did, but this effective dose was about 50-fold higher than that of FK506 (B1). FK506 and cyclosporin A had the same effects on the other eight vma mutants ( vma1, vma2, vma4, vma5, vma6, vma11, vma12, and vma13) as on the vma3 mutant (data not shown). The mutant lacking a VPH1 gene, which encodes a 110-kDa integral membrane subunit of the vacuolar membrane H-ATPase (34) , became more sensitive to Cain the presence of 1 µg/ml FK506 like the vma mutants (Fig. 1). These results suggested that immunosuppressants FK506 and cyclosporin A inhibit a cellular function(s), which regulates intracellular Caand pH homeostasis.


Figure 1: The effect of FK506 on calcium sensitivity of the vph1 mutant. Cell growth was scored using a 96-well microtiter dish as described under ``Materials and Methods.'' Cells were cultured in YPD pH 5.0 medium containing various concentrations of CaClwith 1 µg/ml FK506 ( FK506) or without FK506 ( No FK506). Strains employed in this experiment were YIT499 ( Wild-type), DV3T-A ( vma3), and RHV102 ( vph1).



Inhibition of Calcineurin by FK506-FKBP-12 and Cyclophilin A-Cyp-18 Complexes Results in Deleterious Effects on the vma Mutants

Binding of FK506 to FKBP-12 (Fkb1p), an intracellular FK506 binding protein, results in inhibition of calcineurin activity (7, 12) . To investigate whether the growth inhibition of the vma mutants by FK506 is due to binding of the drug to FKBP-12, we constructed a vma3 fkb1 double mutant that lacks Vma3p and FKBP-12. While the addition of 1 µg/ml FK506 inhibited growth of the vma3 single mutant on YPD pH 5.0 medium containing 5 mM CaCl, the same concentration of FK506 no longer inhibited the growth of the vma3 fkb1 double mutant on the medium containing 25 mM CaCl(B2). We also found that a cyclosporin A binding protein, Cyp-18 (Cyp1p), is required to observe cyclosporin A-dependent growth inhibition of the vma mutant. B3 shows that cyclosporin A has no effect on growth of the vma3 cyp1 mutant, which lacks Vma3p and Cyp-18. Thus, each immunosuppressant binds to the respective cytosolic binding protein to form a complex, leading to growth inhibition of the vma mutants.

To know whether growth inhibition of the vma mutants by FK506 and cyclosporin A is due to inhibition of the calcineurin activity, we examined growth of the mutant that lacks both vacuolar membrane H-ATPase and an essential subunit (Cnb1p) of calcineurin. First, we constructed vma3 cnb1 double mutants with the VMA3 gene on a URA3-harboring plasmid and tested the growth of the strain after elimination of the plasmid using a plate containing FOA. We found that none of the eight strains constructed grow on FOA plate, showing synthetic lethal interaction between VMA3 and CNB1 in this condition (Fig. 2). Second, we examined growth of the double mutant by tetrad analysis. A diploid strain heterozygous for vma3 and homozygous for cnb1 was subjected to sporulation and tetrad dissection on several plates. The double mutants grew from spore neither on YPD nor SD. The double mutants could grow but very slowly on YPD pH 5.0 medium (data not shown). These results indicated that calcineurin plays an important role for growth cooperating with vacuolar membrane H-ATPase in yeast cells.


Figure 2: Calcineurin is essential for growth in the vma3 mutant. Yeast strains were spread onto the surface of YPD pH 5.0 agar medium ( A) or FOA medium ( B) and incubated at 30 °C for 4 days. Strains anti-clockwise from the top were YIT499, DV3TA-A2, DCNB1, MCY300-1, DV3CN1-1, DV3CN1-2, DV3CN1-3, DV3CN1-4, DV3CN1-5, DV3CN1-6, DV3CN1-7, and DV3CN1-8. All the strains from DV3CN1-1 to DV3CN1-8 contained pVMA3-RS316 carrying both VMA3 and URA3 genes.



Loss of Cell Viability of the vma3 Mutant Due to Deregulation of CaHomeostasis by Inhibition of Calcineurin Activity

Ohya et al. (17) reported that the vma mutant cells dramatically decrease their viability within 2 h in YPD medium containing 100 mM CaCl. If inhibition of calcineurin by FK506 results in deregulation of [Ca] homeostasis, the vma mutants would similarly lose their viability in the presence of FK506 and a small amount of CaCl. To test this possibility, we examined cell viability of the vma3 mutant in YPD pH 5.0 medium supplemented with 5 mM CaCland 1 µg/ml FK506. In the presence of 1 µg/ml FK506, most of the vma3 mutant cells rapidly lose viability in YPD medium supplemented with 5 mM CaCl, while the mutant cells were still viable even after 8 h in the absence of FK506 (Fig. 3). Most of the vma3 fkb1 double mutant cells were viable for 17 h in the medium containing 25 mM CaCland 1 µg/ml FK506 (data not shown). Thus, inhibition of calcineurin activity in the vma mutants causes rapid lose of viability in the medium containing 5 mM CaCl.


Figure 3: Cell viability of the vma3 mutant in the presence of FK506 and CaCl. Exponentially growing cells were inoculated into YPD pH 5.0 medium with/without FK506 and/or CaCl, incubated for the indicated time, harvested, washed three times with distilled water, and plated on YPD pH 5.0 plates. After incubation at 30 °C for 3 days, colony numbers were counted.



Inhibition of Calcineurin Decreases Intracellular Free CaConcentration in the vma3 Mutant Cells

To know how calcineurin regulates intracellular Cahomeostasis in the vma mutant cells, we measured [Ca] in the vma cells using a Ca-specific indicator, fura-2 (9) . Fura-2 was electroporated into the exponentially growing vma3 cells under the condition that remains 80-90% of cell viability. Little accumulation of fura-2 was observed into vacuole of the vma3 mutant, probably because the vma3 mutant lacks vacuolar H-ATPase activity. Typical color images of [Ca] in fura-2-loaded cells of the vma3 and vma3 fkb1 double mutants are shown in Fig. 4A. Only [Ca] of the vma3 mutant cultured in YPD pH 5.0 + FK506 (2 µg/ml) for 1 h was more blue-shifted (lower concentration) than that of the others. The average [Ca] of vma3 mutant cells cultured with FK506 (The mean ± S.D. is 448 ± 177 nM, p < 0.0001) was lower than that of the cells without FK506 (651 ± 174 nM, p < 0.0001) (Fig. 4 B). No significant difference in the vma3 fkb1 mutants was observed by the addition of FK506 (562 ± 117 nM with FK506, p < 0.0001; 563 ± 172 nM without FK506, p < 0.0001) (data not shown). These results indicated that loss of calcineurin activity results in decrease of [Ca] in the vma3 mutant cells.


Figure 4: Inhibition of calcineurin by FK506-FKBP-12 complex results in the decrease of [Ca] of vma3 mutant. A, typical color images of calculated [Ca] from 340:380 nm ratio of fura-2-loaded cells. Exponentially growing cells (1 10cells/ml) were harvested, suspended in YPD pH 5.0 medium ( No FK506) or YPD pH 5.0 medium containing 2 µg/ml FK506 ( FK506 1 h), and incubated at 30 °C for 1 h. B, graphical presentation of [Ca] for individual vma3 mutant cells with ( blue bar) and/or without ( red-hatched bar) 2 µg/ml FK506.



Inhibition of Calcineurin Induces CaSequestration into an Intracellular Compartment

Since loss of calcineurin activity in the vma mutant cell results in lower [Ca], cytosolic Camight be sequestered somewhere into a certain compartment or transported to the outside of the cells. We then measured the intracellular distribution of Capools in vma3 mutant cells with and without FK506. The yeast vacuole accumulates over 95% of the total cell-associated calcium (15) , the majority of which is nonexchangeable in a pulse-chase experiment (35) . In our experiment, 88% of the total Capool in wild-type cells cultured in YPD pH 5.0 medium was nonexchangeable. In the vma3 mutant cultured in YPD pH 5.0 medium, the nonexchangeable Capool decreased to 20%, since the vma3 mutant lacks Cauptake activity into vacuole due to loss of the vacuolar H-ATPase activity (Fig. 5). In the presence of FK506 (1 µg/ml), the nonexchangeable Capool dramatically increased, being 8.9-fold higher than that without FK506. The exchangeable Capool was also increased slightly (1.8-fold). Under these conditions, no quinacrine accumulation in vacuoles of vma3 mutant cells was observed with or without FK506 (data not shown), indicating that vacuolar acidification no longer occurred in the vma3 mutant. In addition, no effect of FK506 on these Capools in the vma3 fkb1 mutant was observed. A similar but smaller FK506-induced alteration of the Capool was observed in the wild-type cells (Fig. 5). These results indicated that calcineurin controls an internal compartment(s) to sequester cytosolic free Ca.


Figure 5: Ca compartmentalization in the wild-type, vma3, and vma3 fkb1 strains. The exchangeable ( shaded) and nonexchangeable ( solid) pools of cell-associated Cawere measured in strains YIT499 ( Wild-type), DV3TA-A2 ( vma3), and DV3F1-8A ( vma3 fkb1). Bars indicate the variation of individual values of the total Capools from the mean ( n = 2).



Inhibition of Calcineurin Causes CaTolerance to the Wild-type Cells

As described above, the inhibition of calcineurin activity results in decrease of [Ca] in the vma cells. If calcineurin regulates Cahomeostasis in wild-type cells, inhibition of calcineurin might increase the Catolerance of wild-type cells. In the absence of FK506, wild-type cells grew in YPD medium supplemented with up to 300 mM CaCl, while in the presence of FK506 (1 µg/ml), the cells grew in YPD medium supplemented with up to 500 mM CaCl(Fig. 6 A). FK506 had no effect on the Casensitivity of the fkb1 cells (Fig. 6 C). The cnb1 mutant was tolerant to 500 mM CaClirrespective to FK506 (Fig. 6 B). Similar results were obtained with cyclosporin A. The effect of FK506 was specific to Catolerance; no effect on Mgsensitivity was observed (data not shown). These results indicated that loss of function of calcineurin by FK506 or the cnb1 mutation leads to Catolerance in wild-type yeast cells probably due to decreasing [Ca].


Figure 6: Inhibition of calcineurin causes wild-type cells to Ca tolerance. The growth phenotypes of YIT499 ( A, Wild-type), DCNB1 ( B, cnb1), and DF1 ( C, fkb1) cultured in YPD medium containing CaClwith 1 µg/ml FK506 ( black diamond) or without FK506 ( dotted box) were determined with a 96-well microtiter dish (see ``Materials and Methods'').



Vacuolar H-ATPase Is Essential for the Cell Growth of the pmr1 Mutant

To investigate whether the maintenance of basal [Ca] is essential for the vegetative growth of the pmr1 mutant, the vma3 pmr1 double mutant carrying VMA3 on a plasmid (pVMA3-RS316) was constructed, and a plasmid-loss experiment was performed. Out of eight strains examined, no strains grew on FOA plates (data not shown). Next, the DV3F1-8A strain ( vma3::TRP1 fkb1::URA3) was crossed with DPMR-A1 strain ( pmr1::HIS3). The diploids obtained were sporulated, and growth of the segregants on YPD pH 5.0 medium was examined by tetrad analysis. Among 36 tetrads examined, none of the viable spores showed a HisTrpphenotype (I), indicating that the vma3::TRP1 pmr1::HIS3 double mutant is inviable. No effect of the fkb1 mutation was observed on the cell growth of the vma3 mutant and the pmr1 mutant. These results implied that the vma3 pmr1 double mutant was inviable on YPD pH 5.0 medium.


DISCUSSION

In this report, we found that calcineurin is essential in a strain lacking the vacuolar H-ATPase. In addition, we present the evidence that calcineurin regulates intracellular Capool(s). We took advantage of analyzing calcineurin function in intracellular Cahomeostasis by using the vma mutants. The vacuole accumulates more than 95% of the total cell-associated calcium in wild-type yeast cells (15, 36) , and therefore it was difficult to detect Ca-sequestering activity into other organelles by using the wild-type strain. The vma mutants, however, have no detectable Cauptake activity into the vacuole in vitro (16, 17) . Consequently, we found dramatic increase of Casequestration by addition of FK506 to the vma mutant cells. Moreover, we could easily detect a decrease of [Ca] by inhibition of the calcineurin activity, since the [Ca] of the vma3 mutant was higher at the beginning than that of the wild-type cells (17) . The function of calcineurin in intracellular Cahomeostasis has hardly been studied in detail in wild-type yeast strains.

Inhibition of calcineurin activity enhances the pH and Casensitivities of the vma mutants. The vacuolar H-ATPase, which plays an important role in ion and pH homeostasis (18) , is essential in strains lacking calcineurin activity. The simple interpretation is that calcineurin regulates the function of some organelle to maintain the intracellular pH and Caconcentration. Inhibition of calcineurin activity in the wild-type cells results in Catolerance, while loss of vacuolar H-ATPase activity results in a Ca-sensitive phenotype, suggesting that calcineurin regulates the cytosolic Caconcentration in the opposite way that is directed by the vacuolar H-ATPase.

The addition of FK506 increases the nonexchangeable Capool of the vma3 cells. Although the vacuole is a major nonexchangeable Capool in the wild-type cells, we do not think that Cawas sequestered into the vacuole of the vma3 cells upon the addition of FK506. We observed no accumulation of quinacrine into the vacuole in this condition, indicating that FK506 did not elicit a proton motive force required for Cainflux into the vacuole. In addition, the Casequestration into the vacuole cannot explain why FK506 increases Casensitivity of the vma mutants. At present, we can not specify the identity of such an organelle. Pmr1p, a member of the SERCA family of CaATPases, plays an important role to transport Cainto Golgi for the normal secretion and glycosylation (22, 23) . The strict control of [Ca] is essential for cell growth in the pmr1 mutant, since the vma3 and pmr1 mutations show a synthetic lethal interaction (I). Therefore, it is possible that Pmr1p sequesters Cainto the Golgi under the control of calcineurin. Another possibility is that Catransport across the endoplasmic reticulum may be regulated by calcineurin. Recently, it was found that Cls2p, a Ca-regulatory membrane protein, is localized on the endoplasmic reticulum membrane (37) . Since FK506 has little effect on the Casensitivity of the cls2 mutant,() Cls2p may function downstream of calcineurin.

The simple model consistent with our results is shown in Fig. 7. When calcineurin is activated by the Casignaling through Ca/calmodulin, Cauptake into an internal compartment is repressed. As a result, [Ca] is increased, the Casignal is amplified, and calcineurin is further activated. Thus, reactivation of calcineurin due to amplification of the Casignal may be important. Or, since increased Cais constitutively transported into intracellular compartment, local enhancement of Casignal may be important. If calcineurin activity is inhibited by immunosuppressants or the cnb1 mutation, the Casequestering system into the internal compartment(s) is derepressed, leading to activation of Cauptake activity into the internal compartment(s). Vacuolar acidification driven by vacuolar H-ATPase is essential for normal cell growth in this condition, since the vacuole is capable of storing Cato rescue the excess storage of Cain the other compartment. The acquisition of Catolerance in wild-type cells treated with FK506 and in cnb1 mutants can be explained by this model: the [Ca] is decreased by inhibition of calcineurin so that the cells can grow in the presence of higher concentrations of Ca.


Figure 7: A working model for the calcineurin function in yeast [Ca] homeostasis.



Since the phenotypes of the vma3 cnb1 mutant and the pmc1 cnb1 mutant are totally different, it is likely that calcineurin regulates some of the vacuolar functions. Myers and Forgac (38) reported that the 50-kDa subunit of the clathrin assembly protein AP-2 (AP50), an N-ethylmaleimide-inhibitable autokinase, is associated with vacuolar H-ATPase in the bovine brain clathrin-coated vesicle. Incubation of the purified vacuolar H-ATPase with [-P]ATP results in the N-ethylmaleimide-sensitive phosphorylation of AP50 and the B-subunit (58 kDa) of the vacuolar H-ATPase. Therefore, the vacuolar H-ATPase activity may be regulated by phosphorylation/dephosphorylation with a certain kinase and calcineurin. Alternatively, the H/Caantiporter could be regulated by calcineurin. These questions will be solved by biochemical analysis of isolated vacuoles in wild-type and cnb1 mutants and mutational analysis of the vma mutants.

  
Table: List of yeast strains


  
Table: The immunosuppressants and their binding proteins enhance the calcium and pH sensitivities of the vma3 mutant

CsA, cyclosporin A; N.D., not determined.


  
Table: Tetrad analysis of heterogeneous diploids (VMA3/vma3::TRP1 FKB1/fkb1::URA3 PMR1/pmr1::HIS3)

The diploid strain, DP1V3F1-501 ( MATa/MAT leu2/leu2 lys2/lys2 his3/his3 trp1/trp1 ura3/ura3 VMA3/vma3::TRP1 FKB1/fkb1::URA3 PMR1/pmr1::HIS3), was obtained by the cross DPMR1-A1 with DV3F1-8A.



FOOTNOTES

*
This work was supported in part by Grants-in-aid for Scientific Research on Priority Areas 06740565 and 04266103 (to Y. O. and Y. A.) from the Ministry of Education, Science, and Culture of Japan and a grant from the International Human Frontier Science Program Organization (to Y. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-3-3812-2111 (ext. 4461); Fax: 81-3-3812-4929.

The abbreviations used are: [Ca], cytosolic free Caconcentration; PCR, polymerase chain reaction; FKBP-12, FK506 binding protein; Cyp-18, cyclophilin A; FOA, 5-fluoroorotic acid; kb, kilobase(s); MES, 4-morpholineethanesulfonic acid.

Y. Takita, Y. Ohya, and Y. Anraku, unpublished observations.


ACKNOWLEDGEMENTS

We thank M. Cyert (Stanford University) for critical reading the manuscript; K. W. Cunningham (The John Hopkins University) and S. Kron (Whitehead Institute) for valuable discussion, Fujisawa Pharmacy (Japan) for FK506; and T. Stevens (Oregon University), P. Hieter (The John Hopkins University), L. Prakash (University of Rochester), R. Hirata (Riken), and M. Cyert (Stanford University) for strains and plasmids.


REFERENCES
  1. Rasmussen, H., and Rasmussen, J. E. (1990) Curr. Top. Cell Regul. 31, 1-109 [Medline] [Order article via Infotrieve]
  2. Anraku, Y., Ohya, Y., and Iida, H. (1991) Biochim. Biophys. Acta 1093, 169-177 [Medline] [Order article via Infotrieve]
  3. Trewavas, A. J., and Gilroy, S. (1991) Trends Genet. 7, 356-361 [Medline] [Order article via Infotrieve]
  4. Gardner, P. (1989) Cell 59, 15-20 [Medline] [Order article via Infotrieve]
  5. Clipstone, N. A., and Crabtree, G. R. (1992) Nature 357, 695-697 [CrossRef][Medline] [Order article via Infotrieve]
  6. O'Keefe, S. J., Tamura, J., Kincaid, R. L., Tocci, M. J., and O'Neill, E. A. (1992) Nature 357, 692-694 [CrossRef][Medline] [Order article via Infotrieve]
  7. Liu, J., Farmer, J. D., Jr., Lane, W. S., Friedman, J., Weissman, I., and Schreiber, S. L. (1991) Cell 66, 807-815 [Medline] [Order article via Infotrieve]
  8. Sigal, N. H., and Dumont, F. J. (1992) Annu. Rev. Immunol. 9, 519-560 [CrossRef]
  9. Iida, H., Yagawa, Y., and Anraku, Y. (1990) J. Biol. Chem. 265, 13391-13399 [Abstract/Free Full Text]
  10. Cyert, M. S., Kunisawa, R., Kaim, D., and Thorner, J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7376-7380 [Abstract]
  11. Cyert, M. S., and Thorner, J. (1992) Mol. Cell. Biol. 12, 3460-3469 [Abstract]
  12. Foor, F., Parent, S. A., Morin, N., Dahl, A. M., Ramadan, N., Chrebet, G., Bostian, K. A., and Nielsen, J. B. (1992) Nature 360, 682-684 [CrossRef][Medline] [Order article via Infotrieve]
  13. Nakamura, T., Liu, Y., Hirata, D., Namba, H., Harada, S., Hirokawa, T., and Miyakawa, T. (1993) EMBO J. 12, 4063-4071 [Abstract]
  14. Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433 [CrossRef][Medline] [Order article via Infotrieve]
  15. Eilam, Y., Lavi, H., and Grossowicz, N. (1985) J. Gen. Microbiol. 131, 623-629
  16. Ohsumi, Y., and Anraku, Y. (1983) J. Biol. Chem. 258, 5614-5617 [Abstract/Free Full Text]
  17. Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977 [Abstract/Free Full Text]
  18. Anraku, Y., Umemoto, N., Hirata, R., and Ohya, Y. (1992) J. Bioenerg. Biomembr. 24, 395-405 [Medline] [Order article via Infotrieve]
  19. Anraku, Y. (1987) in Plant Vacuoles (Marin, B., ed), pp. 255-265, Plenum Publishing Corp., New York
  20. Klionsky, D. J., Nelson, H., and Nelson, N. (1992) J. Biol. Chem. 267, 3416-3422 [Abstract/Free Full Text]
  21. Cunningham, K. W., and Fink, G. R. (1994) J. Cell Biol. 124, 351-363 [Abstract]
  22. Rudolph, H. K., Antebi, A., Fink, G. R., Buckley, C. M., Dorman, T. E., LeVitre, J., Davidow, L. S., Mao, J., and Moir, D. T. (1989) Cell. 58, 133-145 [Medline] [Order article via Infotrieve]
  23. Antebi, A., and Fink, G. R. (1992) Mol. Biol. Cell 3, 633-654 [Abstract]
  24. Sherman, F., Hicks, J. B., and Fink, G. R. (1986) Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  25. Sikorski, R. S., and Boeke, J. D. (1991) Methods Enzymol. 194, 302-318 [Medline] [Order article via Infotrieve]
  26. Hirata, R., Ohsumi, Y., Nakano, A., Kawasaki, H., Suzuki, K., and Anraku, Y. (1990) J. Biol. Chem. 265, 6726-6733 [Abstract/Free Full Text]
  27. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27 [Abstract/Free Full Text]
  28. Maniatis, T., Fritsch, E, F., and Sambrook, J. (1982) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Jones, J. J., and Prakash, L. (1990) Yeast 6, 363-366 [Medline] [Order article via Infotrieve]
  30. Umemoto, N., Yoshihisa, T., Hirata, R., and Anraku, Y. (1990) J. Biol. Chem. 265, 18447-18453 [Abstract/Free Full Text]
  31. Tanida, I., Yanagita, M., Maki, N., Namiyama, F., Kobatashi, T., Hayano, T., Takahashi, N., and Suzuki, M. (1991) Transplant. Proc. 23, 2856-2861 [Medline] [Order article via Infotrieve]
  32. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J. Biol. Chem. 260, 3440-3450 [Abstract]
  33. Poenie, M., Alderton, J., Tsien, R. Y., and Steinhardt, R. A. (1985) Nature 315, 147-149 [Medline] [Order article via Infotrieve]
  34. Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. (1992) J. Biol. Chem. 267, 14294-14303 [Abstract/Free Full Text]
  35. Eilam, Y. (1982) Biochim. Biophys. Acta 687, 8-16 [Medline] [Order article via Infotrieve]
  36. Halachimi, D., and Eilam, Y. (1993) FEBS lett. 316, 73-78 [CrossRef][Medline] [Order article via Infotrieve]
  37. Takita, Y., Ohya, Y., and Anraku, Y. (1995) Mol. Gen. Genet. 246, 269-281 [Medline] [Order article via Infotrieve]
  38. Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186 [Abstract/Free Full Text]

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