From the
Saccharomyces cerevisiae VMA genes, encoding essential
components for the expression of vacuolar membrane
H
Intracellular Ca
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
In eukaryotic cells, a very low basal level of
[Ca
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
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.
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. His
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.
His
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
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
In this report, we found that calcineurin is essential in a
strain lacking the vacuolar H
Inhibition of calcineurin activity enhances
the pH and Ca
The addition of FK506 increases
the nonexchangeable Ca
The simple
model consistent with our results is shown in Fig. 7. When
calcineurin is activated by the Ca
CsA, cyclosporin A; N.D., not determined.
The
diploid strain, DP1V3F1-501 ( MATa/MAT
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.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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
Ca
nor 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 Ca
in the vma3 mutant cells. Consequently, FK506 induces an 8.9-fold elevation of
a nonexchangeable Ca
pool. These results suggest that
calcineurin controls calcium homeostasis by repression of
Ca
flux 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
.
plays 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 Ca
channels on the endoplasmic
reticulum and the plasma membrane open
(4) to induce elevation
of cytosolic free Ca
concentration
([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) .
]
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 Ca
signaling in the mating response and
in the regulation of cation concentration, little is known about the
function of calcineurin in cytosolic Ca
homeostasis.
]
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 Ca
pool, accumulating over 95% of the total
calcium associated with cells
(15) . In vitro studies
have indicated that Ca
is transported into the
vacuole by Ca
/H
antiporter 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 Ca
uptake
(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 Ca
in the medium
(17) . These results strongly suggest that
the vacuolar membrane H
-ATPase plays an indispensable
role in intracellular Ca
homeostasis
(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 Ca
into 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.
-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
Ca
homeostasis in yeast cells.
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) .
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).
transformants 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).
transformants 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
10
cells/ml), suspended with 2
YPD medium (2%
Bacto-yeast extract Difco, 4% Bacto-peptone, and 4% glucose) to give a
final concentration of 1
10
cells/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. A
was 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
10
cells/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
10
cells/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
Ca
Intracellular exchangeable
and nonexchangeable CaPools
pools were measured as
described by Cunningham and Fink
(21) with some modification.
Yeast cultures growing exponentially (1-1.5
10
cells/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
Ca
was 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 Ca
pools 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 CaCl
and incubated an additional 20 min
before filtration. The radioactivity released from the cells by this
equilibration procedure was the exchangeable Ca
pool,
which was calculated as the difference between the total
cell-associated Ca
pool and the nonexchangeable
Ca
pool. All measurements were performed in duplicate
and averaged.
Immunosuppressants Enhance the Calcium and pH
Sensitivities of Cells Lacking Vacuolar Membrane
H
To study the effects of FK506
on growth of cells lacking the vacuolar membrane
H-ATPase
-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.
(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
Ca
sensitivities 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 Ca
in 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 Ca
and 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.
-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 Ca
Ohya et al.
(17) reported that the vma mutant cells dramatically
decrease their viability within 2 h in YPD medium containing 100
mM CaClHomeostasis by Inhibition of
Calcineurin Activity
. 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 CaCl
and
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 CaCl
and 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 Ca
To know how calcineurin regulates intracellular
CaConcentration in the vma3 Mutant
Cells
homeostasis 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
10
cells/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
Ca
Since loss of calcineurin activity in the
vma mutant cell results in lower
[CaSequestration into an Intracellular
Compartment
]
, cytosolic
Ca
might be sequestered somewhere into a certain
compartment or transported to the outside of the cells. We then
measured the intracellular distribution of Ca
pools
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 Ca
pool 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 Ca
pool decreased to 20%,
since the vma3 mutant lacks Ca
uptake
activity into vacuole due to loss of the vacuolar
H
-ATPase activity (Fig. 5). In the presence of
FK506 (1 µg/ml), the nonexchangeable Ca
pool
dramatically increased, being 8.9-fold higher than that without FK506.
The exchangeable Ca
pool 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 Ca
pools in the vma3 fkb1 mutant was observed. A similar but smaller FK506-induced
alteration of the Ca
pool 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 Ca
were measured in strains
YIT499 ( Wild-type), DV3TA-A2 ( vma3), and
DV3F1-8A ( vma3 fkb1). Bars indicate the
variation of individual values of the total Ca
pools
from the mean ( n = 2).
Inhibition of Calcineurin Causes
Ca
As described above, the inhibition of calcineurin
activity results in decrease of
[CaTolerance to the Wild-type
Cells
]
in the vma cells. If calcineurin regulates Ca
homeostasis
in wild-type cells, inhibition of calcineurin might increase the
Ca
tolerance 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 Ca
sensitivity of the fkb1 cells (Fig. 6 C). The cnb1 mutant was
tolerant to 500 mM CaCl
irrespective to FK506
(Fig. 6 B). Similar results were obtained with
cyclosporin A. The effect of FK506 was specific to Ca
tolerance; no effect on Mg
sensitivity was
observed (data not shown). These results indicated that loss of
function of calcineurin by FK506 or the cnb1 mutation leads to
Ca
tolerance 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 CaCl
with 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
To
investigate whether the maintenance of basal
[Ca-ATPase Is
Essential for the Cell Growth of the pmr1 Mutant
]
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 His
Trp
phenotype
(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.
-ATPase. In addition, we
present the evidence that calcineurin regulates intracellular
Ca
pool(s). We took advantage of analyzing
calcineurin function in intracellular Ca
homeostasis
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 Ca
uptake activity into the vacuole in
vitro (16, 17) . Consequently, we found dramatic
increase of Ca
sequestration 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
Ca
homeostasis has hardly been studied in detail in
wild-type yeast strains.
sensitivities 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 Ca
concentration. Inhibition of
calcineurin activity in the wild-type cells results in Ca
tolerance, while loss of vacuolar H
-ATPase
activity results in a Ca
-sensitive phenotype,
suggesting that calcineurin regulates the cytosolic Ca
concentration in the opposite way that is directed by the
vacuolar H
-ATPase.
pool of the vma3 cells. Although the vacuole is a major nonexchangeable
Ca
pool in the wild-type cells, we do not think that
Ca
was 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 Ca
influx into the vacuole. In addition, the Ca
sequestration into the vacuole cannot explain why FK506 increases
Ca
sensitivity of the vma mutants. At
present, we can not specify the identity of such an organelle. Pmr1p, a
member of the SERCA family of Ca
ATPases, plays an
important role to transport Ca
into 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 Ca
into the Golgi under the control of
calcineurin. Another possibility is that Ca
transport
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 Ca
sensitivity of the cls2 mutant,
(
)
Cls2p may function downstream of calcineurin.
signaling through
Ca
/calmodulin, Ca
uptake into an
internal compartment is repressed. As a result,
[Ca
]
is increased, the
Ca
signal is amplified, and calcineurin is further
activated. Thus, reactivation of calcineurin due to amplification of
the Ca
signal may be important. Or, since increased
Ca
is constitutively transported into intracellular
compartment, local enhancement of Ca
signal may be
important. If calcineurin activity is inhibited by immunosuppressants
or the cnb1 mutation, the Ca
sequestering
system into the internal compartment(s) is derepressed, leading to
activation of Ca
uptake 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 Ca
to rescue the excess storage of Ca
in the other
compartment. The acquisition of Ca
tolerance 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
/Ca
antiporter 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: The immunosuppressants and their binding
proteins enhance the calcium and pH sensitivities of the vma3 mutant
Table: Tetrad analysis of heterogeneous
diploids (VMA3/vma3::TRP1 FKB1/fkb1::URA3 PMR1/pmr1::HIS3)
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.
]
, cytosolic free
Ca
concentration; PCR, polymerase chain reaction;
FKBP-12, FK506 binding protein; Cyp-18, cyclophilin A; FOA,
5-fluoroorotic acid; kb, kilobase(s); MES, 4-morpholineethanesulfonic
acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.