 |
INTRODUCTION |
Like all eukaryotes, the yeast Saccharomyces cerevisiae
normally maintains a resting cytosolic Ca2+ concentration
of 50-200 nM (1-3). This tight regulation of
intracellular Ca2+ is required to control the complex
signaling pathways mediated by cytosolic Ca2+-sensing
proteins such as calmodulin. Remarkably, yeast cells can maintain
intracellular Ca2+ homeostasis in the presence of
environmental Ca2+ concentrations ranging from <1
µM to >100 mM (4). The vacuole is thought to
play a key role in maintaining Ca2+ tolerance over this
wide range because it contains >90% of the total cellular
Ca2+ (5, 6). Accordingly, many different vacuolar mutations result in an inability to grow in the presence of high concentrations of extracellular Ca2+ (7-13).
Currently, two Ca2+ transporters have been described which
act to sequester Ca2+ in the vacuole. The first of these is
the vacuolar Ca2+ ATPase encoded by the PMC1
gene, a homolog of the mammalian PMCA plasma membrane family of
Ca2+ ATPases. The loss of Pmc1p results in an
inability to grow in the presence of high environmental
Ca2+ (7). The second protein known to be involved in
vacuolar Ca2+ transport is the
H+/Ca2+ exchanger encoded by the
VCX1 (HUM1) gene (14, 15). Although mutants that
do not express Vcx1p show little or no decrease in Ca2+
tolerance, the combination of pmc1
and vcx1
mutations leads to a more severe Ca2+-sensitive phenotype
than the loss of either transporter alone. Both the expression and
function of these two vacuolar Ca2+ transporters are
regulated by calcineurin, a highly conserved protein phosphatase that
is activated by Ca2+/calmodulin. As in mammalian cells, the
activation of yeast calcineurin can be blocked by the immunosupressant
drugs cyclosporin A (CsA)1
and FK506 (16, 17). Although the functional relationship between these
two vacuolar Ca2+ transporters is complex, it has been
reported that calcineurin activation stimulates Pmc1p function and
inhibits Vcx1p function (14, 15).
Several other genes encoding potential Ca2+ ATPases have
been identified within the yeast genome (18); however, the only member of this group demonstrated to play a role in Ca2+ transport
is encoded by the PMR1 gene. Pmr1p is related to the SERCA
family of Ca2+ ATPases and has been shown to reside in the
Golgi apparatus of S. cerevisiae (19-22). Although Pmr1p
and Pmc1p both act to partition Ca2+ into distinct cellular
compartments, their roles in Ca2+ homeostasis do not appear
to be equivalent. First, cells lacking Pmc1p are sensitive to high
environmental Ca2+, whereas cells lacking Pmr1p cannot grow
under low Ca2+ conditions. In addition, the total cellular
Ca2+ level in a pmc1
strain is 2-3-fold
lower than normal, but the total cellular Ca2+ level in the
pmr1
mutant is 4-5-fold higher than normal. These different phenotypes suggest that the vacuole and the Golgi apparatus normally carry out distinct roles in Ca2+ homeostasis.
Genetic screens have identified at least 60 different genes involved in
vacuolar protein localization (23). Among these, the class C vacuolar
protein sorting mutants (which include the vps11,
vps16, vps18, and vps33 mutants)
result in the most severe defects in vacuolar biogenesis. For example,
strains carrying the vps33
mutation lack a
morphologically distinguishable vacuole but instead accumulate small
vesicular and Golgi-like structures (24-26). These anomalous
compartments may result from the inability to dock and/or fuse late
transport vesicles from the biosynthetic, endocytic, and autophagic
pathways with the vacuole (27). A vps33
strain was also
found to secrete >90% of soluble vacuolar proteins such as
carboxypeptidase Y and to mislocalize nearly 50% of the vacuolar
membrane protein
-mannosidase to the cell surface (24).
In this study we asked how the severe defects in vacuolar biogenesis
associated with the vps33
mutation affect cellular
Ca2+ homeostasis. We found that the vps33
strain was sensitive to both high and low levels of environmental
Ca2+ and was unable to regulate cytosolic Ca2+
levels properly when exposed to a sudden, large increase in
environmental Ca2+. Despite its defect in vacuolar
biogenesis, we found that the vps33
strain contains more
total cellular Ca2+ than a wild type strain. To determine
whether other intracellular compartments compensate for reduced
vacuolar Ca2+ storage, we examined whether the
Golgi-localized Ca2+ ATPase Pmr1p plays a significant role
in Ca2+ homeostasis in the vps33
strain. We
found that PMR1 expression is elevated in the
vps33
strain. We also found that a
vps33
/pmr1
strain is hypersensitive to high
extracellular Ca2+, and the combination of certain
mutations effecting both vacuolar and Golgi Ca2+ transport
results in synthetic lethality. These results indicate that the Golgi
apparatus plays a significant role in maintaining Ca2+
homeostasis when vacuolar biogenesis is compromised.
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MATERIALS AND METHODS |
Strains Used--
Strains used in this study are listed in Table
I. The PMC1 and
VCX1 genes were disrupted using the one-step gene
replacement method (28). A 1.62-kb fragment of the PMC1 gene
was generated by PCR using wild type yeast genomic DNA as template. The
forward primer used was 5'-ATCGGTACCA CTTGGATTGC AT-3', and
the reverse primer was 5'-CATGGATCCT GCCATCCTCA-3'. These
primers contained KpnI and BamHI restriction
endonuclease sites respectively (underlined). The PCR product was
digested with KpnI and BamHI and cloned into a
pBluescript II KS (+) plasmid. The 1.06-kb segment of the
PMC1 gene was then removed by digestion with
AflIII and EcoRI and replaced by the
TRP1 gene taken from pJJ280 plasmid (29). A
KpnI/NotI fragment containing the disrupted
pmc1
::TRP1 fragment was then used to transform
yeast. Trp+ colonies were selected, and the correct gene
replacement was confirmed by PCR.
Similarly, a 2.04-kb fragment of the VCX1 gene was generated
by PCR using genomic DNA as template. The forward primer used was
5'-CGTGGTACCT TGTCATCCTCAC-3', and the reverse primer was 5'-GCTAGGATCC GCTAAAATAG G-3'. Again, these primers
contained KpnI and BamHI restriction endonuclease
sites, respectively (underlined). The fragment was digested with these
enzymes and cloned into a pBluescript II KS (+) plasmid. A 1.56-kb
fragment was removed from the VCX1 DNA by digestion with
HincII and HindIII endonucleases and replaced
with a fragment containing the URA3 gene obtained from
pJJ244 (29). A KpnI/BamHI fragment containing the
disrupted vcx1
::URA3 fragment from this plasmid
was used to transform yeast. The replacement of wild type
VCX1 was confirmed by PCR analysis. Other genetic
manipulations were carried out by standard methods (30).
Culture Media--
Bacterial strains were grown on standard
media (31). Yeast strains were maintained on YP medium containing 2%
D-glucose (YPD) or synthetic minimal medium containing 2%
D-glucose (SMD) and other supplements as required (30).
Growth media were routinely buffered with 40 mM MES-Tris,
pH 5.5.
Determination of Ca2+ Concentration in
Media--
EGTA was used to reduce the Ca2+ concentration
of buffered media. Because YPD and SMD media contain divalent cations
other than Ca2+, the effective concentrations of
Mg2+, Mn2+, Fe2+, K+,
and Na+ were considered when calculating free
Ca2+ concentrations. Known quantities of CaCl2
stock solutions were added, and the resulting free Ca2+
concentrations were calculated based on the total concentration of
Ca2+ as well as other cations, pH, and temperature of the
medium. These calculations were done using the Maxchelator 1.2 program.
Measurements of Total Cellular Ca2+,
Mg2+, Na+, K+, and Phosphate
Levels--
50-100 A600 units of yeast growing
in YPD supplemented with CaCl2 or EGTA were harvested by
centrifugation at 5,000 × g for 5 min. The cell
pellets were washed with fresh YP and transferred to microcentrifuge
tubes whose mass had previously been determined gravimetrically to an
accuracy of 0.1 mg on an analytical balance. The tubes were centrifuged
at 15,000 × g for 5 min, and the supernatants were
removed carefully. The tubes were then respun, and any remaining supernatant was again removed. The tubes containing the pellets were
weighed to determine the wet weight of the pellet, and the pellets were
then dried to completion in a Savant SpeedVac system. The tubes were
then weighed again to determine the dry weight of the pellet. 1 M HCl was added to the dry pellets, and the capped microcentrifuge tubes were vortexed and incubated on a rocker for at
least 24 h. Thereafter each sample was centrifuged briefly in a
microcentrifuge, and multiple aliquots of each supernatant were taken
for ion measurements. Ca2+, Na+, and
K+ measurements of aliquots were carried out with an
Eppendorf EFOX-5070 flame photometer; Mg2+ levels of
aliquots were determined using a Varian AA-20 atomic absorption
spectrophotometer. Cellular ion concentrations were then calculated
based on the dry weight of the samples and dilution factors. Total
combined orthophosphate and polyphosphate levels (referred to as total
inorganic phosphate) were determined in the 1 M HCl
hydrolysate described above using an acid molybdate-based diagnostic
kit (Sigma). The phosphorus levels measured represent the sum of the
acid-hydrolyzed polyphosphate and the inorganic phosphate present
(32).
Measurement of Cytosolic Free Ca2+
Concentration--
A pEVP11-based plasmid containing a functional
apoaequorin gene (pAEQ) was transformed into yeast using the
LEU2 gene as selectable marker (1). This plasmid was a gift
from Patrick Masson. Cells containing the pAEQ plasmid were grown in
SMD medium containing other necessary supplements and were harvested in
the logarithmic growth phase. 10 A600 units of
cells were resuspended in 0.2 ml of aequorin test medium, which
consists of SMD medium (which contains 1 mM
Ca2+) supplemented with 2 mM EGTA and 20 mM MES-Tris, pH 6.5. The free Ca2+
concentration of this medium was calculated to be 6 µM.
To convert the apoaequorin to aequorin, 10 µl of 590 µM
coelenterazine (dissolved in methanol) was added, and the cells were
incubated for 20 min at room temperature. They were then centrifuged
briefly in a microcentrifuge, and the supernatant containing excess
coelenterazine was removed. The cells were washed again in 0.5 ml of
aequorin test medium, and the cells were then resuspended in test
medium and incubated at room temperature for 20 min before initiating
the experiment. A Berthold Lumat 9050 luminometer was used to collect
aequorin light emission data at 200-ms intervals. The data were
downloaded directly to a computer using the MS Windows Terminal
software and transferred to Microsoft Excel 5.0 for analysis.
To determine the concentration of cytosolic Ca2+ using the
aequorin reporter system, it was necessary to determine: 1) the total amount of reconstituted aequorin available for light emission and 2)
the relationship between Ca2+ concentration and light
emission (33). The total amount of reconstituted aequorin was
determined routinely in a crude extract of each strain by measuring the
maximum light emission (Lmax) value in the
presence of a saturating concentration of Ca2+. To prepare
the crude extract, 2 A600 units of cells in 0.2 ml of aequorin standard buffer (100 mM MES-Tris, pH 6.5;
150 mM KCl; 20 mM NaCl; 5 mM
MgCl2; and 2 mM phenylmethylsulfonyl fluoride) were lysed by agitation with glass beads at 4 °C. A 25-µl aliquot was placed in the luminometer, and the Lmax of
this sample was induced by injecting 25 µl of a 50 mM
CaCl2 solution. The Lmax value was
generally between 0.5 and 1.0 × 107 relative light
units/s. The protein concentration of cell lysates was also measured
using a Bio-Rad protein assay kit. A correction factor based upon the
Lmax value/unit of protein was determined for
each strain, and this value was used to correct for minor differences
in the concentration of aequorin in different strains.
To determine the relationship between the free Ca2+
concentration and aequorin-based light emission, a standard curve was
prepared using a cell lysate as described (33). Briefly, increasing
concentrations of CaCl2 were added to a crude extract of
wild type cells prepared in aequorin standard buffer. To determine the
cytosolic Ca2+ concentration within intact cells, both the
L observed in intact cells and the
Lmax emission observed in a crude extract of the same cells were determined. The ratio between these values
(L:Lmax) was then used to estimate
the cytosolic free Ca2+ concentration from our standard
curve. In no case was the L value in an experiment greater
than 2-3% of the Lmax value. Thus, the absolute amount of reconstituted aequorin was not limiting in any of
these experiments.
45Ca2+ Uptake and Release--
To
determine the rate of Ca2+ uptake by different mutant
strains, cells were grown in SMD medium to approximately 1.0 A600/ml. Cells were harvested and resuspended in
a buffer containing 40 mM MES-Tris, pH 6.5, and 20 mM D-glucose. An aliquot of
45Ca2+ (NEN Life Science Products) was then
added, and aliquots were filtered through 0.45-µm Millipore filters
on a 12-position Millipore vacuum manifold at the indicated times. The
filtered cells were washed immediately with two 5-ml aliquots of
ice-cold blocking solution (150 mM NaCl, 20 mM
MgCl2, and 2 mM LaCl3). The
cell-associated counts on the filter were then determined by
scintillation counting. To calculate absolute Ca2+ levels,
cpm were converted to mmol of Ca2+/kg dry mass based upon
total cellular Ca2+ measurements as determined by flame
photometry under identical growth conditions.
Cells for Ca2+ exchange experiments were grown in YPD
medium to a density of 0.05 A600. The medium was
then supplemented with 45Ca2+, and the cells
were grown to a cell density of 0.5-1 A600/ml. The cells were then harvested by centrifugation at 4,000 × g for 5 min, washed, and resuspended in fresh YPD
supplemented with 50 mM CaCl2. At the indicated
times, aliquots were removed, filtered, washed, and processed for
scintillation counting as described above.
Northern Analysis--
RNA extraction and Northern analysis were
carried out as described previously (34). Strains were grown in YPD
medium in the presence of 1 mM EGTA (estimated to result in
0.01 mM free Ca2+) or 50 mM calcium
to 1 A600/ml. A 0.56-kb region of the
PMR1 gene was amplified by PCR using the primers DB-483
(5'-GGCCCCAATGAAATAACCGT AG-3') and DB-484 (5'-CCTGTTCCTAC GACGATACCC
T-3'). The ACT1 probe was prepared by PCR amplification
using the primers DB-154 (5'-GCGCG GAATT CAACG TTCCA GCCTT CTAC-3') and
DB-155 (5'-GGATG GAACA AAGCT TCTGG-3'). All probes were labeled with
[
-32P]dATP using the random hexamer method.
Radioactivity in specific hybrids was quantitated using a
PhosphorImager (Molecular Dynamics). After quantitating the
radioactivity associated with PMR1 mRNA, the membranes
were hybridized with the ACT1 probe. After background correction, the PMR1 signal of each sample was corrected
with the ACT1 mRNA control. These corrected values were
then normalized to the wild type strain grown under low
Ca2+ conditions.
 |
RESULTS |
Sensitivity of Yeast Vacuolar Mutants to Different Environmental
Ca2+Concentrations--
We initially compared the
Ca2+ tolerance of yeast strains containing knockouts of
genes involved in vacuolar Ca2+ transport (pmc1
,
vcx1
, or pmc1
/vcx1
) vacuolar biogenesis (vps33
) or a combination of both classes
(vps33
/pmc1
/vcx1
). Each strain was streaked onto
YPD plates supplemented with increasing concentrations of
CaCl2 or with 10 mM EGTA and incubated at
30 °C for 48 h. The wild type, pmc1
, vcx1
, and
pmc1
/vcx1
strains grew similarly on standard YPD
plates (buffered to pH 5.5) containing 0.3 mM
Ca2+ (Fig. 1A),
whereas the colony size of the vps33
and
vps33
/pmc1
/vcx1
strains was slightly smaller. The
wild type, pmc1
, and vcx1
strains also grew
similarly on YPD medium supplemented with 100 mM
CaCl2, whereas the growth rate of the
pmc1
/vcx1
double mutant was reduced significantly on
this medium (Fig. 1B). In contrast, neither the
vps33
strain nor the vps33
/pmc1
/vcx1
strain was able to form visible colonies under these growth conditions
during the 48-h incubation period.

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Fig. 1.
Effect of different concentrations of
extracellular Ca2+ on the growth of vacuolar mutants.
The growth of the indicated strains is shown on standard YPD (0.3 mM Ca2+, panel A), YPD plus 100 mM CaCl2 (panel B), YPD plus 200 mM CaCl2 (panel C), and YPD plus 10 mM EGTA (panel D). The plates were incubated at
30 °C for 48 h. WT, wild type.
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|
When the YPD plates were supplemented with 200 mM
CaCl2, both the wild type and vcx1
strains
grew somewhat more slowly than on YPD plates supplemented with 100 mM CaCl2. The pmc1
/vcx1
double
mutant was unable to grow under these conditions, whereas the
pmc1
strain grew much more slowly than the wild type
strain (Fig. 1C). A further doubling of the Ca2+
concentration in the YPD plate to 400 mM completely
inhibited growth of the pmc1
strain but not the growth of
the wild type and vcx1
strains (not shown). None of these
strains was inhibited by the addition of either 400 mM NaCl
or 400 mM KCl to the YPD plates, indicating that the
increased osmolarity associated with 200 mM
CaCl2 did not cause the growth sensitivity described above. We conclude that strains harboring the vps33
mutation
show greater sensitivity to high extracellular Ca2+ than
strains carrying the pmc1
mutation, the
vcx1
mutation, or both mutations together. Overall, the
rank order of Ca2+ sensitivity observed for these strains
was: vps33
/pmc1
/vcx1
and vps33
strains > pmc1
/vcx1
strain > pmc1
strain > vcx1
and wild type strains.
We also examined whether the growth of these strains was sensitive to
inhibition by the chelating agent EGTA. We found that pmc1
, vcx1
, and pmc1
/vcx1
strains grew similarly to the wild type strain on YPD plates buffered
to pH 5.5 and supplemented with 10 mM EGTA (Fig.
1D). In contrast, the growth of the vps33
and
vps33
/pmc1
/vcx1
strains was severely inhibited
under these conditions, suggesting that they require a higher minimal
level of environmental Ca2+ for efficient growth than the
other strains. However, not only Ca2+ but other cations
such as Zn2+, Fe2+, and Mn2+ are
also complexed effectively by EGTA. To confirm that low environmental Ca2+ was responsible for EGTA sensitivity, we supplemented
EGTA-pretreated media with different divalent cations to determine the
component(s) required for growth of the vps33
strain. We
found that the addition of Ca2+ could restore a significant
amount of growth in YPD medium treated with EGTA, whereas several other
cations (Mg2+, Mn2+, Fe2+,
Zn2+, and Cu2+) could not (data not shown).
These results lead us to conclude that the vps33
and
vps33
/pmc1
/vcx1
strains are more sensitive to
either high or low levels of environmental Ca2+ than the
wild type, pmc1
, vcx1
, and
pmc1
/vcx1
strains.
Measurement of Rapid Changes in the Cytosolic Ca2+
Concentration upon External Ca2+ Challenge--
Yeast
cells, like mammalian cells, have been reported to maintain cytosolic
free Ca2+ levels in the range of 50-200 nM
(1-3). To determine how the above mutations affect the ability of
yeast to maintain cytosolic Ca2+ homeostasis, we introduced
a plasmid encoding a cytosolic form of apoaequorin into each strain
(1). Apoaequorin can be converted to aequorin by incubating the strains
with the membrane-permeant cofactor coelenterazine. Once active
aequorin is generated, it is capable of emitting light as a function of
the free Ca2+ concentration present in the cytosol (33). In
the experiments described here, the aequorin-dependent
light emission of each strain was sampled throughout the experiment at
200-ms intervals. To determine the cytosolic Ca2+
concentration as a function of light emission, a standard curve was
prepared using crude extracts from the wild type strain where the light
emission at each Ca2+ concentration was correlated to the
Lmax each sample was capable of discharging
(Fig. 2A). Using this method,
the relative light units/s emitted from the wild type strain routinely
corresponded to a resting free cytosolic Ca2+ concentration
of ~75 nM when cells were incubated in a medium containing low (~6 µM) free Ca2+ (for
further details, see "Materials and Methods").

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Fig. 2.
Measurement of rapid changes in cytosolic
free Ca2+ levels after a Ca2+
shock. Panel A, standard curve correlating free
Ca2+ concentration to aequorin-dependent light
emission as measured in crude extracts. Panel B, changes in
the cytosolic free Ca2+ concentration were measured in
strains containing aequorin after the addition of 50 mM
CaCl2 to the medium. Light emission was monitored over a
2-min period from wild type, pmc1 /vcx1 ,
vps33 , and vps33 /pmc1 /vcx1 strains.
The Ca2+ shock was initiated by injecting 50 mM
CaCl2 into the test medium after measuring the basal light
emission for 10 s (for further details, see "Materials and
Methods").
|
|
To determine how various mutations affect the ability of these strains
to respond to a sudden increase in extracellular Ca2+, 50 mM CaCl2 was injected rapidly into the cell
suspension while the cytosolic aequorin-dependent light
emission was continuously monitored. We found that the light emission
of the wild type strain increased rapidly and reached a peak level
corresponding to ~300 nM cytosolic Ca2+
within 5 s (Fig. 2B). The Ca2+
concentration decreased rapidly thereafter and returned to a new
steady-state free cytosolic Ca2+ concentration of ~80-85
nM within 90 s.
The light emission measured in the pmc1
/vcx1
strain
corresponded to a basal cytosolic Ca2+ concentration of
75-80 nM. When 50 mM CaCl2 was
injected, the light emission reached a peak value corresponding to
~385 nM cytosolic free Ca2+, which was
somewhat higher than the peak observed with the wild type strain. The
recovery phase of the pmc1
/vcx1
strain was also much
weaker than the wild type control. The post-shock steady-state cytosolic Ca2+ concentration was ~310 nM,
which was 4-fold higher than the steady-state cytosolic
Ca2+ concentration observed in the wild type strain after
the same Ca2+ shock. This suggests that the loss of the
Pmc1p and Vcx1p vacuolar Ca2+ transporters severely
compromises the ability of this strain to return its cytosolic
Ca2+ concentration to a low resting level after exposure to
elevated extracellular Ca2+.
We next examined the response of strains carrying the
vps33
mutation to Ca2+ shock. We found that
the initial resting cytosolic Ca2+ concentration was ~165
nM, which was 2-fold higher than the wild type strain. The
basal cytosolic Ca2+ level measured in the
vps33
/pmc1
/vcx1
strain was ~210 nM, which was almost 3-fold higher than the wild type strain. When the
vps33
strain was exposed to Ca2+ shock, the
maximum light emission was nearly 100-fold higher than observed with
the wild type strain and corresponded to a peak cytosolic
Ca2+ concentration of ~1.75 µM (Fig.
2B). This level was 5-fold higher than the peak observed
with the wild type strain. Like the pmc1
/vcx1
strain,
the recovery of the vps33
strain from the peak cytosolic Ca2+ level was much weaker than the wild type control and
reached a new steady-state level at ~470 nM (6-fold
higher than the wild type strain). The
vps33
/pmc1
/vcx1
strain exhibited a high peak of
cytosolic Ca2+ which corresponded to ~1.5
µM, which was somewhat lower than was observed with the
vps33
strain. However, the recovery of this strain from
the peak level was even weaker than the vps33
strain and
reached a new steady-state level of ~660 nM (more than 8-fold higher than the wild type strain). The weaker recovery of this
strain may indicate that a low level of residual function of the Pmc1p
and/or the Vcx1p transporters remains within the vesicles that
accumulate in the vps33
strain. When taken together, these results indicate that strains carrying the vps33
mutation are severely compromised in their ability to regulate basal
cytosolic Ca2+ levels and are unable to sequester
efficiently the cytosolic Ca2+ that enters the cell after
an acute Ca2+ shock.
Measurement of Total Cellular Ca2+, Mg2+,
and Phosphate Levels in Yeast Vacuolar Mutants--
A large fraction
of total cellular Ca2+, Mg2+, and polyphosphate
normally resides within the vacuole (6, 8, 35, 36). To determine how
these various vacuolar mutations affect the capacity to store these
compounds within the vacuole, we measured their total cellular levels
(Fig. 3). We did not detect a significant change in the level of Mg2+ in the
pmc1
/vcx1
strain, although a small (22%) decrease in total cellular inorganic phosphate (orthophosphate and polyphosphate) was observed. In contrast, the total cellular Ca2+ level
was reduced nearly 2-fold in the pmc1
/vcx1
strain.

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Fig. 3.
The vps33 mutation causes
a large decrease in cellular Mg2+ and phosphate but does
not reduce the amount of cellular Ca2+. Cultures of
the indicated strains were grown in standard YPD medium, and the
relative amounts of Mg2+ (panel A), inorganic
phosphate (panel B), and Ca2+ (panel
C) were determined as described under "Materials and Methods."
WT, wild type.
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|
The cellular levels of these three compounds were significantly
different in strains carrying the vps33
mutation. We
found that the total amount of cellular Mg2+ was 3-fold
lower in both the vps33
and
vps33
/pmc1
/vcx1
strains. Similarly, the total
inorganic phosphate level was reduced more than 4-fold in the
vps33
strain and 6-fold in the
vps33
/pmc1
/vcx1
/ strain. Thus, strains carrying the
vps33
mutation exhibited a severe reduction in the
cellular content of Mg2+ and inorganic phosphate,
consistent with a reduced capacity to store these ions within the
vacuole of strains carrying the vps33
mutation.
Because >90% of total cellular Ca2+ is normally stored
within the vacuole (5, 6), we expected the vps33
strain
also to contain a much lower level of total Ca2+. However,
we found that both the vps33
and
vps33
/pmc1
/vcx1
/ strains contained 15-20% more
cellular Ca2+ than the wild type strain. Thus, the vacuolar
biogenesis defect associated with the vps33
mutation
resulted in a net increase in total cellular Ca2+, and this
phenotype was epistatic to the decrease in total cellular Ca2+ observed in the pmc1
/vcx1
mutant.
When taken in conjunction with the observation that the vacuolar
storage of Mg2+ and inorganic phosphate is compromised in
strains carrying the vps33
mutation, these results
suggest that another intracellular compartment is capable of
compensating for the defects in Ca2+ storage and
homeostasis in strains carrying the vps33
mutation.
Membrane Permeability of the Vacuolar Mutants--
The results
described above indicate that the vps33
mutation has
effects on Ca2+ homeostasis which differ significantly from
the combined loss of the Pmc1p and Vcx1p vacuolar Ca2+
transporters. One possible explanation for the higher level of Ca2+ observed is that the rate of Ca2+ uptake
in the vps33
strain is increased. To test this
possibility, we measured the rate of 45Ca2+
uptake in each strain (Fig. 4). A
CaCl2 solution containing the radionuclide was added to
cells at a final concentration of 1 mM. Aliquots were then
collected at intervals over a period of 90 s to determine the rate
of Ca2+ uptake. All four strains (wild type,
pmc1
/vcx1
, vps33
, and vps33
/pmc1
/vcx1
) showed a similar rate of
Ca2+ uptake, indicating that the vps33
mutation does not significantly alter the rate of Ca2+
uptake under the conditions examined (1 mM extracellular
Ca2+). If the vps33
mutation altered the
plasma membrane permeability in a more general, nonspecific way, it is
likely that the concentration of other intracellular ions would also be
altered. To examine this possibility, we measured the steady-state
concentrations of the monovalent K+ and Na+
ions in each strain when grown in YPD medium (Fig.
5). We found that none of the strains had
any significant differences in the total cellular concentrations of
either cation. Taken together, these results indicate that the membrane
permeabilities of Ca2+, K+, and Na+
are not altered significantly in strains carrying the
vps33
mutation under the conditions examined.

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Fig. 4.
Measurement of the rate of
45Ca2+ uptake. The uptake of
45Ca2+ was measured in the indicated strains
over a time interval of 90 s in cells growing in SMD medium.
Squares, wild type; diamonds,
pmc1 /vcx1 ; circles, vps33 ;
triangles, vps33 /pmc1 /vcx1 .
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Fig. 5.
Measurement of total cellular Na+
and K+ levels. Cultures of the indicated strains were
grown in standard YPD medium, and the relative amounts of
K+(panel A) and Na+ (panel
B) were determined as described under "Materials and Methods."
WT, wild type.
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45Ca2+ Exchange in the Vacuolar
Mutants--
The total cellular Ca2+ found in yeast cells
exists in two distinct forms, termed the exchangeable and
nonexchangeable pools (6, 8). The exchangeable pool represents
Ca2+ that can readily leave the cell, whereas the
nonexchangeable pool is thought to represent a more stable pool of
Ca2+ located primarily within the vacuole in a complex with
polyphosphate. To determine the partitioning of cellular
Ca2+ between the exchangeable and nonexchangeable pools in
the vps33
strains, we measured
45Ca2+ efflux. Strains were grown in YPD medium
containing 45Ca2+ for four generations. After
washing and resuspending the cells in fresh YPD medium containing 50 mM CaCl2, the amount of
45Ca2+ that remained associated with cells from
each strain was determined at various times (Fig.
6A). We found that the wild
type and pmc1
/vcx1
strains quickly exchanged a small
portion of the total cellular Ca2+ during the first 15 min
and subsequently exchanged Ca2+ at a much slower rate. In
contrast, both strains carrying the vps33
mutation
exhibited a much longer period of Ca2+ exchange which
extended for 90 min for the vps33
/pmc1
/vcx1
strain
and 210 min for the vps33
strain. This indicates that most of the Ca2+ within strains carrying the
vps33
mutation does not reside within a nonexchangeable
pool. Although the larger size of the exchangeable Ca2+
pool may partially account for the increased period of time required to
release the exchangeable Ca2+ pool in these strains, other
factors may also be involved.

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Fig. 6.
Measurement of 45Ca2+
efflux. The indicated strains were grown for four generations in
YPD medium supplemented with 45Ca2+. To
initiate Ca2+ release, the strains were harvested, washed,
and resuspended in YPD supplemented with 50 mM
CaCl2. The amount of 45Ca2+ that
remained cell-associated was determined at the indicated times and
converted to total cellular Ca2+ as described under
"Materials and Methods." Panel A, absolute amounts of
cell-associated Ca2+. Squares, wild type;
diamonds, pmc1 /vcx1 ; circles,
vps33 ; and triangles,
vps33 /pmc1 /vcx1 . Panel B,
nonexchangeable Ca2+ pools. Panel C,
exchangeable Ca2+ pools. WT, wild type.
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Because the strains carrying the vps33
mutation exhibited
a prolonged time of release of their exchangeable pools, we compared the nonexchangeable and exchangeable Ca2+ pools in each
strain after Ca2+ efflux was allowed to proceed for 210 min
(Fig. 6, B and C). Under these conditions, we
found that the wild type strain contained 4.9 mmol of
Ca2+/kg dry mass in its nonexchangeable pool. In contrast,
the pmc1
/vcx1
strain retained only 0.5 mmol of
Ca2+/kg of dry mass after 210 min of efflux. Similarly, the
vps33
strain held 1.0 mmol of Ca2+/kg dry
mass, and the vps33
/pmc1
/vcx1
strain held 0.6 mmol
of Ca2+/kg dry mass in their nonexchangeable pools. Thus,
the nonexchangeable pool in each of these mutant strains is 5-10-fold
smaller than in the wild type strain, indicating that all three mutant
strains are severely compromised in their ability to store
Ca2+ within the vacuolar nonexchangeable pool.
When we calculated the amount of Ca2+ that was readily
mobilized during 210 min of efflux, we found that the exchangeable pool in the wild type strain contained 1.2 mmol of Ca2+/kg dry
mass. This pool held 6.9 mmol of Ca2+/kg dry mass in the
vps33
strain and 8.1 mmol of Ca2+/kg dry mass
in the vps33
/pmc1
/vcx1
strain. Thus, the absolute amount of Ca2+ in the exchangeable pool in these strains
was 6-7-fold larger than in the wild type strain. In contrast, the
amount of Ca2+ in the exchangeable pool in the
pmc1
/vcx1
strain was 1.8 mmol of Ca2+/kg
dry mass, which was only 1.5-fold higher than the wild type strain.
These results indicate that although the nonexchangeable Ca2+ pools within the three vacuolar mutant strains are
similar, the exchangeable pools found in the vps33
and
vps33
/pmc1
/vcx1
strains are roughly
4-fold larger than those found in the pmc1
/vcx1
strain.
Sensitivity of Vacuolar Mutants to Cyclosporin A--
Several
studies have found that the loss of calcineurin function leads to a
significant increase in the steady-state level of cellular
Ca2+ (7, 14, 37-39). To determine how the
vps33
strain responds to such an increase in
intracellular Ca2+, we compared the growth of these strains
on YPD plates (pH 5.5) with and without 20 µg/ml CsA (Fig.
7). In the absence of CsA, the
vps33
and the vps33
/pmc1
/vcx1
strains
again had a slightly slower growth rate than the other strains.
However, when CsA was added to the plates the growth of the
vps33
/pmc1
/vcx1
strain was completely blocked,
whereas the vps33
strain showed a severe growth defect
compared with plates lacking CsA. In contrast, growth of the wild type
and pmc1
/vcx1
strains was unaffected by the presence
of CsA. These results are consistent with the possibility that the
vps33
mutation reduces the ability of these strains to
sequester adequately the increased intracellular Ca2+ that
accumulates upon the inhibition of calcineurin function.

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Fig. 7.
Sensitivity of vacuolar mutants to CsA.
The indicated strains were streaked on YPD (panel A) or YPD
supplemented with 20 µg/ml CsA (panel B). WT,
wild type.
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The Golgi Ca2+ ATPase Pmr1p Participates in the
Maintenance of Cellular Ca2+ Homeostasis during
Ca2+ Stress--
The results presented above clearly
demonstrate that the vps33
mutation severely disrupts
intracellular Ca2+ homeostasis. Despite the severe defects
in vacuolar structure and Ca2+ sequestration which result
from this mutation, they remain viable and accumulate a normal amount
of total cellular Ca2+. This raised the possibility that
other intracellular organelles may compensate for the loss of vacuolar
Ca2+ storage in these strains. Besides the vacuole, two
compartments within the secretory pathway have also been implicated in
Ca2+ storage in yeast. The PMR1 gene encodes a
Ca2+ ATPase that has been localized to the Golgi apparatus
(19, 21, 22) and was also recently reported to influence the rate of
degradation of proteins within the endoplasmic reticulum (40). Given
this well defined role of Pmr1p as a Ca2+ ATPase within a
non-vacuolar compartment, we next tested whether Pmr1p may be involved
in maintaining Ca2+ homeostasis in strains defective in
vacuolar biogenesis.
First, we examined PMR1 mRNA levels to determine whether
its expression changes in response to either the concentration of environmental Ca2+ or mutations that effect
Ca2+ homeostasis. To provide the broadest range of
environmental Ca2+ concentrations during this experiment,
strains were grown in YPD containing 1 mM EGTA (calculated
to reduce the free Ca2+ concentration to approximately 0.01 mM) or in YPD supplemented with 50 mM
CaCl2. RNA was extracted from each strain, and the level of
PMR1 mRNA was determined (relative to an ACT1
control). In the wild type strain, we found that the relative level of
PMR1 mRNA increased 1.4-fold as extracellular
Ca2+ increased (Fig. 8). In
the pmr1
/vcx1
strain, we found that the
PMR1 mRNA level was slightly elevated in the low
Ca2+ medium and was increased to 1.6-fold above the wild
type control when the environmental Ca2+ was increased.
Finally, the PMR1 mRNA level in the vps33
strain was 1.6-fold higher than the wild type strain when grown in the presence of low Ca2+ and was increased to 2.2-fold higher
than the wild type control when grown in the presence of 50 mM Ca2+. These results indicate that
PMR1 gene expression increases moderately as a function of
the Ca2+ stress on a wild type strain or as a consequence
of mutations that effect the maintenance of intracellular
Ca2+ homeostasis.

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Fig. 8.
Northern analysis of PMR1
mRNA isolated from strains grown in YPD containing 1 mM EGTA (calculated to yield 0.01 mM free
Ca2+) or YPD containing 50 mM
CaCl2. The abundance of PMR1 mRNA was
corrected for the recovery of ACT1 mRNA in each strain
and normalized to the PMR1 mRNA level measured in the
wild type (WT) strain grown under low Ca2+
conditions.
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To address further the role of Pmr1p in maintaining Ca2+
homeostasis in strains carrying the vps33
mutation, we
examined the progeny of a cross between a pmr1
strain and
a vps33
/pmc1
/vcx1
strain. A total of 36 tetrads was
dissected, and the genotype of the 107 viable spores was determined. We
found that all but three possible combinations of mutations were
obtained. The nonviable combinations, which all contained the
pmr1
mutation, were: pmr1
/pmc1
/vcx1
, pmr1
/vps33
/pmc1
, and
pmr1
/vps33
/pmc1
/vcx1
. This indicates that the
loss of both Ca2+ transporters located in the vacuole
(pmc1
and vcx1
) in conjunction with the
Golgi apparatus Ca2+ transporter (pmr1
) is
lethal. Although strains lacking both the vacuolar Ca2+
ATPase (Pmc1p) and Golgi apparatus Ca2+ ATPase (Pmr1p) were
viable, the introduction of mutations that further compromised
Ca2+ homeostasis (either the vps33
or the
vcx1
mutation) apparently resulted in a lethal imbalance
in Ca2+ homeostasis. These results indicate that specific
combinations of both vacuolar and Golgi mutations lead to
insurmountable defects in Ca2+ homeostasis.
Previous studies reported that disruption of the PMR1 gene
does not confer sensitivity to elevated levels of environmental Ca2+ (19-21). This led to the conclusion that the Golgi
apparatus does not play a significant role in maintaining cellular
Ca2+ homeostasis under conditions of Ca2+
stress. To determine whether the Golgi apparatus plays a more significant role in this process when vacuolar Ca2+ storage
is compromised, we next examined the ability of the
pmr1
/vps33
strain to grow in the presence of elevated
environmental Ca2+ (Fig. 9).
This strain grew somewhat slower than the vps33
strain on
standard YPD medium. Although the vps33
and
vps33
/pmc1
/vcx1
strains were capable of growth on
YPD plates containing 50 mM CaCl2, growth of
the pmr1
/vps33
strain was completely inhibited. These
results indicate that the Golgi apparatus acts to compensate for the
defective Ca2+ homeostasis associated with the
vps33
strain.

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Fig. 9.
The pmr1 mutation
increases Ca2+ sensitivity in conjunction with the
vps33 mutation. WT, wild
type.
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Finally, we examined whether strains carrying the pmr1
mutation alone also exhibited a growth defect in the presence of high environmental Ca2+ (Fig.
10). We found that each of the four
strains examined (wild type, pmr1
, pmc1
,
and vcx1
) grew with similar rates on plates containing
100 mM CaCl2. However, we found that the
pmr1
and pmc1
strains were unable to grow
on plates containing 500 mM CaCl2, whereas the
wild type and vcx1
strains did grow under these
conditions. These results indicate that in cells with intact vacuolar
function, Pmr1p plays a more important role in maintaining Ca2+ homeostasis upon exposure to extreme Ca2+
stress than the vacuolar Vcx1p transporter.

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Fig. 10.
Pmr1p plays a role in Ca2+
homeostasis in strains with intact vacuolar function under conditions
of extreme Ca2+ stress. WT, wild type.
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DISCUSSION |
Wild type strains of S. cerevisiae are capable of
maintaining intracellular Ca2+ levels within a narrow range
when faced with extracellular Ca2+ concentrations ranging
from <1 µM to >100 mM. Consistent with the
fact that the yeast vacuole normally contains >90% of the total
cellular Ca2+, mutations in Ca2+ transporters
which limit vacuolar Ca2+ uptake have been shown to cause a
2-3-fold reduction in the total cellular Ca2+ levels (7,
14, 15). Similarly, we observed a 2-fold decrease in total cellular
Ca2+ in the pmc1
/vcx1
strain. In contrast,
we found that strains carrying the vps33
vacuolar
biogenesis mutation have total cellular Ca2+ levels that
are slightly higher than the wild type strain. This result was
surprising based upon the severe defects in vacuolar biogenesis caused
by mutations in this gene (24-26) in conjunction with our finding that
the steady-state levels of two other substances normally stored
primarily within the vacuole (Mg2+ and polyphosphate) were
greatly reduced. Because the vps33
/pmc1
/vcx1
strain
(which lacks both known vacuolar Ca2+ transporters) also
had this high level of total cellular Ca2+, the increased
accumulation of Ca2+ cannot be attributed to the residual
function of these transporters in a prevacuolar compartment. Instead,
our results suggest that the loss of most (or all) vacuolar
Ca2+ storage in strains carrying the vps33
mutation leads to the redistribution of a significant portion of
intracellular Ca2+ into the Golgi apparatus and possibly
other intracellular compartments as well.
The Golgi apparatus contains the only non-vacuolar Ca2+
ATPase (Pmr1p) that has been characterized in yeast (19-22). Because the pmr1
strain was not previously found to be sensitive
to elevated extracellular Ca2+, it was not thought to play
a significant role in maintaining cellular Ca2+
homeostasis. However, we found that a pmr1
/vps33
strain is more sensitive to elevated extracellular Ca2+
than the vps33
strain alone, and PMR1 gene
expression is elevated in the vps33
strain. In addition,
we found that certain combinations of mutations affecting both vacuolar
and Golgi Ca2+ transport
(pmr1
/pmc1
/vcx1
,
pmr1
/vps33
/pmc1
, and
pmr1
/vps33
/pmc1
/vcx1
) resulted in synthetic
lethality. Taken together, these results indicate that the Golgi
apparatus of yeast plays a significant role in cellular
Ca2+ homeostasis through a Pmr1p-dependent
mechanism when vacuolar Ca2+ storage is compromised. We
also found that a pmr1
strain with normal vacuolar
function is sensitive to high levels of Ca2+ in the growth
medium. Given the fact that the Golgi has not previously been observed
to play a role in Ca2+ homeostasis under other growth
conditions, Golgi Ca2+ sequestration may only play a
significant role in cellular Ca2+ homeostasis when the
cytosolic Ca2+ load exceeds the capacity of the vacuolar
Ca2+ storage system.
Although our study clearly implicates Pmr1p in the maintenance of
Ca2+ homeostasis in vps33
strains, we
observed only a 2-fold increase in PMR1 transcription.
Although a larger increase might have been expected, it is possible
that PMR1 expression is regulated primarily at a
post-transcriptional level. In this way a significant increase in Pmr1p
activity could occur without a concomitant increase in mRNA
abundance (or protein abundance if the regulation is exerted at a
post-translational level). Alternatively, Pmr1p may be present and
active under all conditions, but the vacuolar Ca2+ uptake
system may sequester cytosolic Ca2+ more efficiently than
the Golgi apparatus under all but the most severe conditions. This
could occur, for example, if the vacuolar transporters were activated
at a lower cytosolic Ca2+ concentration than Pmr1p. By
either mechanism, a high level of Golgi Ca2+ storage would
not be observed under most growth conditions that did not subject the
cells to high Ca2+ stress. Such an overlapping hierarchy of
transporter activation to control Ca2+ homeostasis (either
at the level of synthesis or function) would be consistent with the
observations obtained in the current study. Such a mechanism would also
explain why Pmr1p was not attributed a role in the maintenance of
cellular Ca2+ homeostasis in previous studies.
Other results obtained in this study are also consistent with a
hierarchical control of Ca2+ homeostasis. First, we found
that strains carrying the vps33
mutation exhibit a
2-3-fold higher basal level of cytosolic Ca2+ when
incubated in a medium containing only 10 µM
Ca2+ (Fig. 2). This finding provides evidence that Pmr1p
function may be activated at a higher cytosolic Ca2+
concentration than the vacuolar Ca2+ transporters. Because
our results suggest that the secondary system utilizing Pmr1p plays a
larger role in Ca2+ homeostasis in strains carrying the
vps33
mutation, it would be expected that the basal
cytosolic Ca2+ would be maintained near the concentration
that activates this transporter. We also found that strains carrying
the vps33
mutation exhibited a severe defect in the
maintenance of cytosolic Ca2+ homeostasis when exposed to
50 mM extracellular CaCl2. Under these
conditions, we found that the cytosolic Ca2+ concentration
of the vps33
strain quickly rose to 1.75 µM, a level that was 6-fold higher than the wild type
strain. Furthermore, the rate of recovery was slower, and the new
steady-state level that was reached was also much higher than the
control strain. Again, these results suggest that this secondary system
of Ca2+ sequestration cannot remove excess Ca2+
from the cytosol as quickly as the vacuolar system. Despite these limitations, this system remains capable of maintaining intracellular Ca2+ homeostasis (at least to the extent required to
maintain growth) in strains carrying the vps33
mutation
when challenged by environmental concentrations as high as 50 mM Ca2+ (see Fig. 9).
Previous studies have shown that strains carrying vps33
mutations mislocalize the vacuolar membrane protein alkaline
phosphatase to the cell surface (24-26). Unfortunately, neither the
extent of the mislocalization of other vacuolar membrane proteins nor the composition of vesicles that accumulate in the vps33
strain has been characterized further. Nevertheless, it is possible
that vacuolar Ca2+ transporters may also be mislocalized to
the cell surface and thus could potentially contribute to the increased
cytosolic Ca2+ levels observed upon exposure to high
extracellular Ca2+. However, we found that the peak
cytosolic Ca2+ level was still 5-fold higher than the wild
type strain in the vps33
/pmc1
/vcx1
strain. This
indicates that the mislocalization of the Pmc1p and Vcx1p transporters
to the plasma membrane is not responsible for most of the elevated
cytosolic Ca2+ observed in strains carrying the
vps33
mutation. Two additional lines of evidence suggest
that the vps33
mutation does not significantly alter the
general permeability of the plasma membrane. First, the steady-state
cellular concentrations of two other cations, K+ and
Na+, were unaffected by the vps33
mutation.
In addition, the rate of 45Ca2+ uptake measured
in strains carrying the vps33
mutation was identical to
that of the wild type strain. Taken together, these results suggest
that the vps33
mutation does not significantly alter the
permeability of the plasma membrane in the vps33
strain. As discussed above, it is more likely that the higher peak in cytosolic
Ca2+ is caused by a reduced capacity to sequester the
Ca2+ into other cellular compartments rapidly.
Several studies have reported that the loss of calcineurin function
increases the total cellular Ca2+ level (7, 14, 37-39).
Our finding that the vps33
strain shows an increased
sensitivity to CsA on standard YPD medium is also consistent with the
model that this secondary system of Ca2+ sequestration is
not capable of transporting Ca2+ from the cytosol into
intracellular compartments as efficiently as the wild type strain. In
addition, it has been shown that the induction of PMR1
expression is prevented by the immunosuppressive drug FK506 (which
functions to inhibit calcineurin activation in a manner analogous to
CsA) (14). Thus, the combined effects of increased cellular
Ca2+ uptake and lack of PMR1 induction could
account for the increased CsA sensitivity that was observed in the
vps33
strains.
The results of this study provide evidence that the Golgi apparatus
plays a significant role in the maintenance of cellular Ca2+ homeostasis under conditions where the accumulation of
cytosolic Ca2+ exceeds the capacity of the vacuole. This
suggests that the vacuolar storage system that normally mediates the
bulk of Ca2+ homeostasis in yeast may have been
superimposed upon another system that is functionally related to the
Ca2+ storage and signaling system found within the
secretory pathway of mammalian cells. Further studies are required to
determine whether other intracellular organelles of yeast (such as
mitochondria) also participate in the maintenance of cellular
Ca2+ homeostasis under conditions of extreme
Ca2+ stress.