1 Department of Microbiology, University of Alabama at Birmingham, Birmingham,
AL 35294, USA
2 Department of Clinical Chemistry, University Medical School, 7624 Peçs,
Hungary
* Author for correspondence (e-mail: dbedwell{at}uab.edu)
Accepted 16 January 2003
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Summary |
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Key words: Yeast, Pmr1p, Ca2+ homeostasis, Vacuole
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Introduction |
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A relatively small number of Ca2+ transporters appear to
maintain cellular Ca2+ homeostasis in yeast. Among these are the
vacuolar Ca2+-ATPase Pmc1p
(Cunningham and Fink, 1994b);
the vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p
(Cunningham and Fink, 1996
;
Pozos et al., 1996
); the
endoplasmic reticulum (ER) Ca2+-ATPase Cod1p/Spf1p
(Bonilla et al., 2002
;
Cronin et al., 2000
;
Cronin et al., 2002
;
Suzuki and Shimma, 1999
); and
the Golgi Ca2+-ATPase Pmr1p
(Antebi and Fink, 1992
;
Rudolph et al., 1989
;
Sorin et al., 1997
).
Remarkably, the action of this small group of Ca2+ transporters
maintains the resting cytosolic Ca2+ level between 50 and 200 nM
when environmental Ca2+ concentrations range from <1 µM to
>100 mM (Batiza et al.,
1996
; Iida et al.,
1990b
; Miseta et al.,
1999b
). When the level of environmental Ca2+ is
elevated, the calmodulin/calcineurin signaling pathway activates the
expression of many genes (including those encoding Pmr1p and Pmc1p) by the
transcription factor Tcn1p/Crz1p. By contrast, the level of Vcx1p is slightly
reduced by high environmental Ca2+, and its activity is further
repressed by calcineurin activation through a post-translational mechanism
(Cunningham and Fink, 1996
;
Matheos et al., 1997
;
Stathopoulos-Gerontides et al.,
1999
; Yoshimoto et al.,
2002
).
The vacuole is the principle Ca2+ storage organelle in yeast,
and normally contains >95% of the total cellular Ca2+
(Dunn et al., 1994;
Eilam et al., 1985
). However,
it has been shown that the loss of the Golgi-localized Ca2+
transporter Pmr1p causes an increased sensitivity to high environmental
Ca2+ when vacuolar Ca2+ transport is compromised,
indicating that the Golgi apparatus also plays an important role in
Ca2+ sequestration (Miseta et
al., 1999b
; Tanida et al.,
1995
). Furthermore, Pmr1p has also been reported to be involved in
maintaining the resting Ca2+ concentration within the ER
(Strayle et al., 1999
),
whereas both Pmr1p and Pmc1p influence Ca2+-dependent functions
within the secretory pathway such as protein degradation in the ER and protein
sorting in the Golgi apparatus (Durr et
al., 1998
). The ability of these transporters to influence
Ca2+-dependent processes in multiple organelles might be due to
their movement through these compartments during the transit to their final
cellular destinations. However, it has been reported that the distribution of
Pmc1p in Golgi fractions increases in a pmr1
strain,
suggesting that its abundance in compartments of the secretory pathway might
be influenced by the lumenal Ca2+ concentration
(Marchi et al., 1999
). These
findings illustrate both the complexity and the sophisticated regulatory
mechanisms that control cellular Ca2+ homeostasis in yeast.
In mammalian cells, free Ca2+ located in the ER serves as a
mobilizable pool that can be released into the cytosol in response to an
appropriate stimulus. The resulting increase in cytosolic Ca2+ can
then activate signaling pathways that alter the expression of many genes in a
coordinated manner (Putney,
1992). In many non-excitable cells, the release of ER
Ca2+ can also induce a store depletion signal that results in the
influx of Ca2+ ions across the plasma membrane in a process termed
capacitative Ca2+ entry (CCE). Recent studies have provided
evidence that yeast cells might also utilize a mechanism that couples
intracellular store depletion to Ca2+ uptake across the plasma
membrane. A pmr1
mutant has been shown to exhibit a higher
rate of Ca2+ uptake than the WT strain
(Antebi and Fink, 1992
;
Halachmi and Eilam, 1996
;
Rudolph et al., 1989
;
Sorin et al., 1997
). This led
to the model that the depletion of Golgi Ca2+ stores can stimulate
Ca2+ uptake into yeast cells in a manner analogous to CCE in
mammalian cells (Csutora et al.,
1999
; Durr et al.,
1998
; Locke et al.,
2000
). In contrast to the pmr1
mutant, a
pmc1
strain exhibits a reduced level of total cellular
Ca2+ (Cunningham and Fink,
1994b
). Since Pmc1p is the only known vacuolar
Ca2+-ATPase in yeast and the vacuole normally contains the bulk of
total cellular Ca2+, this suggests that Ca2+ uptake
across the plasma membrane is coupled to the ability of the cell to remove it
efficiently from the cytosol.
In the current study, we examined how a reduced level of divalent cations
in the growth medium influences cellular Ca2+ homeostasis in the
pmr1 strain. We found a large increase in Ca2+
uptake and accumulation under these conditions, which led to activation of the
calcineurin signaling pathway. Consistent with this high level of
Ca2+ uptake, we found that the PMC1 gene was required for
growth of the pmr1
mutant under these growth conditions. Our
observation that cellular Ca2+ uptake increases as the
concentration of environmental Ca2+ decreases suggests that an
extracellular Ca2+ sensor is capable of coupling Ca2+
uptake to extracellular Ca2+ levels. Finally, we found that
conditions of cellular Ca2+ stress result in a vacuolar
fragmentation phenotype in both WT and mutant yeast strains. This might serve
as an adaptive mechanism to maintain cellular Ca2+ homeostasis
under these stress conditions.
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Materials and Methods |
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Culture media
Bacterial strains were grown on standard media
(Miller, 1992). 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
(Burke et al., 2000
). Culture
media were routinely buffered with 40 mM Mes-Tris, pH 5.5.
Total cellular Ca2+ measurements
Total cellular Ca2+ was measured using flame photometry as
described previously (Miseta et al.,
1999a). Cells were grown in 30-40 ml of YPD or the same medium
supplemented with either 1 mM EGTA or 50 mM CaCl2. Cultures were
harvested at a cell density of
1 OD600/ml, harvested by
centrifugation and washed with 20 ml of YPD. Cells were then transferred to
previously weighed microfuge tubes and harvested by centrifugation in a
micro-centrifuge (16,000 g) for 5 minutes. After aspirating
the supernatant, a second spin was conducted for 3 minutes. Samples were
weighed, then dried in a Speedvac. The dried samples were weighed again and
resuspended in HCl for flame photometric measurements.
Ca2+ uptake
Ca2+ uptake measurements were similar to a method described
previously (Halachmi and Eilam,
1996). Cells were grown in YPD to 0.7-1.0 OD600/ml,
harvested, washed twice with ddH2O, then re-suspended in buffer
containing 25 mM Mes-Tris, pH 6.0 supplemented with 20 mM glucose to 1
OD600/ml and incubated at 30°C for 10 minutes. Uptake was
initiated by the addition of 1 µCi/ml 45Ca2+. At the
indicated time-points, 1 ml aliquots were collected by filtration through 0.45
µm membrane filters (Gelman Sciences) using a vacuum manifold. Membranes
were immediately washed with two 5 ml aliquots of ice-cold wash buffer (20 mM
MgCl2, 0.2 mM LaCl3), dried, and the cell-associated
radioactivity was measured by liquid scintillation counting.
Northern analysis
RNA extraction and northern blot analysis were carried out as described
previously (Bonetti et al.,
1995). Strains were grown overnight in YPD medium or YPD
supplemented with either 1 mM EGTA or 50 mM CaCl2 to
1
OD600/ml. A 0.44 kb probe for the PMC1 mRNA was generated
by PCR using primers DB490 (5'-ATGTCTAGACAAGACGAAAA-3') and DB491
(5'-ATACTGTGGAGGTTGCATCC-3'). As control, an ACT1 probe
was generated using the primers DB154
(5'-GCGCGGAATTCAACGTTCCAGCCTTCTACG-3') and DB155
(5'-GGATGGAACAAAGCTTCTGG-3'). Probes were labeled with
[
-32P]dATP using the random hexamer method
(Sambrook et al., 1989
). The
specific band representing the PMC1 mRNA was confirmed by its absence
in RNA extracted from the pmc1
strain YDB0224. Gels were
quantitated by PhosphorImager analysis (Molecular Dynamics). The relative mRNA
levels were normalized using the ACT1 mRNA as internal control after
background correction.
Aequorin assay
Aequorin assays were carried out as described earlier
(Miseta et al., 1999a). The
two-micron-based plasmid pDB617 expressing a functional apoaequorin gene
(pAEQ) was transformed into yeast. Cells containing pAEQ were grown
in SMD medium and harvested in the early logarithmic growth phase (0.5-1.0
A600 units/ml). 10 A600 units of cells were harvested
and re-suspended in 0.2 ml of aequorin test medium, which consists of SMD
medium (which normally contains 1 mM Ca2+) supplemented with 2 mM
EGTA and 40 mM MES-Tris, pH 6.5. To convert the apoaequorin to aequorin, 20
µl of 590 µM coelenterazine (dissolved in methanol) was added, and the
cells were incubated for 20 minutes at room temperature. The cells were then
briefly centrifuged, and the supernatant containing excess coelenterazine was
removed. The cell pellets were washed again in 0.5 ml of aequorin test medium,
re-suspended to a cell density of 1 OD600/0.1 ml and incubated at
room temperature for 20 minutes before initiating the experiment. Bafilomycin
A1 (5 µM) was added from a 100 µM stock solution (dissolved
in DMSO) 10 minutes prior to the measurements
(Abe and Horikoshi, 1995
).
After detecting the baseline light emission, 100 mM Ca2+ was
administered into the chamber to generate a Ca2+ shock. A Berthold
Lumat 9050 luminometer was used to collect aequorin light emission (L) data at
200 millisecond intervals. The data were downloaded directly to a computer and
transferred to Microsoft Excel 5.0 for analysis. After measuring maximal light
emission from crude cell extracts upon Ca2+ addition
(Lmax), L/Lmax values were plotted on a standard curve
to estimate the free cytosolic Ca2+ concentrations, as previously
described (Miseta et al.,
1999a
).
Light microscopy
Cells expressing the Pmc1p-GFP fusion protein and/or stained with FM 4-64
were collected, re-suspended in fresh YPD medium, or YPD medium supplemented
with Ca2+ or EGTA, to 10 OD600 units/ml and 10 µl of
the culture was mounted on slides coated with concanavalin A, covered with a
coverslip and viewed immediately. FM 4-64 staining was carried out as
published (Vida and Emr, 1995)
with minor modifications. Briefly, yeast cells were grown in the indicated
media to mid-log phase, 2 OD600 units of cells were collected and
re-suspended in 100 µl of YPD (Ca2+ was omitted at this step
because it decreased total fluorescence). A 1 µl aliquot of an FM 4-64
stock (4 mM in DMSO) was added to the cells, and they were stained for 10-15
minutes at 30°C. Cells were collected and re-suspended in 200 µl fresh
media supplemented with Ca2+ or EGTA and incubated at 30°C for
40-50 minutes. Light microscopy was conducted using a Leitz Orthoplan
microscope with epifluorescence optics and Hoffman Modulation Contrast optics.
The images were acquired with a Photometrics CH250 liquid-cooled CCD
high-resolution monochromatic camera (Roper Scientific; Tucson, AZ) and
analyzed by IPLab Spectrum software from Scanalytics (Fairfax, VA).
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Results |
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The reduced availability of divalent cations leads to increased
PMC1 mRNA levels and increased total cellular Ca2+ in the
pmr1 strain
Previous studies have shown that PMC1 expression increases through
a calcineurin-dependent mechanism as the level of environmental
Ca2+ increases (Cunningham and
Fink, 1994b; Marchi et al.,
1999
). However, the results described above suggest that the
normal pattern of PMC1 expression is altered in the
pmr1
strain. To understand these findings better, we next
examined the level of PMC1 mRNA in the WT and pmr1
strains using northern blot analysis (Fig.
2A). As expected, we found that the level of PMC1 mRNA in
the WT strain increased with increasing environmental Ca2+. The
PMC1 mRNA level in the pmr1
strain was only slightly
higher than the WT strain when grown in either YPD (which contains 0.3 mM
Ca2+) or YPD supplemented with 50 mM CaCl2. By contrast,
the steady-state level of PMC1 mRNA in the pmr1
strain was
5-fold higher than normal when these strains were grown in YPD
medium containing 1 mM EGTA. In fact, the PMC1 mRNA level in the
pmr1
strain was higher in cells grown in the presence of a
reduced level of divalent cations than under any other condition tested. These
results confirm that the pattern of PMC1 expression is significantly
altered in the pmr1
strain.
|
Since the expression of the PMC1 gene is controlled by calcineurin
activity (Cunningham and Fink,
1994b; Marchi et al.,
1999
), the results above suggest that the pmr1
strain might experience Ca2+ stress even when grown in the presence
of low environmental Ca2+. To test this possibility, we compared
the total cellular Ca2+ levels in WT and pmr1
strains when grown under different environmental Ca2+ conditions.
Consistent with previous studies (Halachmi
and Eilam, 1996
; Sorin et al.,
1997
), we found that the level of cellular Ca2+ in the
pmr1
strain was 1.4-fold higher than the WT strain when grown
in YPD and 1.5-fold higher than WT in YPD containing 50 mM CaCl2.
By contrast, we found that the pmr1
strain contained
7.5-fold more total cell Ca2+ than the WT strain when grown in
YPD medium supplemented with 1 mM EGTA
(Fig. 2B). This level of total
cellular Ca2+ was actually higher than the level measured when this
strain was grown in YPD supplemented with 50 mM CaCl2. These
results confirm that the pmr1
strain exhibits excessive
Ca2+ accumulation when grown in media containing a reduced level of
divalent cations, which results in the transcriptional activation of genes
controlled by the calmodulin/calcineurin signaling pathway.
Ca2+ uptake and accumulation are further increased in the
pmr1/pmc1
strain
The results of previous studies have indicated that Pmc1p contributes to
the filling of ER Ca2+ stores in a pmr1 strain
(Bonilla et al., 2002
;
Durr et al., 1998
). Given our
finding that total cellular Ca2+ is significantly higher in a
pmr1
strain when the level of divalent cations in the growth
medium is reduced, we next examined Ca2+ uptake by the
pmr1
/pmc1
strain when grown in YPD medium.
Consistent with previous reports, we found that the rate of Ca2+
uptake in the pmr1
strain was 1.8-fold higher than the WT
strain (Halachmi and Eilam,
1996
; Sorin et al.,
1997
), whereas the pmc1
strain had a
Ca2+ uptake rate that was
20% lower than normal
(Fig. 3A). By contrast,
Ca2+ uptake in the pmr1
/pmc1
mutant
was 3.5-fold higher than the WT strain (and almost 2-fold higher than the
pmr1
strain).
|
We next examined total cellular Ca2+ levels following the growth
of these strains in YPD medium. We found that the total cellular
Ca2+ level in the pmc1 strain was roughly 2-fold
lower than the WT strain, as previously reported
(Cunningham and Fink, 1994b
).
By contrast, the pmr1
/pmc1
strain contained
3.8-fold more Ca2+ than the WT strain, and 2.2-fold more total
cellular Ca2+ than the pmr1
strain
(Fig. 3B). These results
demonstrate that the pmc1
mutation further exacerbates the
Ca2+ hyper-accumulation phenotype of the pmr1
strain and suggest that the growth defect observed when the
pmr1
/pmc1
strain is grown in a low
Ca2+ environment is caused by an excessive cellular Ca2+
load in combination with a reduced ability to sequester this excess
Ca2+ adequately into intracellular compartments.
The Ca2+/H+ Exchanger Vcx1p maintains
Ca2+ homeostasis in the pmr1/pmc1
mutant
It was previously shown that Vcx1p activity is downregulated upon
calcineurin activation, suggesting that this protein does not play a
significant role in Ca2+ sequestration under conditions of high
Ca2+ stress (Cunningham and
Fink, 1996; Pozos et al.,
1996
). Consistent with this conclusion, we previously demonstrated
that the presence of a vcx1
mutation did not have any effect
on the level of total cellular Ca2+ when the growth medium was
supplemented with more than 5 mM CaCl2
(Miseta et al., 1999a
).
However, some residual Vcx1p activity remains under such repressing
conditions, since a pmc1
/vcx1
strain is more
sensitive to high extracellular Ca2+ than a pmc1
mutant (Cunningham and Fink,
1994a
; Miseta et al.,
1999a
; Pozos et al.,
1996
). To gain further insight into the role of Vcx1p in the
maintenance of Ca2+ homeostasis under conditions of Ca2+
stress, we measured total cellular Ca2+ levels in the
pmc1
and pmc1
/vcx1
strains. We
found that the total cellular Ca2+ level in the
vcx1
/pmc1
strain was
20% lower than the
pmc1
mutant when these strains were grown in YPD medium. When
grown in YPD supplemented with 50 mM CaCl2, the total cellular
Ca2+ level increased in both strains, but the level measured in the
vcx1
/pmc1
strain was
36% lower than the
pmc1
strain (Fig.
4A). These results directly implicate Vcx1p in Ca2+
sequestration in the pmc1
mutant under conditions of high
Ca2+ stress, and suggest that a functionally significant level of
Vcx1p activity is maintained in this strain when grown in the presence of high
extracellular Ca2+.
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The Vcx1p Ca2+/H+ exchanger utilizes the proton
gradient across the vacuolar membrane to help maintain cytosolic
Ca2+ levels within a narrow physiological range. The vacuolar
proton gradient is maintained by the vacuolar H+-ATPase
(Forster and Kane, 2000),
which is sensitive to the inhibitor bafilomycin A1. We previously
used a cytosolic aequorin reporter system to characterize how bafilomycin
A1 influenced the regulation of cytosolic Ca2+ levels
(Miseta et al., 1999a
). We
found that Vcx1p plays a key role in rapidly restoring basal cytosolic
Ca2+ levels following a rise in the cytosolic Ca2+
concentration. Since the pmr1
/pmc1
mutant
lacks the two major Ca2+-ATPases involved in the maintenance of
cellular Ca2+ homeostasis, we next used bafilomycin A1
and the aequorin reporter system to examine the role of Vcx1p in controlling
cytosolic Ca2+ levels in the
pmr1
/pmc1
mutant. We found that bafilomycin
A1 treatment increased the cytosolic Ca2+ level in this
strain significantly (Fig. 4B).
Using a standardization procedure described previously
(Miseta et al., 1999a
), we
found that the cytosolic Ca2+ level in the
pmr1
/pmc1
mutant increased from
l60 nM to
260 nM following bafilomycin A1 treatment. By contrast, a
similar treatment did not alter the basal cytosolic Ca2+ levels in
WT, pmr1
and pmc1
strains (data not shown).
These results suggest that Vcx1p plays an important role in the maintenance of
the resting cytosolic Ca2+ level in the
pmr1
/pmc1
strain.
To examine further the role of Vcx1p in Ca2+ homeostasis of the
pmr1/pmc1
strain, we next tested the ability
of Vcx1p to regulate cytosolic Ca2+ levels following the exposure
of this strain to a 100 mM CaCl2 shock
(Fig. 4B). Immediately
following the addition of this Ca2+ bolus, the cytosolic
Ca2+ level rapidly increased to
300 nM. In the absence of
bafilomycin A1 treatment, we found that the
pmr1
/pmc1
mutant could successfully recover
from this rapid increase in cytosolic Ca2+, with the resting
cytosolic Ca2+ level returning to
180 nM within 30 seconds. By
contrast, a brief pre-treatment with bafilomycin A1 prior to the
Ca2+ shock completely eliminated the ability of this strain to
compensate for this abrupt increase in cytosolic Ca2+. These
results demonstrate that Ca2+/H+ exchange by Vcx1p plays
a key role in the maintenance of cellular Ca2+ homeostasis in the
pmr1
/pmc1
strain.
The pmr1/pmc1
strain exhibits vacuolar
fragmentation
Previous studies have suggested that newly synthesized Pmc1p contributes to
the maintenance of Ca2+ levels within the secretory pathway during
its transit to the vacuole in the pmr1 mutant
(Bonilla et al., 2002
;
Durr et al., 1998
;
Locke et al., 2000
) (this
study). Subcellular fractionation of a pmr1
strain has shown
that Pmc1p is present not only in vacuolar fractions, but also overlaps
fractions containing Golgi markers (Marchi
et al., 1999
). These results suggest that a significant amount of
Pmc1p may be retained in the Golgi apparatus in the pmr1
mutant. To determine whether the Golgi localization of Pmc1p could be
visualized directly in yeast cells, we constructed a Pmc1p-GFP fusion protein.
Following the integration of this construct into the nuclear genome of
pmc1
and pmr1
/pmc1
strains, we
found that the Pmc1p-GFP fusion restored normal Pmc1p function, as indicated
by its ability to complement both the Ca2+ sensitivity of the
pmc1
mutant and the EGTA sensitivity of the
pmr1
/pmc1
mutant (data not shown). Thus, these
complemented pmc1
, and
pmr1
/pmc1
strains were functionally equivalent
to WT and pmr1
strains, respectively.
We first asked whether the majority of the Pmc1p-GFP fusion protein
co-localized with vacuoles as indicated by FM 4-64, a vital stain that
accumulates in the yeast vacuolar membrane
(Vida and Emr, 1995). As shown
in Fig. 5, we observed complete
co-localization of these two markers in the expected vacuolar pattern in WT
cells grown in YPD medium. Whereas FM 4-64 and Pmc1p-GFP fluorescence also
co-localized completely in the pmr1
strain, we found that both
markers produced an identical pattern of highly fragmented staining. FM 4-64
staining was also used to examine the vacuolar morphology in the
pmc1
and pmr1
/pmc1
strains.
The vacuolar morphology was normal in the pmc1
strain. By
contrast, the pmr1
/pmc1
strain exhibited a
highly fragmented, frequently tubular, pattern of fluorescence. No Pmc1p-GFP
fluorescence could be detected that was distinct from the FM 4-64 staining in
either the WT or pmr1
strain, indicating that any accumulation
in the Golgi apparatus (or another subcellular location) was below the
resolution of this assay. However, these results demonstrate that significant
vacuolar fragmentation occurs in strains containing the pmr1
mutation when grown in standard YPD medium. On the basis of our finding that
the pmr1
and pmr1
/pmc1
strains
both undergo Ca2+ stress under these conditions, these results
suggest that Ca2+ stress might induce vacuolar fragmentation.
|
To test this hypothesis, we next used the Pmc1p-GFP fusion protein to
monitor vacuolar morphology in the WT and pmr1 strains grown
in media containing different Ca2+ concentrations
(Fig. 6). We found that WT
cells grown in low and intermediate environmental Ca2+
concentrations (YPD supplemented with 1 mM EGTA, YPD alone, or YPD
supplemented with 5 mM CaCl2) exhibited normal vacuolar morphology
(Fig. 6A). However, this strain
exhibited an increasing degree of vacuolar fragmentation when grown in media
containing 50 or 200 mM CaCl2. We found that 70-80% of WT cells
grown YPD supplemented with 200 mM CaCl2 contained four or more
vacuoles, while 70-80% of cells grown in standard YPD contained three or fewer
vacuoles. Furthermore, as the number of the vacuolar structures increased,
their size decreased. This vacuolar fragmentation phenotype was independent of
osmotic stress, since WT cells grown in YPD medium supplemented with 300 mM
NaCl2 did not exhibit any significant change in vacuolar morphology
(data not shown).
|
As shown above, we found that the pmr1 strain contained
highly fragmented vacuoles when grown on YPD medium
(Fig. 6B). This fragmented
vacuole phenotype was even more severe when grown in the presence of 1 mM
EGTA, consistent with our finding that this strain undergoes excessive
Ca2+ stress under these conditions. Growth of the
pmr1
strain in YPD supplemented with 5-50 mM CaCl2
resulted in a much less severe alteration in vacuolar morphology. Increasing
the extracellular CaCl2 concentration to 200 mM CaCl2
led to a fragmented vacuolar morphology similar to that observed in WT cells
under these conditions. Overall, these results indicate that vacuolar
fragmentation is observed in both WT and pmr1
strains in
conjunction with an elevated level of cellular Ca2+ stress.
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Discussion |
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A previous study found that PMC1 expression from a multicopy
plasmid was capable of suppressing phenotypes associated with the
pmr1 mutation (Durr et
al., 1998
). This suggested that Pmc1p contributes to the filling
of Ca2+ stores within the ER or Golgi apparatus during its transit
through the secretory pathway. In support of this hypothesis, a
pmr1
/pmc1
strain was also shown to exhibit a
more severe unfolded protein response (UPR) than a pmr1
strain, suggesting that the loss of Pmc1p function causes a further depletion
of ER Ca2+ that inhibits protein folding
(Bonilla et al., 2002
). It was
also shown that the depletion of Golgi Ca2+ stores induces cellular
Ca2+ uptake by a high-affinity Ca2+ uptake transporter
encoded by the CCH1 and MID1 genes. These results led to the
hypothesis that yeast cells possess a mechanism that couples the level of
Ca2+ in the Golgi apparatus to Ca2+ uptake that is
analogous to the CCE response in mammals
(Locke et al., 2000
). This
model predicts that a pmr1
strain grows poorly in an
environment containing a reduced level of divalent cations because a limiting
level of extracellular Ca2+ (and/or Mn2+) further
reduces its ability to transport these cations efficiently into the cell so
that it can replenish the depleted pool of these ions in the Golgi
apparatus.
Our finding that Ca2+ accumulation is significantly increased in
the pmr1 strain when the availability of divalent cations is
reduced cannot be explained solely by a CCE-like mechanism
(Csutora et al., 1999
;
Locke et al., 2000
), since it
is highly unlikely that the Ca2+ level in the Golgi apparatus will
be further depleted under conditions where the level of total cellular
Ca2+ is threefold higher. We also found that the
pmr1
strain exhibited less vacuolar fragmentation in the
presence of 5 mM or 50 mM CaCl2 than either higher or lower
concentrations (see Fig. 6),
suggesting that a moderate increase in extracellular Ca2+ can
reduce the rate of cellular uptake (and the resulting level of Ca2+
stress). When taken together, these results suggest that a mechanism exists
that can couple the rate of cellular Ca2+ uptake to the
extracellular Ca2+ concentration. The results of a previous study
also support the existence of an extracellular Ca2+-sensing
mechanism. We have shown that the loss of the major isoform of
phosphoglucomutase (encoded by the PGM2 gene) causes a large increase
in Ca2+ uptake and accumulation when a pgm2
strain
is grown in YP medium containing galactose as carbon source
(Fu et al., 2000
).
Furthermore, we found that a pgm2
/pmr1
strain
is unable to grow in YP galactose medium, presumably because the combination
of these mutations leads to excessive Ca2+ uptake and accumulation
that results in an inhibition of cell growth. However, we found that growth of
the pgm2
/pmr1
strain could be restored on YP
galactose medium when 100 mM CaCl2 was added to the growth medium
(Fu et al., 2000
). In light of
our current results, we propose that this increase in the concentration of
CaCl2 in the growth medium can restore growth of the
pgm2
/pmr1
strain by reducing Ca2+
uptake and accumulation through the action of an extracellular
Ca2+-sensing mechanism.
While several mechanisms could be used to monitor the level of
extracellular Ca2+, the most straightforward method would utilize a
Ca2+ sensor on the cell surface
(Fig. 7). This cell-surface
Ca2+ sensor could be functionally equivalent to the extracellular
Ca2+-sensing receptor of mammalian cells, which can respond to
extremely small changes in the free Ca2+ concentration in the blood
(Brown et al., 1995;
Hebert et al., 1997
). The
results of the current study provide strong evidence that an extracellular
Ca2+-sensing mechanism can also play an important role in coupling
the level of environmental Ca2+ to cellular Ca2+ uptake,
homeostasis and signaling in yeast. To our knowledge, such a mechanism has not
been proposed previously for yeast cells. This might be due to the fact that
this mechanism works in conjunction with other redundant processes that
together tightly control cellular Ca2+ homeostasis. Such
overlapping mechanisms could explain why yeast mutants that maintain
abnormally high levels of Ca2+ uptake and accumulation (such as the
pmr1
and pgm2
strains) were necessary to
obtain evidence of this novel control mechanism. These mutant strains have
shed new light on the mechanisms that couple cellular Ca2+ uptake
and accumulation. For example, the inability of the pmr1
strain to grow in the presence of a reduced level of divalent cations allowed
us to show that excessive Ca2+ accumulation is responsible for this
growth defect. This problem is further exacerbated in the
pmr1
/pmc1
strain in two ways. First, the loss
of Pmc1p further aggravates the reduced ability to properly fill the ER store
depletion caused by the pmr1
mutation. Second, the
pmc1
mutation diminishes the ability of the cell to sequester
excess Ca2+ in the vacuole. As a result, the cytosolic
Ca2+ load becomes more severe in the
pmr1
/pmc1
strain, whereas its ability to
sequester excess Ca2+ adequately into the vacuole is decreased.
Together, these consequences result in an inability to grow in a
Ca2+-depleted environment.
|
In order to understand better the functional interplay between
Ca2+ transporters in yeast, we also examined the role of Vcx1p in
maintaining cytosolic Ca2+ homeostasis in the
pmr1/pmc1
strain. We found that Vcx1p plays an
important role in Ca2+ homeostasis over a much broader range of
extracellular Ca2+ concentrations than previously appreciated
(Pozos et al., 1996
). Genetic
studies have suggested that Vcx1p might play a less important role in
Ca2+ homeostasis than the Ca2+-ATPases Pmc1p and Pmr1p.
Consistent with this conclusion, we have observed that a
pmr1
/vcx1
strain is no more sensitive to
chelating agents than a pmr1
strain. This result indicated
that Pmc1p alone is sufficient to sequester the high cellular Ca2+
that accumulates under these conditions, and suggested that Vcx1p plays only a
secondary role in Ca2+ homeostasis (R. Kellermayer and D. Bedwell,
unpublished). However, in the current study, we show that a
pmr1
/pmc1
mutant can successfully cope with a
considerable level of Ca2+ stress, whereas cellular Ca2+
homeostasis is completely disrupted when its vacuolar proton gradient is
disrupted by bafilomycin A1. Since it has been shown that vacuolar
vesicles derived from a strain lacking Vcx1p do not possess any
Ca2+/H+ exchange activity
(Pozos et al., 1996
), these
findings strongly indicate that Vcx1p plays the predominant role in
maintaining Ca2+ homeostasis in the
pmr1
/pmc1
mutant. In addition, the previous
observation that the combination of the pmr1
,
pmc1
and vcx1
mutations together causes
synthetic lethality reinforces the importance of Vcx1p in Ca2+
regulation in the absence of these two Ca2+-ATPases
(Cunningham and Fink, 1996
;
Miseta et al., 1999a
). These
results suggest that the Pmr1p, Pmc1p and Vcx1p Ca2+ transporters
are all capable of independently maintaining cellular Ca2+
homeostasis in many yeast strains. Apparently, subtle differences in the
extent of Vcx1p inhibition by calcineurin are responsible for conflicting
reports regarding the viability of pmr1
/pmc1
strains in different genetic backgrounds
(Cunningham and Fink, 1996
;
Locke et al., 2000
) (this
report).
We also used a Pmc1p-GFP fusion protein to show that vacuolar fragmentation
coincides with Ca2+ stress. Our finding that vacuolar fragmentation
occurs under diverse conditions that lead to cellular Ca2+ stress
in both WT and mutant strains strongly suggests a direct relationship between
these two events. The data presented does not allow us to determine whether it
is high Ca2+ in the cytosol or vacuole that leads to this
fragmentation phenomenon. However, in other experiments we found that a
pmc1/vcx1
strain exhibits fragmented vacuoles
in media containing 50 mM CaCl2 in a manner similar to the WT
strain (R. Kellermayer and D. Bedwell, unpublished). Since this mutant
accumulates much less Ca2+ in the vacuole than the WT strain
(Cunningham and Fink, 1996
;
Miseta et al., 1999a
;
Pozos et al., 1996
), these
results strongly suggest that an elevated cytosolic Ca2+ causes
vacuolar fragmentation in S. cerevisiae.
A recent study examined a large collection of knockout strains from the
Saccharomyces Genome Deletion Project for defects in homotypic
vacuolar fusion (Seeley et al.,
2002). Surprisingly, 714 out of 4828 deletion strains examined
(
15%) exhibited alterations in vacuole morphology. After excluding a
large number of genes thought to influence vacuole morphology by indirect
means, 137 genes (
3%) were chosen as candidate VAM genes that
were thought to play a direct role in vacuolar morphology. Among these were a
variety of genes that encoded proteins previously related to homotypic
vacuolar fusion, including fusion catalysts, enzymes of lipid metabolism,
SNARES, GTPases and their effectors, protein kinases, phosphatases, and
cytoskeletal proteins. Another group of genes encoded proteins involved in
cation transport (including the PMR1 gene). Because vacuolar
fragmentation was associated with the deletion of these genes, it was reasoned
that the loss of these gene products caused defects in vacuolar fusion,
resulting in the vacuolar fragmentation phenotype. However, on the basis of
the strong correlation between cellular Ca2+ stress and vacuolar
fragmentation we observed in both WT and mutant yeast strains, we propose that
an elevation of cytosolic Ca2+ might lead to vacuolar fragmentation
as a regulatory response to aid in Ca2+ sequestration in WT yeast,
rather than simply being the result of a defect in vacuolar fusion associated
with high cytosolic Ca2+. On a per unit volume basis, multiple
small vacuoles provide a greater surface area than fewer, larger vacuoles.
This increased surface/volume ratio could aid in accommodating the function of
the increased number of Pmc1p transporters in the vacuolar membrane that are
induced by calcineurin activation, thus allowing vacuolar Ca2+
sequestration to proceed more efficiently.
In both the current study and a prior study of genes that influence
homotypic vacuolar fusion (Seeley et al.,
2002), it was shown that a pmr1
strain exhibits a
vacuolar fragmentation phenotype. By contrast, another study found that a
pmr1
mutation reversed the vacuolar fragmentation phenotype
associated with oxidative stress in a sod1
strain
(Corson et al., 1999
). These
results led to the conclusion that oxidative stress associated with the
sod1
mutation altered cellular iron homeostasis, leading to
oxidative damage that somehow led to vacuolar fragmentation. It was proposed
that the pmr1
mutation suppressed this vacuolar fragmentation
phenotype by raising the concentration of Mn2+ in the cytosol,
which acted to scavenge free radicals and reduce oxidative stress. Thus,
pmr1
mutations have been associated with both the induction
and reversal of vacuolar fragmentation. These markedly different effects
suggest the existence of a complex regulatory mechanism that allows the
cytosolic levels of different divalent cations to influence vacuolar
morphology in distinct ways. Further studies are needed to determine how this
complex physiological adaptation is carried out.
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Acknowledgments |
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References |
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