From the Departments of Nutritional Sciences and
§ Physiology, University of Missouri, Columbia, Missouri
65211
Received for publication, January 17, 2003
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ABSTRACT |
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Zinc is an essential nutrient but toxic to cells
with overaccumulation. For this reason, intracellular zinc levels are
tightly controlled. In the yeast Saccharomyces cerevisiae,
the Zrc1 and Cot1 proteins have been implicated in the storage and
detoxification of excess zinc in the vacuole. Surprisingly,
transcription of ZRC1 is induced in zinc-limited cells by
the zinc-responsive transcription factor Zap1. We show here that this
increase in ZRC1 expression is a novel mechanism of zinc
homeostasis and stress tolerance. Zinc-limited cells also express high
levels of the plasma membrane zinc uptake transporters. As a
consequence, when zinc-limited cells are resupplied with small amounts
of zinc, large quantities quickly accumulate in the cell, a condition
we refer to as "zinc shock." We show here that ZRC1 and
its induction in zinc-limited cells are required for resistance to this
zinc shock. Experiments using the zinc-responsive fluorophore FuraZin-1
as an indicator of vacuolar zinc levels indicated that Zrc1 is required
for the rapid transport of zinc into the vacuole during zinc shock. We also present evidence that cytosolic zinc rises to higher levels in
cells unable to sequester this excess zinc. Thus, the increase in ZRC1 expression occurs prior to the zinc shock stress
for which this induction is important. We propose that this
"proactive" strategy of homeostatic regulation, such as we document
here for ZRC1, may represent a common but largely
unrecognized phenomenon.
All organisms face constantly changing nutrient
availability and environmental stresses. As a consequence, organisms
have regulatory mechanisms that maintain nutrient homeostasis and deal with damage caused by stress. For example, deficiency of an essential nutrient often induces expression of the genes involved in acquiring that nutrient. Stresses such as heat shock or reactive oxygen species
increase expression of genes whose products reverse the resultant
cellular damage. As a rule, these regulatory systems are reactionary in
nature, responding to the stress to alter levels of gene expression
and/or protein activity. An alternative strategy would be to increase
expression of the required genes prior to, rather than in response to
the specific stress. We designate this latter strategy as
"proactive" regulation to distinguish it from the commonly
recognized "reactive" responses. In this report, we describe an
apparent proactive mechanism of zinc homeostasis in the yeast
Saccharomyces cerevisiae, the up-regulation of a metal
tolerance gene in zinc-deficient cells as protection against "zinc shock."
Zinc is an essential nutrient required for many processes. Its chemical
properties make this metal a useful catalytic and/or structural
cofactor in many proteins. Despite its importance however, excess zinc
is toxic. Zinc toxicity may involve competition with other metal ions
for the active sites of enzymes or intracellular transport proteins.
For this reason, organisms have evolved with mechanisms of zinc
homeostasis that tightly control the intracellular level of zinc as
extracellular concentrations change. An indication of the exquisite
precision of this control was recently obtained in studies of
Escherichia coli, where regulatory systems controlling zinc
uptake and efflux apparently strive to maintain the free intracellular
zinc concentration at less than one atom per cell (1). Several reports
suggest that eukaryotic cells also maintain very low levels of
cytoplasmic labile zinc under steady-state conditions (2-4).
Studies of yeast have revealed several components of zinc homeostasis
in this organism (5). Among these, the ZRC1 gene encodes a
potential transporter protein of the cation diffusion facilitator (CDF)
family (6). This family also includes bacterial, plant, and mammalian
proteins involved in zinc efflux and compartmentalization. ZRC1 is known to contribute to zinc tolerance (7, 8), and the Zrc1 protein was localized to the yeast vacuole membrane (9-11). Our recent in vitro studies provided evidence that Zrc1
directly mediates vacuolar zinc transport, most likely via a
zinc/H+ antiport mechanism (11). The COT1 gene
encodes a related protein that may act analogously to Zrc1 in cobalt
detoxification (8, 12). Cot1 also contributes to zinc detoxification
(13) and both Zrc1 and Cot1 appear to be required for sequestration of zinc in an intracellular storage compartment for later use under zinc-limiting conditions (14).
During the transition from zinc-replete to zinc-limiting conditions,
many genes are induced to maintain adequate supplies of zinc for cell
growth. Three such genes, ZRT1, ZRT2, and
FET4 encode plasma membrane transporters responsible for
zinc uptake under deficient conditions (15-17). All three genes are
targets of the Zap1 transcriptional activator (18), a central player in
zinc homeostasis. Zap1 is active in zinc-limited cells and is repressed
by high cytoplasmic/nuclear zinc (19). When active, Zap1p binds to an
11-bp sequence, the zinc response element
(ZRE),1 in the promoters of
its target genes (20, 21).
DNA microarray analysis has suggested that at least 46 genes in yeast
are direct targets of Zap1 regulation (21). Surprisingly, this group
and others (22) noted ZRC1 among these Zap1 target genes.
These data raise an intriguing question. Why would a transporter involved in zinc storage and detoxification be up-regulated under zinc-limiting conditions? One potential explanation was suggested by
our previous studies of zinc uptake by zinc-deficient cells. Due to the
induction of ZRT1, ZRT2, and FET4, the
maximum capacity for zinc uptake by zinc-limited cells is ~100-fold
greater than in zinc-replete cells (15, 16). Therefore, when even a low concentration of zinc is added back to zinc-limited cells, the metal is
rapidly overaccumulated, a condition we refer to as zinc shock. In this
report we show that the increased expression of ZRC1 in
zinc-limited cells is essential for the cell's ability to tolerate the
stress of zinc shock.
Yeast Strains and Growth Conditions--
Four different media
were used for yeast cultures. YPD and synthetic-defined (S.D.) medium
with 2% glucose (23) are zinc-replete and contain no strong chelators.
Low zinc medium (LZM) and chelex-treated synthetic-defined (CSD) medium
were prepared as previously described (14, 21). LZM is zinc limiting
because of the inclusion of 1 mM EDTA and 20 mM
citrate as divalent cation chelators. CSD is zinc limiting because zinc
is removed from the medium with the chelex-100 ion exchange resin. Zinc
supplemented into CSD is much more bioavailable than in LZM due to the
absence of strong chelators in CSD. Cell density determinations and
DNA and Protein Manipulations--
To construct the
YEpzrc1mZRE plasmid, overlap PCR was used to generate
transversion mutations in the ZRE sequence of the ZRC1 promoter (24). The resulting fragment was inserted into
BlpI-, BstXI-digested YCpZRC1 (11) by gap repair.
Construction of YEpZRC1-lacZ was previously described (21).
YEpzrc1mZRE-lacZ was constructed by amplifying the
ZRC1 promoter from YEpzrc1mZRE and inserting the
fragment into YEp353 by gap repair. All plasmid constructs were
verified by DNA sequencing. Total protein was extracted from yeast, and
immunoblot analysis was performed as previously described (25).
Anti-Vma1 antibody was obtained from Molecular Probes.
Zinc Uptake and Accumulation Assays--
Zinc uptake was assayed
as described (15). Standard zinc uptake assays were performed in LZM
lacking EDTA (LZM-EDTA) to increase the bioavailability of the zinc
tracer. To determine cell-associated zinc after growth in
65Zn2+-containing medium, 1-ml aliquots of
cells were collected on glass fiber filters (Whatman) and washed twice
with 5 ml of wash buffer (20 mM sodium citrate, pH 4, 1 mM EDTA). Radioactivity retained on the filters was
quantified with a gamma counter. 65Zn2+ uptake
by cells loaded with FuraZin-1 (see below) was assayed in the same
buffer used for assays of fluorophore fluorescence to allow direct
comparison of total cell and vacuolar zinc accumulation under these conditions.
Measurement of Vacuole Zinc Content with FuraZin-1--
Yeast
cultures (250 ml) were grown to log phase in LZM + 2 µM
zinc. Cells were harvested, washed twice with phosphate-buffered saline
and resuspended in phosphate-buffered saline at a final density of
3 × 108 cells/ml. 50 µg of FuraZin-1 acetoxymethyl
(AM) ester was dissolved in 16.6 µl of a 20% Pluronic solution (both
obtained from Molecular Probes) and the solution diluted 4-fold with
Me2SO to give a stock solution of 1.25 mM fluorophore and 5% Pluronic. Fluorophore was added to a
1-ml aliquot of the cell suspensions to give a final concentration of
25 µM. Another 1-ml aliquot of each strain was treated in
parallel but without exposure to fluorophore. The cell suspensions were
incubated at 30 °C in the dark for 1 h with agitation. The
cells were recovered by centrifugation, washed three times with 5 ml of
chilled zinc uptake buffer (10 mM MES-Tris, pH 6.5, 4 mM MgCl2, 2% glucose) and 1 mM
EDTA and then incubated at 30 °C for another 30 min. This step
allowed the cells to redistribute cytoplasmic fluorophore to the
vacuole and complete its hydrolysis. The cells were then chilled and
washed twice with MES-Tris uptake buffer ( ZRC1 and COT1 Are Functionally Redundant for Steady-state Zinc
Tolerance--
A previous study indicated that both Zrc1 and Cot1
contribute to tolerance of excess zinc (13). In the yeast strain
INVSC2, wild-type and cot1 cells were resistant to zinc
concentrations up to ~5 mM. Mutant zrc1 cells
tolerated up to 1 mM, but the zrc1 cot1 mutant
was not viable at this zinc concentration. In our strain background
(W303), we obtained qualitatively similar results (Fig.
1). Wild-type cells showed tolerance to
up to 6 mM zinc. The zrc1 mutation caused a
slight defect in zinc tolerance, reducing the inhibitory concentration
by 1-3 mM. The cot1 mutation had little effect,
only lowering the maximum tolerable concentration to 5 mM.
In contrast to these minor effects, the zrc1 cot1 double mutant strain showed a ~50-fold reduction in the tolerable zinc concentration (0.07 mM) relative to the single mutants.
These observations demonstrated that Zrc1 and Cot1 are functionally redundant for protection against high levels of zinc under steady-state conditions. However, even with the loss of both genes, yeast was still
tolerant of a relatively high concentration of zinc (70 µM).
ZRC1 Is Induced in Low Zinc by Zap1--
Previous studies
suggested a role for Zrc1 in zinc-limited cells. Specifically,
expression of the ZRC1 gene is induced in zinc-limited cells
in a Zap1-dependent manner (21, 22). Furthermore, the
ZRC1 promoter contains a potential ZRE that is functional when inserted into a heterologous promoter (21). To further examine
this regulation, a mutant ZRC1 promoter-lacZ fusion was generated in which all nucleotides in the putative ZRE were altered by
transversion mutations. Strains bearing wild-type or ZRE mutant reporter plasmids were cultured in LZM supplemented with a range of
zinc levels and then assayed for
Transcriptional control of ZRC1 was also reflected in
altered protein levels. We examined the accumulation of protein
expressed from a functional (data not shown) epitope-tagged allele of
ZRC1 regulated by its own promoter and integrated into its
native chromosomal location. Immunoblot analysis showed that two
closely spaced bands representing forms of the Zrc1 protein accumulated
to higher levels in zinc-deficient cells (LZM + 0.3-10
µM zinc) compared with zinc-replete cells (Fig.
2B). No bands of this size were detected in protein from a
control strain expressing untagged Zrc1 (data not shown). The level of
a loading control protein, Vma1, was minimally affected by zinc status.
ZRC1 and COT1 Are Not Required for Steady-state Zinc-limited
Growth--
The Zap1-dependent regulation of
ZRC1 suggested that the Zrc1 protein is required during zinc
deficiency. Supporting this hypothesis, it was previously reported that
zrc1 mutants grew poorly compared with wild type under
zinc-limiting conditions (22). Contrary to that previous result,
however, we found no defect in zinc-limited growth (LZM + 0.3-10
µM zinc) for zrc1, cot1, or
zrc1 cot1 cells (Fig.
3A). The explanation for the
apparent discrepancy between our results and the prior study will be
presented under "Discussion." The sensitivity of the zrc1
cot1 mutant to high zinc (LZM + 30-1000 µM zinc) is
consistent with the redundancy of Zrc1 and Cot1 in steady-state zinc
tolerance. To determine the effect of these mutations on zinc
accumulation, we measured the total zinc content of wild-type and
mutant strains after growth over a range of added zinc. Again, at zinc
concentrations of 10 µM or less, these mutations had no
effect on cell zinc content (Fig. 3B). In medium
supplemented with 30 µM zinc or more, all three mutant
strains accumulated less zinc than wild type, and the zrc1
cot1 double mutant accumulated less than either single mutant.
Notably, these effects of zrc1 and cot1 mutations
on growth and zinc accumulation were observed only at zinc
concentrations in LZM of greater than 10 µM. This
concentration was previously identified as the transition point between
zinc deficiency and repletion (14). This can also be seen as such here
because for the wild-type strain, the maximum growth rate is observed
at 10 µM zinc and higher. Thus, we found no evidence that
Zrc1 or Cot1 are required for zinc-limited growth or alter zinc
homeostasis in zinc-limited cells.
To test for an effect of high ZRC1 activity on zinc
homeostasis in zinc-deficient cells, we overexpressed the gene from a multicopy plasmid and measured the effect on total cellular zinc accumulation. We confirmed an increase in Zrc1 protein level due to
overexpression in both high and low zinc media (data not shown). As
shown in Fig. 4A,
overexpression of ZRC1 significantly increased cellular zinc
content when cells were grown under zinc-replete conditions.
Importantly, it had no effect in zinc-deficient conditions (Fig.
4A, inset). The effect of this increased zinc
accumulation on cytoplasmic zinc availability was then determined.
Methods to directly measure cytoplasmic labile zinc are not currently available, but we have previously shown that an indirect assessment of
this parameter can be made using a Zap1-regulated reporter gene (14).
ZRE-lacZ reporter activity was significantly increased in cells that
overexpressed ZRC1, indicating reduced cytoplasmic labile
zinc. This observation is consistent with a model whereby Zrc1
expression results in zinc transport from the cytoplasmic/nuclear compartment into an organelle. Again however, an effect of Zrc1 was
only seen in cells that were zinc-replete. In summary, the experiments
shown in Figs. 3 and 4 revealed no evidence for an effect of Zrc1 on
zinc homeostasis in yeast cells during growth under steady-state
zinc-deficient conditions. Thus, while it is not possible to rule out a
more subtle role not revealed by these assays, these experiments did
not provide an obvious explanation for why ZRC1 is
up-regulated in zinc-deficient cells.
ZRC1 and COT1 Protect Zinc-deficient Cells from Zinc Shock--
In
the experiments shown in Fig. 1, zinc-replete cells were used to
inoculate plates. Zinc-replete cells have repressed high-affinity zinc
uptake systems and do not accumulate excess zinc when inoculated into
fresh medium. In contrast, when zinc-limited cells are resupplied with
zinc, they rapidly accumulate large quantities because of the high
activity of the plasma membrane zinc transporters (15). We refer to
this condition as zinc shock. During zinc shock, newly acquired
zinc rapidly enters the cytoplasm where it can accumulate to high
levels. We hypothesized that yeast detoxify this excess zinc by rapid
transport into an intracellular compartment. If this is true, the high
expression of the ZRC1 zinc tolerance gene in zinc-deficient
cells may be required to mediate the rapid sequestration of excess
cytoplasmic zinc during zinc shock. This model predicts that a
zrc1 mutant will be hypersensitive to zinc shock; a
prediction we confirmed (Fig.
5A). The medium used in this
experiment, CSD, is made zinc limiting by treatment with a chelating
resin. Because CSD contains no strong chelators, the availability of
added zinc is many orders of magnitude higher than for equivalent
concentrations of total zinc in LZM. When zinc-limited zrc1
mutants (i.e. pregrown in LZM + 1 µM
ZnCl2) were inoculated into CSD medium supplemented with as
little as 1 µM zinc, zrc1 cells failed to
grow. In contrast, neither the wild-type nor the cot1 mutant
showed any growth defect up to 10 µM zinc (the increase
in growth yield observed for the wild-type and cot1 strains
with increased zinc is due to zinc-limitation in CSD with less than 1 µM added zinc). The zrc1 cot1 mutant was even
more sensitive to zinc shock than the zrc1 mutant,
indicating Cot1 also contributes to zinc shock tolerance. None of these
cell types were zinc sensitive when inoculated from zinc-replete
cultures (i.e. pregrown in LZM + 1 mM
ZnCl2) (Fig. 5B). Thus, the sensitivity is a
consequence of the transition from zinc-limited to zinc-replete conditions. The retarded growth of the zrc1 cot1 mutant when
inoculated from zinc-replete under zinc-limiting conditions does not
correspond to zinc toxicity (because growth at higher zinc
concentrations is similar to wild-type), and may be due to the low
vacuolar zinc stores in this mutant (14).
While the Zrt1, Zrt2, and Fet4 plasma membrane uptake transporters are
induced in zinc-limited cells, Zrt1 is the major pathway of zinc
uptake; mutation of ZRT1 reduces zinc uptake in
zinc-deficient cells by Effect of zrc1 and cot1 Mutations on Intracellular Zinc
Compartmentalization--
To assess if zinc sequestration is altered
in zrc1 and cot1 mutants during zinc shock, we
first assayed the effects of these mutations on zinc accumulation.
Accumulation of substrate on the cytoplasmic side of the plasma
membrane can directly inhibit the transporters responsible for uptake,
for example via trans-inhibition (26, 27). Data from zinc
uptake experiments (15) predicted that in the absence of any
intracellular compartmentalization during zinc shock, cytoplasmic zinc
levels would rise into the millimolar range within minutes. Under these
conditions, mutant strains unable to sequester zinc might exhibit
impaired zinc uptake due to trans-inhibition. To test this
prediction, zinc accumulation by wild-type, zrc1,
cot1, and zrc1 cot1 mutants under zinc shock conditions
was compared; i.e. zinc-deficient cells were transferred to
an uptake buffer that contained 1 µM
65Zn2+ and lacked strong metal ion chelators.
In each strain, the initial rates of zinc accumulation over the first 1 min were indistinguishable (Fig.
6A). Thus, zrc1 and
cot1 mutations do not affect zinc uptake activity in cells
maintained under zinc-limiting conditions. A time course of uptake
indicated that, although accumulation in wild-type and cot1
mutant cells continued almost linearly for 20 min, accumulation by
zrc1 and zrc1 cot1 mutants began to plateau before 5 min (Fig. 6B). This observation was consistent with
a failure of the zrc1 mutants to properly sequester zinc,
leading to the direct inhibition of plasma membrane transporters by
high cytoplasmic zinc concentrations. Subsequent experiments to
directly assess vacuolar zinc compartmentation during zinc shock
supported this interpretation (see below). No contribution of Cot1 was
observed under the conditions described in the legend to Fig.
6B (growth of cells in LZM + 1 µM zinc). To
further examine the potential role of COT1 during zinc
shock, we used this impairment of zinc accumulation as an indirect
assay to examine the contribution of Zrc1 and Cot1 to zinc homeostasis
in cells grown over a range of zinc concentrations. Again, the
mutations had no affect on the initial rate of zinc uptake as measured
over 1 min (data not shown). Over a longer period (5 min), the
zrc1 mutation reduced zinc accumulation by cells grown in
zinc-limiting media (LZM + 0.3-10 µM zinc) (Fig.
6C). An effect of the cot1 mutation on zinc accumulation was observed, but only in the absence of Zrc1 and in cells
grown at zinc concentrations greater than 10 µM.
Therefore, both Zrc1 and Cot1 appear to contribute equally to zinc
storage and detoxification in zinc-replete cells, but in zinc-deficient cells undergoing zinc shock, Zrc1 alone plays the dominant role.
We suspected that the decreased zinc uptake and growth inhibition
phenotypes of zrc1 strains subjected to zinc shock were a
consequence of a defect in vacuolar zinc uptake. To test this prediction directly, we used the ratiometric fluorescent
indicator FuraZin-1 to non-invasively assay changes in vacuolar zinc
content in living cells during zinc shock. When cells were loaded with the acetoxymethyl (AM) ester of FuraZin-1 and then treated with glucose
(as described under "Materials and Methods"), the fluorophore accumulated predominantly in the vacuole (Fig.
7A). Counts of randomly
selected cell fields indicated that >99% of the cells that had taken
up FuraZin-1 had sequestered the fluorophore in the vacuole, and this
distribution was independent of strain genotype (Fig. 7A and
data not shown). This location of FuraZin-1 in yeast allowed us to use
simple fluorimetric assays of fluorophore speciation in cell
suspensions to assay the vacuole zinc content during zinc shock. In
cells grown in zinc-deficient medium, excitation wavelength scans
showed that the fluorophore was predominantly in the zinc-free form,
with an excitation maximum at 380 nm and a fluorescence ratio
(F325 nm/F380 nm) of ~0.2. Under conditions of zinc shock, there was a rapid increase in the zinc-bound form (with
an excitation maximum at 325 nm) of the fluorophore as indicated by the
increase in the F325 nm/F380 nm ratio (Fig. 7B). No change in this signal was observed during incubation
in the absence of zinc (not shown), and the zinc-induced ratiometric change was reversed by the addition of TPEN, a cell-permeant zinc chelator (Fig. 7D). A rapid increase in signal could also be
induced by treating cells with the zinc ionophore pyrithione to allow free diffusion of zinc into the yeast cell (Fig. 7E). Taken
together, these observations indicated that the ratiometric change in
fluorescence observed during zinc shock resulted from an increase in
the zinc content of the vacuole compartment.
When zinc-deficient zrc1 mutant cells were loaded with
FuraZin-1 and subjected to zinc shock, an increase in the fluorophore signal was also observed but at a much reduced rate in comparison to
the wild-type (Fig. 7B). This difference was not due to an inability of the fluorophore within the zrc1 cells to
respond to zinc, as exposure to zinc-pyrithione eliminated the
difference between the strains (Fig. 7E). The observed
difference in fluorophore signal was also not attributable to a
difference in zinc uptake by the cells (Fig. 6B); under the
conditions used in this experiment (cell growth in LZM + 2 µM zinc and transfer to 100 µM zinc), the
initial rate of zinc uptake was the same (Fig. 7C).
Therefore, these data provide direct evidence that during zinc shock,
Zrc1 is required for sequestering zinc into the vacuole compartment. In
addition, these data strongly suggest that during zinc shock, the
zrc1 mutant accumulated a larger proportion of zinc in the cytoplasm. This overaccumulation of zinc in the cytoplasm, and perhaps
a resultant increase in zinc accumulated by other compartments (e.g. the mitochondria) may be responsible for the growth
defect observed in the zrc1 mutant following zinc shock
(Fig. 5A).
ZRC1 Induction by Zap1 Is Required for Zinc Shock
Tolerance--
The observation that Zrc1 was largely responsible for
zinc sequestration during zinc shock suggested that the Zap1-mediated induction of this gene might be important for zinc shock tolerance. To
test this hypothesis, we generated a strain in which the
ZRC1 promoter with an inactivated ZRE (Fig. 2A)
controlled expression of the chromosomal ZRC1 locus. This
allele, designated zrc1mZRE, did not alter the
zinc tolerance of zinc-replete cells grown on high zinc (data not
shown). This observation is consistent with the lack of an effect of
the ZRE mutation on basal, Zap1-independent expression of the promoter
(Fig. 2A). When the effect of the
zrc1mZRE mutant gene on zinc shock was tested,
the results showed that the loss of ZRC1 induction by low
zinc substantially decreased tolerance (Fig.
8). In fact, the
zrc1mZRE mutant was almost as sensitive to zinc
shock as the complete zrc1 deletion mutant. Moreover,
mutation of the ZRE impaired zinc accumulation during zinc shock (Fig.
6B), suggesting that the absence of ZRC1
induction also impaired the compartmentalization of cytoplasmic zinc.
Consistent with this interpretation, a zrc1mZRE
mutant strain was also observed to accumulate less vacuolar zinc during
zinc shock, as measured by FuraZin-1 fluorescence (Fig. 7B).
Therefore, the induction of ZRC1 by Zap1 under
zinc-deficient conditions is essential to protect cells against zinc
shock.
ZRC1 and COT1 encode putative zinc
transporters of the CDF family that are required for metal ion
tolerance. Whereas previous reports suggested that ZRC1 and
COT1 primarily confer tolerance to zinc and cobalt ions
respectively (8, 12), it is now clear that under certain conditions,
these genes are functionally redundant with respect to zinc. Zrc1 and
Cot1 are both important determinants of zinc detoxification in
zinc-replete yeast grown in high zinc, and both genes were implicated
in the vacuolar storage of zinc in zinc-replete cells (13, 14). Given
these roles of Zrc1 in zinc-replete or excess conditions, it was
surprising to find that ZRC1 is a Zap1 target gene and
induced under zinc-limiting conditions (21, 22). This regulation is
inconsistent with either of the previously recognized functions of Zrc1
in zinc homeostasis. One possible explanation is that zinc-limited
cells require more Zrc1 activity to maintain a flux of zinc through the
vacuole, perhaps to provide for zinc-dependent processes
within that compartment (14, 22). As yet, we have found no evidence to
support this hypothesis. Cells mutant in ZRC1 (and/or
COT1) showed no defect in growth (see below) or zinc
accumulation under zinc-limiting conditions. This result was also
obtained in time course studies comparing growth of these mutants and
wild-type cells throughout lag and exponential growth phases (data not
shown). It is conceivable that the deleterious effects of these
mutations in zinc-limited cells may be too subtle to detect by these
assays. However, additional assays also found no evidence that Zrc1 is capable of zinc transport when cytosolic zinc levels are low. Overexpressing Zrc1 increased zinc accumulation and reduced labile cytosolic zinc levels in zinc-replete cells but had no impact on cells
grown in low zinc (i.e. LZM + If not active in zinc-limited cells, why then is ZRC1 a Zap1
target gene? In this report, we have clearly shown the crucial importance of Zrc1 activity and its regulation by Zap1 in tolerance to
zinc shock. Zinc shock occurs when zinc-limited cells, which have a
high latent capacity for zinc uptake, are resupplied with zinc. Large
amounts of zinc then pass across the plasma membrane and accumulate in
the cytoplasm. The cell responds to this zinc excess by down-regulating
the transcription of ZRT1, ZRT2, and FET4 (15, 16), and by inactivating the Zrt1 protein via
zinc-induced endocytosis (28). However, these processes occur too
slowly to prevent uptake of large quantities of zinc; we calculate that in the absence of any organellar sequestration, the cytosolic zinc
levels would rise into the millimolar range within minutes. If
cytosolic zinc is not rapidly sequestered into the vacuole, growth
arrest may result from which the cells are unable to recover. Several
lines of evidence are presented here to support this model. First,
zrc1 and zrc1 cot1 mutants are extremely
sensitive to conditions of zinc shock. Second, our data indicate that
zinc sequestration in the vacuole was disrupted in zrc1 and
cot1 mutants undergoing zinc shock. Third, zinc-limited
zrc1 mutant cells are protected from zinc shock if they are
unable to express the major inducible zinc uptake system, Zrt1. The
induction of ZRC1 expression by Zap1 is a critical part of
zinc shock resistance. Mutating the ZRE element in the promoter
disrupted induction in zinc-deficient cells but did not affect basal,
zinc-replete expression. Mutation of the ZRE also rendered the mutant
cells almost as incapable of tolerating zinc shock as a full deletion
of the ZRC1 gene, and this sensitivity was associated with a
defect in vacuolar zinc uptake during zinc shock.
We suggest that tolerance to zinc shock is the major role of Zrc1 in
zinc homeostasis, and not resistance to high steady-state extracellular
zinc levels per se. This is evident when the levels of zinc
required to elicit zinc toxicity under these conditions are compared.
Under steady-state conditions of high zinc, Zrc1 is required for
resistance to more than 4 mM zinc. However, under conditions of zinc shock, Zrc1 is required to tolerate as little as 1 µM zinc, i.e. a 4000-fold lower concentration.
Transitions from zinc-deficient to moderately zinc-replete conditions
are likely to occur frequently in nature, while millimolar
concentrations of free zinc would be rarely encountered.
In this work we used a novel zinc indicator (FuraZin-1) to
non-invasively determine the effect of Zrc1 on zinc homeostasis in
living cells. Under appropriate conditions, ratiometric probes such as
FuraZin-1 can be used to estimate in vivo ion concentrations with a standard equation (29). However, this equation is not applicable
in the present case because the high concentration of fluorophore
required in the vacuole to detect changes in zinc levels perturbs the
labile vacuolar zinc pool (30). A more accurate approach to estimate
vacuolar zinc levels considers the total concentration of fluorophore
loaded into that organelle. By measuring indicator fluorescence
in vitro and comparing with values obtained in
vivo, we calculated that FuraZin-1 routinely reached 1 mM in the vacuole of loaded cells. Saturation of this
concentration of fluorophore with zinc-pyrithione resulted in a maximum
F325/F380 ratio of ~2 (Fig. 7E).
Given that the fluorophore was ~50% saturated (F325/F380 ratio of 1) in wild-type cells
within 6 min of zinc treatment (Fig. 7B), these results
indicate that vacuolar zinc levels quickly rise to the millimolar range
during zinc shock. These data suggest that vacuolar sequestration
during zinc shock is very efficient in removing cytosolic labile zinc.
In contrast to our results, Miyabe et al. (22) presented
data that suggested that Zrc1 was required for growth under
steady-state zinc-limited conditions. These authors grew wild-type and
zrc1 mutant cells in a low zinc medium for a period of time
and then plated those cells onto agar plates to determine the number of viable cells from the colonies that formed. Fewer colonies formed for
the zrc1 mutant than the wild type suggesting that growth in
the zinc-limited medium was impaired. However, we have determined that
the reduced plating efficiency of the zrc1 mutant in these experiments was due to zinc shock; the medium used for cell counting, YPD, is zinc-rich (~10 µM total zinc (data not shown)
with no strong chelators present). When we replicated their
experiments, we found that zinc-deficient zrc1 mutant cells
did not grow when plated on YPD, but that these cells were viable when
plated on a zinc-limiting medium (data not shown). Thus, the results of the previous study suggesting Zrc1 was essential for growth under steady-state zinc-deficient conditions are in fact explained by the
zinc shock effect.
While Zrc1 and Cot1 are closely related proteins, and both affect zinc
homeostasis, Zrc1 clearly plays the major role in zinc shock tolerance.
The minor tolerance that Cot1 provides is perhaps explained by its lack
of regulation by Zap1 (21). It is intriguing then that the
COT1 gene may be a target of the Aft1 transcription factor
(31). Aft1 activates transcription of genes in yeast in response to
iron deficiency. Aft1 targets include the high affinity iron uptake
system that is responsible for iron accumulation under limiting
conditions. By analogy to Zrc1, perhaps Cot1 levels increase in
iron-limited cells to protect those cells from the high influx of iron
that occurs upon iron repletion. However, we found no effect of the
cot1 mutation on tolerance to such "iron shock"
conditions.2 Alternatively,
increased Cot1 activity could help suppress the toxicity of other metal
ions that may be accumulated by relatively nonspecific cation uptake
systems induced in iron-limited cells (10, 17, 32).
To our knowledge, this report represents the first well documented
example of a proactive mechanism of homeostasis. However, we note other
reports of potential proactive adaptation to environmental change. For
example, the PHM genes of yeast are required for
synthesis of polyphosphate, the vacuolar storage form of inorganic
phosphate (Pi) (33). The PHM genes are
co-induced with Pho84, the high affinity phosphate uptake transporter,
under phosphate-deficient conditions. However, yeast cells do not
accumulate polyphosphate under steady-state phosphate-deficient
conditions (34), so the reason for PHM gene induction under
these conditions is not clear. We note that most phm
mutations impair Pi uptake by deficient cells (33), a
phenotype that could be attributed to trans-inhibition of
Pho84 following the accumulation of cytoplasmic Pi. If this is true, then the induction of the PHM genes may be required
for the rapid sequestration of Pi in the vacuole during the
transition from phosphate-deficient to replete conditions.
phm mutants do show impaired polyphosphate synthesis under
these conditions, but it is not known if this phenotype is associated
with sensitivity to "phosphate shock" conditions. The
CopB gene of Enterococcus hirae is another
potential example of the proactive induction of a metal ion
detoxification system. CopB and CopA encode
Cu+-transporting P-type ATPases. Whereas both genes
are present in the same operon, their products have different roles:
CopA mediates Cu+ uptake, while CopB mediates efflux.
Expression of both CopA and CopB is induced under copper-deficient
conditions. Analogous to zinc shock, it was speculated that CopB
induction by Cu+ deficiency is a proactive adaptation to
the potential for a rapid increase in Cu+ availability
(35). In plants, the induction of many genes conferring tolerance to UV
irradiation and low temperature is linked to the circadian clock. It
has been proposed that this regulation allows the anticipation of
cyclically predictable stresses, such as cold nights or increased light
intensity during daylight hours (36, 37). Similarly, zinc shock is a
predictable stress faced by zinc-deficient yeast. These few examples
suggest that the phenomenon of proactive homeostatic regulation is not
restricted to zinc homeostasis in yeast, but may in fact represent a
common and largely unrecognized feature of gene expression in many organisms.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactosidase assays were performed as previously described (14).
S. cerevisiae strains CM100, 102, 103, 104, and
142 were also previously described (14). All newly constructed strains
are isogenic to CM100. CM141 (MAT
zrt1::LEU2
zrc1::HIS3) was derived from a cross of ZHY1 (15) and CM102. The CM146 (MATa zrc1mZRE) strain
was constructed by integrating the insert of YCpzrc1mZRE at
the chromosomal ZRC1 locus. Transformants were selected by complementation of the zinc-sensitive phenotype of a zrc1
cot1 double mutant strain (CM104), and the presence of the
mutation was verified using PCR. The cot1 mutation was then
removed by backcrossing to a wild-type strain.
EDTA). Cell pellets were
resuspended in 2 ml of uptake buffer and maintained on ice prior to
zinc uptake assays. Fluorimetric assays of fluorophore speciation were
performed in a Hitachi F3010 spectrofluorimeter. To start the assay, an
aliquot of loaded cells (100 µl) was added to 4 ml of MES-Tris uptake
buffer and 100 µM zinc. The temperature of the cuvette
was maintained at 30 °C using a recirculating water bath. With the
instrument set at maximum scan speed, the excitation wavelength was
varied from 250 to 450 nm, and the intensity of emission at 500 nm was
recorded. Spectra were recorded at the start of the assay and at 2-min
intervals for up to 6 min. To correct for the effects of fluorophore
leakage during the experiment, immediately before each measurement a
1-ml aliquot of the assay was removed and filtered through a 0.45-µm syringe filter. After completion of the experiment, unloaded cells were
added to samples of the filtered buffer to give the same cell density
as present in the initial suspensions. Spectra of these samples were
immediately recorded and the curves subtracted from the original
spectra. This procedure provided a one-step correction for both
cellular autofluorescence and leakage of fluorophore from the cells
during the experiment. The corrected traces were scanned to obtain
emission intensities at the excitation wavelengths of 325 and 380 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Zinc sensitivity of zrc1 and
cot1 mutant strains. CM100
(Wild-type), CM102 (zrc1), CM103
(cot1), and CM104 (zrc1 cot1) strains were grown
in zinc-replete S.D. medium to stationary phase. 5-µl aliquots
(2 × 105 cells) were applied to S.D. medium plates
supplemented with the indicated concentration of zinc. Plates were
incubated at 30 °C for 2 days before photography.
-galactosidase activity (LZM is
zinc limiting because it contains EDTA, which chelates most zinc in the
medium rendering it unavailable to cells). The wild-type promoter was
strongly induced under zinc-limited conditions (Fig. 2A). Added zinc reduced
expression to ~20% of the maximal level. This basal expression of
ZRC1 is Zap1-independent (21, 22). Consistent with this
conclusion, mutation of the ZRE completely eliminated zinc-responsive
regulation without affecting basal expression. This result supports the
contention that ZRC1 is transcriptionally regulated by Zap1
through this ZRE. Furthermore, the ZRE mutant promoter provided a
useful reagent for subsequent studies (see below).
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Fig. 2.
Zinc-regulated expression of
ZRC1. A, ZRC1 promoter
activity over a range of zinc concentrations. Wild-type cells (DY1457)
carrying YEpZRC1-lacZ or YEpzrc1mZRE-lacZ were grown to
late log phase in LZM with the indicated zinc concentration and assayed
for -galactosidase activity. Symbols are the means of three
replicates, with one standard deviation less than the width of the
symbol in all cases. B, immunoblot analysis of CM142, which
expresses hemagglutinin epitope-tagged Zrc1 from its own promoter.
Protein was extracted from cells grown in LZM supplemented with zinc at
the indicated concentration. 5 µg of protein was fractionated on a
10% acrylamide gel and blotted to nitrocellulose. The blot was probed
with anti-hemagglutinin (Zrc1) and anti-Vma1 monoclonal antibodies
(Molecular Probes), followed by secondary horseradish
peroxidase-labeled anti-mouse antibody (Pierce). Signal was detected
using ECL (Amersham Biosciences).
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Fig. 3.
Effects of zinc availability on growth and
zinc accumulation by zrc1 and cot1
mutants. A, effect of zinc availability on growth. WT,
zrc1, cot1, and zrc1 cot1 strains (see
Fig. 1) were grown to saturation in zinc-replete S.D. medium. LZM
containing the indicated zinc concentration was inoculated at an
initial absorbance at 600 nm (A600) of 0.01. Cultures were incubated for 18 h and final
A600 determined. B, effects on
cell-associated zinc. S.D. cultures of the above strains were used to
inoculate LZM containing 65Zn2+ isotope and
non-radioactive zinc to give the total zinc concentration indicated.
Cultures were grown to late log phase and cell-associated zinc was
quantified. Data points are the means of three replicates, and
error bars represent ±1 S.D.
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Fig. 4.
Effect of ZRC1
overexpression on zinc accumulation and cytoplasmic zinc
availability. A, effect of ZRC1 overexpression on
cell-associated zinc. Wild-type cells (CM100) carrying either the
pFL44-S (Control) or YEpZRC1 (ZRC1
O/E) plasmids were used to inoculate aliquots of LZM,
which contained 65Zn2+ isotope and additional
cold zinc to give the total zinc concentration indicated. Cultures were
grown to late log phase and cell-associated
65Zn2+ was quantified. B, effect of
ZRC1 overexpression on Zap1 activity. Wild-type cells
(CM100) carrying a Zap1-regulated reporter plasmid (pDG2-1) and either
pFL44-S (Control) or YEpZRC1 (ZRC1
O/E) plasmids were grown to late log phase in LZM
with the indicated zinc concentration and assayed for -galactosidase
activity. Data points are the means of three replicates, and
error bars represent ±1 S.D.
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Fig. 5.
Effects of zrc1,
cot1, and zrt1 mutations on zinc shock
sensitivity. Panels A and B, effect of
zrc1 and cot1 mutations on growth after zinc
shock. WT, zrc1, cot1, and zrc1 cot1
strains (see Fig. 1) were grown in low zinc (LZM + 1 µM
zinc) or replete zinc (LZM + 1 mM zinc) media. Aliquots of
CSD medium supplemented with the indicated zinc concentration were then
inoculated with zinc-limited (panel A) or replete
(panel B) cells to an initial A600 of
0.01. After 18 h of incubation, A600 of the
cultures was determined. C, effect of the zrt1
mutation on zinc shock. Overnight cultures of the indicated genotypes
were grown in zinc-deficient conditions. Because mutation of
ZRT1 altered zinc requirements for growth in LZM, the
strains were grown in media that caused similar levels of zinc
deficiency, as judged by the expression of a zinc-responsive ZRE-lacZ
reporter gene (LZM with 1 µM added zinc for CM100
(WT) and CM102 (zrc1), or 300 µM
for ZHY1 (zrt1) and CM141 (zrt1 zrc1), data not
shown). CSD medium with (1 µM) or without added zinc was
then inoculated with zinc-deficient cells of the four strains to an
initial A600 of 0.01. The cultures were
incubated for 18 h and final A600
determined. For all experiments shown, data points are the means of
three replicates, and error bars represent ±1 S.D.
80% (16). Therefore, we predicted that zinc
shock results largely from the high activity of Zrt1 in deficient
cells. To test this prediction, we examined the effect of a
zrt1 mutation on the zinc sensitivity of a zrc1
mutant. As before, zinc-deficient cells were inoculated into CSD medium
containing no zinc or 1 µM added zinc. The
zrt1 mutation completely suppressed the zinc sensitivity
associated with the zrc1 mutation (Fig. 5C).
These data indicate that the high level of zinc accumulation mediated by Zrt1 in zinc-limited cells is responsible for the zinc sensitivity of zrc1 mutants undergoing zinc shock. Poor growth of the
zrt1 and zrt1 zrc1 mutants without added zinc is
likely due to the impaired zinc uptake in these strains.
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Fig. 6.
Effects of zrc1 and
cot1 mutations on zinc uptake and accumulation during
zinc shock. A, initial rate of zinc uptake by zinc-deficient
wild-type, zrc1, cot1, and zrc1 cot1
strains (see Fig. 1). Strains were grown in LZM + 1 µM
ZnCl2 and assayed for zinc uptake after 1 min in EDTA-free
buffer with 0.5 µM 65Zn2+.
B, time course of zinc uptake by zinc-deficient
zrc1, cot1, and zrc1mZRE
(CM146) mutant strains. Strains were grown in LZM + 1 µM
ZnCl2 and assayed for zinc uptake with 1 µM
65Zn2+. A single representative experiment is
shown. C, effect of initial zinc status on sustained zinc
accumulation by zrc1 and cot1 mutant strains.
Strains were grown to log phase in LZM containing the indicated zinc
concentration. Cells were then assayed for zinc uptake with 10 µM 65Zn2+ for 5 min. For
A and C, values represent the means of four
experiments, and error bars show standard deviation.
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Fig. 7.
Measurement of vacuolar zinc accumulation
in vivo. A, intracellular distribution of FuraZin-1 in
yeast cells. CM100 (WT) and CM102 (zrc1) cells
were grown in LZM + 2 µM zinc and loaded with
cell-permeant FuraZin-1 as described under "Materials and Methods."
Live cells were fixed to concanavalin-coated slides, and intracellular
fluorophore was visualized using the DAPI filter of an epifluorescence
microscope (Epi). Whole cells were visualized using Nomarski
differential interference contrast (Nom). Images are
overlayed for comparison (Overlay). B, change in
vacuolar fluorophore signal during zinc shock. CM100 (WT),
CM102 (zrc1), and CM146 (zrc1mZRE)
cells were loaded with FuraZin-1 as described under "Materials and
Methods." To start the assay, aliquots of cells were added to
Tris-MES zinc uptake buffer containing 100 µM zinc. At
the indicated times after addition, fluorophore speciation in
vivo was determined as described under "Materials and Methods"
and plotted as relative fluorescence intensity
(F325/F380). C, whole cell zinc
uptake by fluorophore-loaded cells. FuraZin-1 loaded cells (see Fig.
7B) were added to Tris-MES buffer containing a total
concentration of 100 µM 65Zn2+
and non-radioactive zinc. At the indicated time intervals, aliquots of
cells were removed and cell-associated radioactivity determined, as
described under "Materials and Methods." D, effect of a
cell-permeant zinc chelator on FuraZin-1 fluorescence.
Fluorophore-loaded wild-type (CM100) cells were added to buffer with
100 µM zinc. Change in fluorescence was followed for 10 min, after which 500 µM
N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine
was added to the cell suspension. E, effect of the zinc
ionophore pyrithione on FuraZin-1 speciation in vivo.
Wild-type (CM100) and zrc1 (CM102)-loaded cells were added
to Tris-MES buffer containing 100 µM zinc and 50 µM pyrithione and change in fluorophore speciation
recorded. For B and C, data points are the
means of three replicates, and error bars represent ±1 S.D.
For D and E, one representative experiment is
shown.
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Fig. 8.
ZRC1 induction by Zap1 is required
for tolerance to zinc shock. CM100 (WT), CM146
(zrc1mZRE), and CM102 (zrc1) strains
were grown to late log phase (Preculture) in zinc-replete
(+, LZM + 1 mM zinc) or zinc-deficient ( , LZM + 1 µM zinc) conditions as indicated. Aliquots of CSD medium
with (+, 1 µM) or without (
) added zinc (treatment)
were then inoculated with replete or deficient cells and incubated for
18 h before determination of final A600.
Data points are the means of three replicates, and error
bars represent ±1 S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
10 µM zinc,
Fig. 4). This lack of activity may be due to an insufficient affinity
of Zrc1 for the available substrate in zinc-limited cells. Given that expression of Zrc1 protein is maximal in zinc-limited cells, the absence of a detectable effect of Zrc1 under these conditions is remarkable.
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ACKNOWLEDGEMENTS |
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We thank Amanda Bird, Tom Lyons and members of the Eide laboratory, Mary Lou Guerinot, Mick Petris, Elizabeth Rogers, and Daniel Schachtman for helpful discussions and their comments on the manuscript.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM56285 (to D. J. E.) and DK37512 (to M. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO 65211. Tel.: 573-882-9686; Fax: 573-882-0185; E-mail: eided@missouri.edu.
Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M300568200
2 C. W. MacDiarmid and D. J. Eide, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ZRE, zinc response element; MES, 4-morpholineethanesulfonic acid; S. D., synthetic-defined medium or standard deviation; CSD, chelex-treated synthetic-defined medium; LZM, low zinc medium; WT, wild type.
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