From the Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132
Received for publication, October 2, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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The sequence of the yeast gene YDR205W
places it within the family of cation diffusion facilitators: membrane
proteins that transport transition metals. Deletion of YDR205W was
reported to result in an increase in unequal sister chromatid
recombination and was named meiotic sister chromatid recombination 2 (MSC2; Thompson, D. A., and Stahl, F. W. (1999)
Genetics 153, 621-641). We report here that a
msc2 strain shows a phenotype of decreased viability in
glycerol-ethanol media at 37 °C. Associated with decreased growth is
an abnormal morphology typified by an increase in size of both cells
and vacuoles. Addition of extracellular Zn2+ completely
suppresses the morphological changes and partially suppresses the
growth defect. Regardless of the concentration of Zn2+ in
the media, the msc2 strain had a higher Zn2+
content than wild type cells. Zinquin staining also revealed that
msc2 had a marked increase in fluorescence compared with the wild type, again reflecting an increase in intracellular
Zn2+. The deletion strain accumulated excess
Zn2+ in nuclei-enriched membrane fractions, and when grown
at 37 °C in glycerol-ethanol media, it showed a decreased expression
of Zn2+-regulated genes. The expression of genes regulated
by either Fe2+ or Cu2+ was not affected. An
epitope-tagged Msc2p was localized to the endoplasmic
reticulum/nucleus. These results suggest that Msc2p affects the
cellular distribution of zinc and, in particular, the zinc content of nuclei.
The cation diffusion facilitator
(CDF)1 family consists of
genes that encode transition metal/H+ antiporters (1). Most
CDF genes were discovered through overexpression experiments, which
resulted in resistance to toxicity from transition metals (2, 3). More
recently, additional CDF members have been identified through sequence
homology. Members of the CDF family are found in all biological
kingdoms, and in eukaryotes, different CDF proteins may be
localized to different organelles. In many cases, the normal function
of CDF proteins is unknown. In a few cases, gene deletion studies or
studies of naturally occurring mutants have defined a physiological
role for CDF proteins. Gene knockout studies in mice have demonstrated
that ZNT3 is responsible for the accumulation of vesicular
Zn2+ in nerve cells (4). A mutation in a homologous gene,
ZNT4, was shown to be responsible for the murine toxic milk syndrome. ZNT4 proved to be required for secretion of Zn2+ into
breast milk (5).
The sequence of at least five genes in the budding yeast
Saccharomyces cerevisiae places these genes within the CDF
family. Two family members, COT1 and ZRC1, are
localized to the vacuole (6) and confer resistance to Co2+
(7) and Zn2+ (8), respectively. Other family members,
MMT1 and MMT2, encode mitochondrial proteins that
were identified through a suppressor screen. Overexpression of either
MMT1 or MMT2 was shown to affect Fe2+
levels in the mitochondria and cytosol (9). However, deletion of these
genes, either alone or in combination, did not affect essential
Fe2+-dependent mitochondrial processes, leaving
their precise role in mitochondrial Fe2+ metabolism unclarified.
The yeast gene YDR205W shows extensive homology to members of the CDF
family, with some notable differences. Most members of the
CDF family contain six transmembrane domains with an extensive histidine-rich cluster in a cytosolic loop. The protein sequence of
YDR205W, however, reveals 12 transmembrane domains and two histidine-rich cytosolic clusters. A deletion in YDR205W was identified in a screen of mutants that affected the rate of meiotic sister chromatid exchange. The gene is referred to as MSC2 (10).
The selection system was based on transposon insertion. When the gene was deleted, no increase in unequal sister chromatid exchange was
detected in the deletion strain. Thus, the role of the gene in
recombination events is unclear (10). We report here the characterization of a msc2 strain. The deletion strain shows
an increased sensitivity to H2O2 and is not
viable at 37 °C in glycerol-ethanol media. We further show that the
deletion strain has an alteration in cellular Zn2+ content
leading to an increase in nuclear Zn2+ content in cells
grown on respiratory substrates.
Strains and Media--
The yeast strains used in this study were
DY150 and DY1457, which were derived from a W303 background. The
diploid DY1640 was derived from a cross between DY150 and an isogenic
strain with the opposite mating type, DY151 (11). The media used
included: 1% yeast extract, 2.0% peptone, and 2.0% glucose
(YPD); 1.0% yeast extract, 2.0% peptone, 2.0% ethanol, and 2.0%
glycerol (YPGE); a synthetic medium of yeast nitrogen base, amino
acids, and glucose (CM); synthetic media with glycerol-ethanol (CMGE);
and low zinc/low iron/low copper CMGE medium (yeast nitrogen base was
from BIO101 Inc.).
Construction of the msc2 Deletion Strain--
A double fusion
PCR technique was used to delete YDR205W, generating the yeast strain
msc2 (12). The primers designed to amplify the 5' end
of YDR205W were 5'-GCAAGCCATTGATCAACGTG-3' and
5'-GTCGTGACTGGGAAAACCCTGGCGAAACACCACCTGCCTCTATC-3'. The primers used to
amplify the 3' end were
5'-TCCTGTGTGAAATTGTTATCCGCTTTACCGCTACAGCCTATAGC-3' and
5'-CATATATGATTCCTGAAGAC-3'. The HIS3 gene was used as
the selectable marker. The double fusion PCR products were transformed into diploid cells (DY1640). The transformed diploid was sporulated, and spores containing the disruption were selected by growth on media
lacking histidine. The deletion of YDR205W was confirmed by PCR.
Cloning of YDR205W and an Epitope-tagged Construct--
A probe
to the YDR205W open reading frame was generated by PCR. Southern
blot colony analysis (13) was used to identify YDR205W in a yeast
genomic library. The genomic library was obtained from the American
Type Culture Collection (ATCC#37323) and transformed into DH5 S1 Ribonuclease Protection Assay--
S1 analysis was performed
as described previously (18). Briefly, 50 ng of RNA was hybridized
overnight at 55 °C with an end-labeled oligonucleotide probe for
ZRT1 or CMD1, which was used as an internal
control. The RNA was digested by the addition of 50 units of S1
ribonuclease. The samples were analyzed by polyacrylamide gel
electrophoresis, and the developed gels were analyzed by PhosphorImager analysis.
Subcellular Fractionation and Zn2+
Measurement--
Cells grown in YPD were transferred to YPGE and grown
for 12 h at 37 °C. Subcellular fractionation and organelle
isolation were performed as described previously (19), with some
modifications. Briefly, spheroplasts were homogenized with a Dounce
homogenizer ("B" pestle). The homogenate was centrifuged at
3,000 × g for 5 min. The 3,000 × g
pellet was resuspended in mitochondria isolation buffer and centrifuged
at 120 × g for 5 min. The supernatant was collected by
centrifugation and found by microscopy to be enriched in nuclei. The
3,000 × g supernatant was centrifuged at 12,000 × g for 10 min and separated into a pellet and supernatant.
The pellet was applied to a 15% Percoll gradient and centrifuged at 59,000 × g for 27 min, and the gradient was
fractionated as described previously (20). The 12,000 × g supernatant was centrifuged at 100,000 × g for 30 min and separated into a pellet and supernatant. Each fraction was analyzed for protein concentration using the BCA
reagent (Pierce) and for Zn2+ content using a PerkinElmer
Life Sciences inductively coupled plasma atomic absorption
spectrometer as described previously (6).
Zinquin Staining, Immunofluorescence, and Western Blot
Analysis--
Zinquin (CF-125) a gift from Dr. Thomas V. O'Halloran
(Northwestern University, Evanston, IL) was used as described
previously (3). Cells grown in YPD were washed and transferred to YPGE. At specified times, cells were incubated with 25 µM
zinquin for 30 min at 37 °C. Cells were washed twice with
phosphate-buffered saline, placed on concanavalin A (1 mg/ml)-coated
slides, and examined by fluorescence microscopy.
For immunofluorescence analysis, cells transformed with the Myc- tagged
construct of Msc2p were prepared as described previously (6). The cells
were stained with a 1:100 dilution of a monoclonal anti-Myc antibody
(Babco Inc.), followed by a 1:200 dilution of an Alexa Fluor
594-conjugated goat anti-mouse antibody (Molecular Probes). Cells
treated with the primary and secondary antibody were incubated with 5 µg/ml 4',6-diamidino-2-phenylindole (Molecular Probes) for 5 min.
Cytoplasm and membrane fractions were isolated from cells transformed
with the FLAG-tagged Msc2p-containing plasmid. Samples were run on a
4-20% SDS-polyacrylamide gel electrophoresis gel (Bio-Rad) and
transferred to nitrocellulose. The blot was probed with a 1:5000
dilution of a mouse anti-FLAG antibody (M2; Sigma) followed by a
1:10,000 dilution of peroxidase-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch). The blot was developed with chemiluminescence reagents
(Renaissance) from PerkinElmer Life Sciences.
Phenotypes of the Alterations in Zn2+ Levels and Distribution in
We examined intracellular Zn2+ levels in msc2
using zinquin. Zinquin is a fluoresceine derivative that fluoresces
when bound to Zn2+ and can be visualized microscopically.
The intensity of the zinquin fluorescence changes as the levels of
Zn2+ change. Wild type cells grown in glycerol-ethanol
and incubated in zinquin show a diffuse cytosolic fluorescence.
Both the level and distribution of fluorescence change dramatically
when intracellular Zn2+ levels are increased. The
Zn2+-regulated transcription factor ZAP1 induces
the transcription of genes involved in Zn2+ transport and
metabolism (22). In the absence of Zn2+, Zap1p
permits the transcription of Zn2+-regulated genes through
its binding to specific DNA sequences termed zinc-responsive elements.
Zap1p regulates the transcription of ZRT1, which encodes a
high affinity Zn2+ transporter. Transformation of wild type
cells with an allele of the Zn transcriptional regulator
ZAP1up results in constitutive expression of
Zn-regulated genes, leading to an increased concentration of cellular
Zn2+ (22). Cells expressing ZAP1up show
increased zinquin fluorescence, with the fluorescence localized to
punctate vesicles. Zn2+-regulated Transcription in the
Decreased expression of the Zn2+ regulon in
glycerol-ethanol-grown msc2 cells was confirmed by S1
analysis using a probe designed to measure ZRT1 transcripts
(Fig. 7). The amount of ZRT1
transcript was reduced in msc2 cells, but not to the same
level as measured by the reporter construct. To test the possibility
that deletion of MSC2 acted downstream of
Zn2+-regulated transcriptional control, both wild type and
msc2 cells were transformed with a plasmid containing the
ZAP1up allele. Transformed wild type and
msc2 cells had similar levels of
ZRT1- Expression and Localization of Msc2p--
We generated an
epitope-tagged (FLAG) Msc2p to determine its location. MSC2
is predicted to encode a protein of 84 kDa. Western analysis showed the
FLAG-tagged Msc2p migrating at a molecular mass of ~75 kDa (Fig.
8A). Genetic studies confirmed
that the epitope marked Msc2p expressed in both low or high copy
plasmids complemented the deletion strain with regard to
H2O2 sensitivity and inability to grow on
glycerol-ethanol at 37 °C (data not shown). Immunofluorescence
studies using the high copy Myc-tagged plasmid indicated that the
protein is localized to the endoplasmic reticulum/nucleus (Fig.
8B), as further demonstrated by the images of the Myc-tagged Msc2p surrounding the 4',6-diamidino-2-phenylindole-stained nucleus (Fig. 8C). The appearance of the Myc-tagged protein in the
structure near the plasma membrane has been seen for other endoplasmic
reticulum proteins (21).
The CDF transporters affect the concentration of transition metals
within cells. Overexpression of CDF genes in bacteria results in
increased transition metal resistance by elevating metal export. Increased expression of CDF genes in eukaryotes may also provide transition metal resistance, although eukaryotes sequester metals in
intracellular compartments rather than exporting them. Sequestration of
metals in vesicles has the effect of lowering their concentration in
cytosol. As reported previously, some of yeast CDF proteins are in
nonendocytic organelles, and the function of these proteins is less
clear. The sequence of MSC2 clearly places it within the CDF
family, although the deduced sequence is much larger than other members
of the family. By direct sequencing of the isolated functional
MSC2 gene, we have confirmed that the sequence reported in
the data base is correct. The size of the epitope-tagged protein (75 kDa) is lower than predicted (84 kDa) but is still larger than the rest
of the CDF members. One explanation for the difference between the
observed and predicted size is that the protein undergoes posttranslational processing. Our localization data indicate that the
protein is localized to an internal membrane system. Overexpressed protein is found in the endoplasmic reticulum/nucleus. We have not been able to detect the protein when expressed by a low copy vector. Thus, the localization of the protein to the nucleus or endoplasmic reticulum is tentative. We have identified a
mammalian homologue of Msc2p, which is of a similar size and shows 63%
homology to Msc2p, but expression of this homologue in yeast does not
complement the msc2 phenotype.
The phenotype of the msc2 cells and suppression of the
phenotype by increased media Zn2+ suggest that Msc2p is
involved in Zn2+ homeostasis. Synthesis of either
MSC2 mRNA or protein, however, is not regulated by
Zn2+ (22).2
Deletion of MSC2 led to a dramatic morphological abnormality and a growth defect in glycerol-ethanol media at 37 °C. Increased media Zn2+ suppressed the morphological abnormality and
reduced the growth deficit of msc2 cells. Surprisingly, when
grown under identical conditions, msc2 cells had higher
Zn2+ levels than control cells. Subcellular fractionation
studies indicated that compared with wild type cells, msc2
cells show decreased cytosolic Zn2+ and increased
membrane-associated Zn2+, specifically in fractions that
were enriched in nuclei.
The increased fluorescence of zinquin observed in msc2 cells
also suggests an increased Zn2+ content. We expected that
the zinquin fluorescence would reflect not only the increased
intracellular Zn2+ but also the localization of the
Zn2+. The intensity of the zinquin fluorescence did
increase in msc2, but the dye was localized in small
punctate vesicles. Previously, we reported that overexpression of a
vacuolar Zn2+ transporter, Zrc1p, protected cells against
Zn2+ toxicity (6). Cells expressing Zrc1p showed an
increase in zinquin fluorescence. The fluorescence did not localize to
the vacuole but was again found in small punctate vesicles, as we observed in this study (data not shown). Our interpretation of these
results is that zinquin-bound Zn2+ is accumulated in
vesicles, regardless of the initial distribution of Zn2+.
Therefore, the vesicular localization of zinquin fluorescence may not
represent the normal distribution of Zn2+ and should be
interpreted with caution. There is a precedent for this interpretation
because in some mammalian cells, fluoresceine derivatives used
for either Ca2+ or pH measurements accumulate in small
vesicles. This accumulation does not reflect the native distribution of
either Ca 2+ or H+ but is due to accumulation
of the dye by a probenacid-inhibitable organic anion transporter (24).
We have not determined whether probenacid will inhibit the vesicular
accumulation of zinquin in yeast.
Measurement of the Zn2+ content of subcellular organelles
suggests that msc2 cells have a higher nuclear
Zn2+ content than wild type cells. This conclusion is
supported by the differential response of Zn2+-regulated
genes in msc2 cells compared with wild type cells. msc2 cells grown in glycerol-ethanol show decreased
expression of Zn2+-regulated genes, which can be overcome
by the constitutive allele of ZAP1
(ZAP1up). This result indicates that deletion
of MSC2 does not prevent Zn2+ from entering the
nucleus but rather reduces the egress of Zn2+. The function
of MSC2 appears analogous to the CDF family members MMT1/MMT2 because these genes regulate the distribution of
Fe2+ between mitochondria and cytosol.
Why does the altered distribution of Zn2+ result in a
growth deficit seen only in glycerol-ethanol at the elevated
temperature? Our data demonstrate that growth on glycerol-ethanol leads
to an increase in transition metal content, particularly in
Zn2+ content. It is thought that increased transition metal
content is a reflection of increased mitochondrial activity necessary for respiratory activity. A number of enzymes, even cytosolic enzymes,
that are necessary for respiratory activity, such as ALD2 or
ALD3, are Zn2+-requiring enzymes. Transcripts
for these enzymes are increased under stress or Zn2+
deprivation (23). It might be expected that under the dual conditions
of stress (due to increased temperature) and respiratory growth,
alterations in cellular Zn2+ metabolism would be
exacerbated. Under these conditions,
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Escherichia coli. A plasmid containing the YDR205W open reading frame and a 1000-base pair upstream region was subcloned into
low and high copy plasmids that contained URA3 as a
selectable marker. A Myc epitope was added to the carboxyl
terminus of YDR205W by PCR. The primers used were
5'-CCCCAAGCTTAGTAAAAGAGTATGTTGAGG-3' and
5'-GCTCTAGATTAATTCAAGTCCTCTTCAGAAATGAGCTTTTGCTCCATATTTGCTATAGGCTGTAGCGG-3'. A FLAG epitope construct was also generated by PCR using the
primers 5'-CCCCAAGCTTAGTAAAAGAGTATGTTGAGG-3' and
5'-AAGGAAAAAAGCGGCCGCTTATCACTTGTCATCGTCATCCTTGTAATCACCACCATTTGCTATAGGCTGTAGCGG-3'. The PCR products were ligated into both low copy and high copy vectors.
-Galactosidase Assay--
A Zn-responsive
-galactosidase
reporter construct containing the upstream sequence of the
ZRT1 gene (14) was a gift from Dr. David Eide
(University of Missouri, Columbia, MO). A Cu-sensitive
-galactosidase reporter construct containing the upstream sequence of CTR1 (15) was a gift from Dr. Dennis Winge
(University of Utah, Salt Lake City, UT). A Fe-sensitive
-galactosidase reporter construct was created from a PCR fragment
of the FET3 promoter region. The PCR primers used to
construct the fragment were 5'-TCCCCCGGGTGCCTTGGCTTGCCTATTTC-3' and
5'-AAAACTGCAGGCATCTAGTTCTAATTTTTTGCTACTCTT-3'. The PCR product was cloned into the SmaI-PstI sites of YEp354, a
LacZ expression vector (16). A plasmid containing the
Zap1up allele was a gift from Dr. Dennis Winge. All
plasmids were transformed into either wild type or msc2
cells. Cells were grown in glucose media or glycerol-ethanol
media with different concentrations of transition metals for 3 or 14 h before the assay.
-Galactosidase activity was assayed as
described previously (17).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
msc2 Strain--
A yeast strain with a
deletion in YDR205W (
msc2) was generated as described
under "Materials and Methods." The deletion strain, msc2, grew well in both YPD and CM at 30 °C and 37 °C.
However, the strain showed a temperature-sensitive growth defect in
YPGE media at 37 °C (Fig.
1A). The deletion strain also
showed an increased sensitivity to H2O2, which
was exacerbated by growth on glycerol-ethanol medium (Fig.
1B). Both the growth defect and the increased sensitivity to
H2O2 were recessive traits because they were
not expressed in the heterozygotic diploid. Microscopic examination of
msc2 cells grown in YPGE at 30 °C revealed that a small
percentage of the population had an abnormal morphology, typified by
the presence of large cells containing giant granules. The majority of
msc2 cells showed the abnormal phenotype upon a shift in
temperature to 37 °C (Fig. 2).
Transformation of msc2 cells with either high or low copy
plasmids containing MSC2 restored both normal growth and
morphology. Increasing the concentration of Zn2+ in the
medium restored normal morphology to msc2 cells. Increased media Zn2+ attenuated the growth defect of msc2
cells (Fig. 3A, a) but did not
restore a normal growth rate (Fig. 3B). Supplementation of YPEG with other transition metals, such as Fe2+ (Fig.
3A, b), Cu2+ (Fig. 3A, c), or
Mn2+ (Fig. 3A, d) had no effect on the growth
defect or morphological abnormality of msc2 cells at
37 °C (data not shown).
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Fig. 1.
Growth of wild type and msc2 cells in YPGE
media and in media supplemented with H2O2.
Cells were spotted on YPGE plates and incubated at 30 °C or 37 °C
for 3 days. DY1640 is a wild type diploid, DY1640/msc2 is a
heterozygote, and 1a, 1b, 1c, and 1d are haploid spores. 1a and 1c are
the cells carrying the deletion of MSC2 as confirmed by
genetic and PCR analysis (A). Similar dilutions of cells
were plated on YPGE plates that contained different concentrations of
H2O2 (B).
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Fig. 2.
Morphology of wild type and msc2 cells grown
in glycerol-ethanol medium. Cells grown in YPD medium were
transferred to YPGE medium and incubated at 37 °C for 12 h.
Wild type cells (A) and msc2 cells (B)
were examined by phase-contrast microscopy and photographed.
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Fig. 3.
Effect of extracellular Zn2+
on the growth of msc2 cells grown at
37 oC. Wild type, msc2
cells, or msc2 cells transformed with high or low copy plasmids
containing MSC2 were spotted on YPGE plates that contained
different amounts of the specified transition metals. The cells were
grown at 37 °C for 3 days and then photographed (A). Wild
type cells and msc2 cells were grown in YPGE media with and
without 5 mM Zn2+ at 37 °C. At selected
times, samples were taken, and the optical density was measured at a
wavelength of 600 nm (B).
msc2--
The deletion phenotype suggests that MSC2
affects intracellular Zn2+ homeostasis. To test this
hypothesis, we measured the transition metal content of wild type and
msc2 cells grown in both glucose-containing and
glycerol-ethanol-containing media. Cells grown at 30 °C in glycerol-
ethanol media had a higher content of Fe2+,
Cu2+, and Zn2+ than did cells grown in
glucose-containing media (Table
I). Although the absolute content
of metals differed in each experiment, the relative difference between
metals was constant. Of the three transition metals assayed, growth in
glycerol-ethanol had a greater affect on Zn2+ than it did
on Fe2+ or Cu2+. The msc2 strain
grown in either glucose or glycerol-ethanol had similar levels of
Fe2+ and Cu2+ when compared with the wild type
parent. However, when grown in glycerol-ethanol, msc2 cells
had a 50% increase in intracellular Zn2+ compared with
wild type cells. Increases in the Zn2+ content of
msc2 cells were seen at several different concentrations of
media Zn2+ (data not shown). Zn2+ content was
not uniformly increased in all msc2 organelles. A membrane
fraction had a higher Zn2+ content than did the cytosol
(Fig. 4). Of all membrane fractions examined, nuclei-enriched fractions had the highest Zn2+
content. A more extensive subcellular fractionation was precluded by
the loss of Zn2+ from cellular organelles.
Metal content of cells grown in CM or CMGE media
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Fig. 4.
Distribution of Zn2+
in subcellular fractions obtained from wild type and
msc2 cells. Wild type and msc2 cells grown in YPD
medium were transferred to YPGE medium and incubated at 37 °C for
12 h. The cells were homogenized, and subcellular fractions were
obtained as described under "Materials and Methods." Each fraction
was analyzed for protein and Zn2+ content.
msc2 cells also show a robust zinquin
fluorescence with the signal concentrated in punctate vesicles (Fig.
5). These data support the observation
that cellular Zn2+ levels are altered in the
msc2 strain. As discussed below, we feel that the
vesicular location of the zinquin fluorescence does not reflect the
normal distribution of Zn2+.
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Fig. 5.
Zinquin fluorescence of wild type and msc2
cells grown in glycerol-ethanol medium. Cells grown in YPD
medium were transferred to YPGE medium. After 12 h of growth at
37 °C, the cells were incubated in the same medium with 25 µM zinquin for 30 min and then examined by fluorescence
microscopy.
msc2
Strain--
We took advantage of the ability of Zap1p to sense
intracellular Zn2+ levels to further demonstrate that
msc2 cells show an alteration in Zn2+
homeostasis. Wild type and msc2 cells were transformed with
a plasmid carrying a Zn2+-regulated
-galactosidase
reporter construct derived from the promoter elements of
ZRT1. As expected, incubation of cells in high
Zn2+ medium resulted in decreased transcription of the
Zn2+ regulon and low levels of
-galactosidase activity
(Fig. 6). Conversely, incubation of wild
type cells in low Zn2+ media resulted in increased
expression of the Zn2+ regulon and increased levels of
-galactosidase. However, incubation of msc2 cells in low
Zn2+ media did not result in increased
-galactosidase
activity, suggesting that Zap1p may be occupied by Zn2+ and
thus transcriptionally inactive. The effect of the MSC2 gene deletion on Zn2+-dependent
-galactosidase
activity was seen only when cells were grown in glycerol-ethanol
medium. No effect was seen in cells grown in glucose- or
galactose-containing media. Alteration in intracellular metal
homeostasis was selective for Zn2+ because reporter
constructs designed to measure cytosolic levels of Cu2+
or Fe2+ showed no alteration in
-galactosidase activity
in cells grown in either glucose or glycerol-ethanol. This result is
consistent with the determination of metal concentrations in wild type
and msc2 cells (Table I), which demonstrated that only
Zn2+ was affected by deletion of MSC2.
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Fig. 6.
Use of
-galactosidase reporter constructs to assay metal
concentration in wild type and msc2 cells. Wild type and
msc2 cells were transformed with plasmids that contained
-galactosidase reporter construct preceded by the upstream
fragment derived from the Fe2+-dependent
promoter of FET3, the Cu2+-dependent
promoter of CTR1, or the
Zn2+-dependent promoter of ZRT1.
Cells were grown in CM or CMGE media with different
concentrations of iron (low, 160 µM BPS; medium, 10 µM FeSO4; high, 100 µM
FeSO4) copper (low, 30 µM BCS; medium,
10 µM CuSO4; high, 200 µM Cu
SO4), or Zn2+ (low, no added Zn2+;
medium, 10 µM ZnSO4; high, 500 µM ZnSO4) for 12 h at 30 °C. The
cells were homogenized, and samples were taken for determination of
protein concentration and
-galactosidase activity.
-galactosidase activity. Similar results were
obtained measuring ZRT1 transcript levels. These results
suggest that deletion of MSC2 alters the transcription of
Zn2+-responsive genes by affecting cellular
Zn2+ levels.
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Fig. 7.
Use of
-galactosidase reporter constructs and S1 analysis
to examine the effect of msc2 and ZAP1up
on expression of
Zn2+-regulated genes. Wild type
and msc2 cells were transformed with a zinc-responsive
element-
-galactosidase fusion construct and either a
ZAP1up plasmid or a control plasmid. The cells were
transferred from CM medium into CMGE medium. After a 3- or 14-h
incubation at 30 °C, cells were homogenized, and samples were taken
for determination of protein and
-galactosidase activity. RNA was
also isolated and used for S1 analysis using either probes against
ZRT1 or CMD1 (calmodulin). The ratio of
CMD1/ZRT1 was determined by PhosphorImager analysis.
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Fig. 8.
Localization and Western analysis of
epitope-tagged Msc2p. Cells (msc2) were transformed
with a high copy plasmid containing a FLAG-tagged construct of Msc2p.
The cells were then examined by Western analysis (A).
Cells (msc2) were transformed with a high copy plasmid
containing a Myc-tagged construct of Msc2p and then examined by
immunofluorescence microscopy as described under "Materials and
Methods." B, cells as viewed by both Normaski and
fluorescent optics. C, two different cells stained
with 4',6-diamidino-2-phenylindole, stained for the Myc-Msc2p, and the
merge of the two.
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
msc2 cells show
increased nuclear zinc, depressing the transcription of
Zn2+-regulated enzymes, and decreased cytosolic
Zn2+, decreasing the activity of Zn2+-requiring
enzymes. Supplementation of media Zn2+ results in an
increase in cellular zinc levels, decreasing transcription of the
Zn2+ regulon while, at the same time, increasing the
absolute concentration of Zn2+ in the cytosol. The
increased cytosolic Zn2+ may populate
Zn2+-requiring proteins. It is known that transition metals
must gain access to the nucleus, both as structural components
(Zn2+ fingers) and as regulatory components (metal-sensing
transcription factors). Zn2+, as a small molecule, would be
expected to diffuse freely between cytosol and nucleus without the need
for a transporter to effect either entry or exit. Our data suggest that
under the dual conditions of increased temperature and respiratory
growth, movement between the nucleus and cytosol becomes rate-limiting,
requiring the participation of a transporter. Based on sequence
homology, we assume that Msc2p is a Zn2+ transporter. The
possibility exists that Msc2p may transport Zn2+
chelates, which become rate-limiting under stress conditions. Whereas further experiments are required to determine the chemical nature of the transported molecule, our data do demonstrate that Msc2p
is a zinc transporter that is localized to an internal membrane and can
effect alterations in cellular Zn2+ homeostasis.
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ACKNOWLEDGEMENTS |
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We thank our colleagues in the Utah Metals group for their help in preparing the manuscript and Drs. J. Gitscher and R. Palmiter for sharing information regarding gene sequences.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health (NIDDK-DK30534 and DK52380) with support for core facilities from a National Institutes of Health National Cancer Institute Cancer Center support grant.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. E-mail:
Kaplan@bioscience.biology.utah.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M008969200
2 L. Li and J. Kaplan, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: CDF, cation diffusion facilitator; YPD, enriched growth media with glucose (1% yeast extract, 2.0% peptone, and 2.0% glucose); YPGE, growth media with glycerol-ethanol (1.0% yeast extract, 2.0% peptone, 2.0% ethanol, and 2.0% glycerol); CM, synthetic medium with glucose; CMGE, synthetic medium with glycerol-ethanol; PCR, polymerase chain reaction.
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