(Received for publication, March 25, 1997)
From the Department of Biological Chemistry, The University of Michigan, Medical School, Ann Arbor, Michigan 48109-0606
Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.
Copper is an essential trace element that is required for a number
of cellular enzymes including cytochrome c oxidase,
Cu/Zn-superoxide dismutase, lysyl oxidase, and
dopamine--monooxygenase (1). Copper also plays a critical role in
the assimilation of iron both in microbial and mammalian systems (2,
3). However, when allowed to accumulate in excess, copper is highly
toxic due to its proclivity to engage in redox reactions which result
in the formation of hydroxyl radical, a reactive species that causes extensive damage to nucleic acids, proteins, and lipids (4, 5).
Furthermore, copper may also be toxic through inappropriate incorporation into proteins, such as the estrogen receptor, which normally bind other metal ligands (6). Therefore, all organisms must be
able to sense both toxic and nutritional levels of copper to allow
sufficient copper to accumulate to drive biochemical reactions, yet
prevent the accumulation to toxic levels. Indeed, the failure to
appropriately establish and maintain copper homeostasis results in at
least two human genetic disorders, Wilson's disease and Menkes disease
(1, 7, 8). A number of cellular regulatory responses that result from
fluctuations in environmental copper levels have been reported
including transcriptional activation or repression, changes in protein
stability, and the modulation of protein trafficking (9-13).
Yeast cells have provided an excellent model system for studies of copper transport, distribution, and detoxification (5, 8, 14). In response to high concentrations of copper, yeast cells activate the transcription of the CUP1 (15-17) and CRS5 (18) genes, which encode copper-sequestering proteins called metallothioneins, as well as the SOD1 gene, encoding Cu/Zn-superoxide dismutase (19). This transcriptional activation involves the copper metalloregulatory transcription factors (MRTFs)1 Ace1p (20) and Amt1p (21) from the bakers' yeast Saccharomyces cerevisiae and the opportunistic pathogenic yeast Candida glabrata, as well as cis-acting promoter regulatory sequences with the consensus sequence HTHXXGCTG (H = A, C or T; X = any residue). The binding of Cu(I) to Ace1p or Amt1p to form a tetra-copper cluster activates their DNA-binding domains via a conformational change, thereby providing a direct link between the toxic copper sensor and the activation of detoxification genes (22, 23).
S. cerevisiae cells acquire copper as Cu(I) under high affinity conditions through the action of a plasma membrane-associated Cu(II)-Fe(III) reductase activity encoded by the FRE1 gene (24, 25) and two high affinity copper transport proteins encoded by the CTR1 (26) and CTR3 (27) genes. Indeed, cells that are defective in high affinity copper transport exhibit a number of phenotypes which can be corrected by exogenous copper that include respiratory deficiency, sensitivity to superoxide generating agents due to a defect in Cu/Zn-superoxide dismutase activity, and severely diminished iron accumulation due to a defective copper-dependent ferroxidase (Fet3) required for high affinity iron transport (28, 29). As would be expected, the genes encoding proteins involved in high affinity copper or iron transport are repressed, at the level of steady-state mRNA, by low concentrations of their respective metals and induced by copper or iron starvation, respectively (10, 24, 27). Furthermore, consistent with the involvement of the FRE1-encoded Cu(II)-Fe(III) reductase in both high affinity copper and iron transport, the expression of this gene is repressed by both copper and iron (24, 25). Insight into the mechanism for iron-dependent transcriptional repression in yeast has been gained by the recent identification and analysis of the iron-responsive DNA-binding protein Aft1 (30), which binds to the consensus sequence PyPuCACCCPu found in the promoters of the FET3, FTR1, FTH1, CCC2, FRE1, and FRE2 genes (31).
Currently, it is unclear how yeast cells sense very low concentrations
of copper and respond by repressing the expression of the
FRE1, CTR1, and CTR3 genes. One
protein implicated in this signaling pathway, Mac1, was discovered
based on significant homology to the amino-terminal 40 amino acid
residues of the copper-activated DNA-binding domains of the Ace1 and
Amt1 proteins (32). Interestingly, mac1 cells exhibit
phenotypes consistent with defective copper transport including
respiratory deficiency, defective Cu(II) and Fe(III) reductase
activity, impaired regulation of FRE1
transcription, hypersensitivity to a variety of stresses including
metal and oxidative stress, and poor growth on rich medium (25, 32). Furthermore, a dominant mutation in a duplicated region rich in cysteine exhibits high levels of Cu(II) reductase activity and FRE1 mRNA that are virtually not repressed by copper
(25). These characteristics of Mac1p, as well as the nuclear
localization of a Mac1-
-galactosidase fusion protein (32), suggests
that Mac1 plays a key role in copper homeostasis.
In this report, we demonstrate that the regulation of FRE1, CTR1, and CTR3 transcription is exquisitely copper-sensitive and specific. We demonstrate that these three genes share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this copper-responsive occupation of the CuREs requires a functional MAC1 gene. Taken together these results show that yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway to acquire this micronutrient from the environment.
Stock solutions at 10-100 mM CuSO4 (100.2%), FeCl2 (99.0%), NH4Fe(SO4)2 (100.4%), CoCl2 (99.0%), CaCl2 (99.0%), MnCl2 (99.0%), (C2H3O2)Pb (99.3%), AgNO3 (99.9%), CdCl2 (99.9%), ZnCl2 (99.9%), NiSO4 (99.9%), MgSO4 (99.9%), HgCl2 (99.9%), NaO2CCH2CH(SAu)CO2Na, AuCl3 (99.9%), and BCS (Aldrich) were freshly prepared. Solutions of AgNO3, HgCl2, CdCl2, ZnCl2, FeCl2, and NH4Fe(SO4)2 were analyzed by atomic absorption spectroscopy using a Perkin-Elmer PEC 3000 spectrophotometer and found to contain no detectable copper ion contamination.
Yeast Strains and Cell GrowthYeast strains used in this
study are listed in Table I. The
ura3::KANr allele was constructed by
deleting 391 bp of the URA3 open reading frame (ORF) and
replacing it with the KANr marker (34). The
mac1::URA3 allele was constructed using the disruption plasmid pmac1::URA3 (generously provided by
Daniel Kosman). This disruption rendered the MAC1
gene nonfunctional as ascertained by the lack of growth on YPE media.
Furthermore, the allele status of each of these loci in the strains
generated in this study was verified using diagnostic polymerase chain
reaction (35).
|
Yeast cells were grown in a modified minimal medium (SD), which was depleted for copper as described previously (10). This depleted medium contains 16 nM copper as determined by atomic absorption spectroscopy while standard synthetic complete media contains 150 nM copper. Copper administration or copper starvation of yeast strains were carried out by adding the indicated amount of CuSO4 or bathocuproinedisulfonate (BCS) to cells grown to mid-logarithmic phase (optical density at 600 nm = 1.1 to 1.3) in this modified SD medium. Under nonselective conditions, yeast cells were grown in YPD (1% yeast extract, 2% bactopeptone, and 2% dextrose).
Plasmids and Gene Analyses MethodsPlasmid
YEp357RTRP1 was constructed by insertion of a blunt-ended
1.7-kilobase TRP1 fragment isolated from p330 (gift of
Robert Fuller) into the StuI and Klenow filled-in
NcoI sites of YEp357R, disrupting the URA3
genetic marker. The oligonucleotides CTR3-A (5-CTCGCGGATCCAGTCATAGCATGAACAATTC-3
) and CTR3-B
(5
-GTCCGGAATTCGAAGCAGTGCTGCTACTGCCTCC-3
) were used to polymerase
chain reaction amplify the CTR3 promoter (1116 bp of the
5
-noncoding region) and the first 10 codons of CTR3 gene
from genomic DNA of strain DTY1, which expresses both the Ctr1p and
Ctr3p copper transporters. The polymerase chain reaction product was
sequenced in its entirety and fused in-frame to the Escherichia
coli lacZ gene using the BamHI and EcoRI
sites of YEp357RTRP1 to generate YEpCTR3-lacZ. A
low-copy number plasmid pRSCTR3-lacZ was also constructed.
To accomplish this construct, a 4.8-kilobase DNA fragment from plasmid
YEpCTR3-lacZ containing the CTR3 sequence from
1116 to +10 fused to the E. coli lacZ gene was inserted
into the BamHI and SmaI sites of pRS314. To perform the RNase protection analyses as described previously (36), two
plasmids for making antisense RNA probes were made. pKSlacZ
was constructed by the insertion of the 233-bp
EcoRV-BclI fragment from the E. coli
lacZ gene into the EcoRV and BamHI sites of
pBluescript II KS. pKSACT1 was made by the insertion of the 132-bp HindIII-EcoRI fragment from the S. cerevisiae ACT1 gene into the HindIII-EcoRI
sites of pBluescript II KS. A series of plasmids containing sequential
deletions from the 5
end of the CTR3 promoter (Fig.
3B) were created from plasmid YEpCTR3-lacZ using
standard protocols (Exo III/Mung Bean Nuclease Deletion Kit,
Stratagene). To assess the capability of the CuREs to mediate copper
repression and copper starvation induction, a series of purified
oligonucleotides (Figs. 4C and 5C) were annealed
pairwise to form double-stranded DNA and then ligated into the
BglII and XhoI sites of a CYC1-lacZ
fusion plasmid pCM64 (generously provided by Charles Moehle).
RNA was extracted by the hot phenol method as described previously (37). Northern blot analyses were carried out by standard protocols (38). The radioactive bands corresponding to specific transcripts were quantitated using a PhosphorImager SP and ImageQuant 3.3 software (Molecular Dynamics). The data derived from the PhosphorImage quantitation were plotted and analyzed using Kaleidagraph software 3.02 (Synergy Software, Reading, PA). DNA isolation and polymerase chain reaction were performed using standard protocols (38, 35). DNA sequencing was carried out using Sequenase according to the manufacturer's protocol (U. S. Biochemical Corp.).
In Vivo Dimethyl Sulfate FootprintingCultures of the
isogenic strains DTY1 (MAC1), DTY205
(MAC1up1), or SLY2 (mac1) containing
YEpCTR3-lacZ were grown to early log-phase in modified SD
media. Untreated and copper-treated (10 nM) cultures were
incubated for 1 h at 30 °C, 375 rpm. In vivo
footprinting was carried out as described previously (39). Isolated DNA
samples were digested by BstYI prior to G + A-specific DNA
cleavage of the dimethyl sulfate-treated DNA. The purified
oligonucleotide used as primer in the extension reactions was CTR3E
(5
-GCCTCCCATATTCATCTTTGTATAGCCC-3
), which hybridizes to
CTR3 gene positions +15 to
14 with respect to A of the
translational start codon.
Previous investigations have demonstrated that
all three yeast genes known to be involved in high affinity copper
transport, CTR1, CTR3, and FRE1, are
repressed by copper at the level of steady-state mRNA (10, 27).
This regulation is independent of the S. cerevisiae copper
MRTF Ace1p, which activates the CUP1, CRS5, and
SOD1 genes in response to micromolar copper concentrations (10).2 To characterize in detail the
regulation of CTR3 by metals, the low-copy number plasmid
pRSCTR3-lacZ was transformed into DTY1, a strain that
expresses both high affinity copper transporters Ctr1p and Ctr3p, and
the level of lacZ steady-state mRNA was assayed by RNase
protection experiments. Among the metal ions tested at many
concentrations, Cu(II), Fe(II), Fe(III), Ag(I), Cd(II), Zn(II), Hg(II),
Pb(II), Co(II), Mn(II), Ni(II), Mg(II), Ca(II), Au(I), and Au(III),
only four were capable of repressing expression of lacZ
mRNA under the control of the CTR3 promoter: copper,
silver, cadmium, and mercury (Fig. 1). Moreover by using
a range of concentrations for each of these four metal ions,
CuSO4 and AgNO3, were most effective for the
repression of CTR3-lacZ transcription. Only 2.0 × 1011 M and 1.4 × 10
11
M CuSO4 and AgNO3, respectively,
was required for half-maximal repression (repression index 50%
(RI50)) of expression from the CTR3 promoter. In
contrast, CdCl2 and HgCl2 repress transcription from the CTR3 promoter, but at concentrations 3 orders of
magnitude greater than for CuSO4 and AgNO3. The
RI50 for CdCl2 and HgCl2 were
4.0 × 10
8 M and 1.7 × 10
8 M, respectively. Although we cannot
eliminate the possibility that the CdCl2 and
HgCl2 solutions contain trace levels of copper, atomic
absorption spectroscopy failed to detect any copper in the
CdCl2 and HgCl2 stock solutions. Therefore, the
repression of CTR3 gene expression exhibits a high degree of
selectivity for copper ions. Moreover, the observation that Ag(I), a
metal which is electronically similar to the reduced form of cupric Cu(II), represses CTR3 gene expression with an efficacy
similar to CuSO4, suggests that cuprous Cu(I) might be the
active form of copper in the signaling process resulting in repressing
CTR3 gene expression.
Time Course of Repression and Activation of CTR3 Gene Expression
Because yeast cells alter the expression of the copper
transport machinery as a function of changing environmental copper levels, we analyzed CTR3-lacZ steady-state mRNA levels
over time in response to either copper repletion or starvation. Using
the low-copy number plasmid pRSCTR3-lacZ in strain DTY1, we
followed the time course of down- and up-regulation of CTR3
gene expression in the presence of copper (1 µM) or BCS
(100 µM), respectively. The derepression of
CTR3-lacZ gene expression is rapid with 91% of the maximal
level of transcript detected 10 min after treatment with BCS (Fig.
2). On the other hand, 79% of the CTR3-lacZ
transcript levels remained detectable after a 10-min exposure to
copper. The time course data observed using the low-copy number plasmid pRSCTR3-lacZ were virtually identical to those observed for
the endogenous CTR3 gene.3 These
data demonstrate that yeast cells respond to changes in environmental
copper levels by rapidly altering steady-state levels of the
CTR3 copper transport mRNA. The temporal differences
observed between derepression and repression of the
CTR3-lacZ reporter gene expression may be due to changes in
the stability of the mRNA or may reflect unidentified differences
in the repression versus the derepression signaling
pathways.
Identification of cis-Acting Elements Necessary for Copper Repression and Copper-starvation Activation of CTR3, CTR1, and FRE1 Gene Expression
To identify cis-acting elements
necessary for the copper repression and copper-starvation-mediated
induction of CTR3 expression, we fused 1116 bp of the
5-noncoding region and the first 10 codons of CTR3 in-frame
with the E. coli lacZ gene on both low- and high-copy number
plasmids pRSCTR3-lacZ and YEpCTR3-lacZ,
respectively. CTR3-lacZ expression from both reporter
plasmids was down-regulated in the presence of copper (approximately
5-fold), and up-regulated in the presence of BCS (approximately 8-fold)
(Fig. 3 and data not shown). To identify the
copper-responsive CTR3 promoter elements, a series of nested
5
deletions of promoter sequences beginning at position
1116 were
created in the high-copy number plasmid YEpCTR3-lacZ (Fig.
3). Removal of the CTR3 upstream region between
1116 and
248 had little effect on the copper and BCS-regulated expression of
the CTR3-lacZ fusion, although the magnitude of the response
decreased progressively. When the CTR3 promoter was further
deleted to position
214, the overall magnitude of the response
decreased by approximately 75% as compared with the parental plasmid,
but the reporter gene was still regulated in response to copper levels.
Further deletion to position
160 was found to completely abolish both
the down- and up-regulation of the reporter gene (Fig. 3). Due to the
observation that the integrity of the region between positions
248
and
160 was essential for driving copper repression and
copper-starvation activation of the CTR3-lacZ fusion, we
examined whether this sequence could regulate a heterologous reporter
gene in a copper-dependent fashion. A double-stranded DNA
oligomer derived from the CTR3 promoter (positions
237 to
173) was inserted in its natural orientation into the minimal
promoter of the iso-1-cytochrome c (CYC1) gene fused to lacZ (40). This fusion promoter was able to
down-regulate (approximately 5-fold) and up-regulate (approximately
9-fold) lacZ mRNA expression in the presence of copper
or BCS, respectively (Fig. 4). Within this 64-bp
CTR3 promoter DNA fragment, we noted the presence of two
copies of a repeated sequence, TTTGCTC, that are similar to the binding
sites for the Ace1 and Amt1 copper MRTFs. To ascertain if this element
plays a role in CTR3 regulation by copper, all seven of
these residues were mutationally altered in either or both repeats and
the cells carrying CTR3-CYC1-lacZ fusion plasmids were
assayed for copper-regulated expression of lacZ mRNA
(Fig. 4C). Multiple point mutations in either or both elements abolished copper responsiveness of the
CTR3-CYC1-lacZ fusions (Fig. 4). Moreover, a
CTR3-CYC1-lacZ promoter fusion plasmid in which the last C
in each TTTGCTC element was changed to G, failed to respond to the
presence of a wide range of copper concentrations or copper-starvation
to repress or activate gene expression (Fig. 4 and data not shown). Our
data do not allow us to establish the reason why a single CuRE cannot
regulate the CTR3-CYC1-lacZ fusion derivative, but does
regulate with a low magnitude a 5
-truncated CTR3
promoter-lacZ derivative which has one element (Fig. 3).
Interestingly, the TTTGCTC element was also observed to be perfectly conserved and repeated in the CTR1 and FRE1 promoters, both of which drive the expression of proteins involved in high affinity copper transport. As shown in Fig. 5, fusion of the regions from the CTR1 or FRE1 promoters encompassing these TTTGCTC repeats mediated copper repression and copper-starvation activation of lacZ mRNA expression. For both the CTR1-CYC1-lacZ and FRE1-CYC1-lacZ promoter fusions, the integrity of the TTTGCTC repeats was essential for copper-responsive gene expression (Fig. 5). Therefore, based upon these studies the TTTGCTC element, denoted CuRE, plays a critical role in copper-regulated gene expression for all three yeast genes encoding components of the high affinity copper transport machinery. Furthermore, the CuREs in CTR1, CTR3, and FRE1 function in both copper-repression and copper-starvation-mediated gene expression.
Yeast High Affinity Copper Transport Genes Require MAC1 for Expression and Regulation by CopperThe repression of
CTR3 gene expression is highly specific for copper and
occurs at exquisitely low extracellular copper concentrations. Since
CTR1 and CTR3 gene repression by copper is
independent of the Ace1p copper MRTF (Ref. 10, and data not shown), we
sought to identify components of the copper-signaling pathway that play a key role in the copper-regulated expression of the high affinity copper transport genes. The S. cerevisiae MAC1 gene encodes
a nuclear protein with a high level of homology to the first 40 amino
acids of the Ace1 and Amt1 copper MRTFs. A hallmark of
mac1 mutants is that virtually all of the pleiotropic
phenotypes associated with these cells can be overcome by exogenous
copper, thereby clearly implicating Mac1p in copper metabolism (32).
Using isogenic strains harboring a wild type MAC1 gene, an
insertionally inactivated mac1 allele (mac1
),
and a dominant gain-of-function allele (MAC1up1), we
ascertained whether MAC1 plays an essential role in
CTR1, CTR3, and FRE1 gene regulation
as a function of cellular copper status by Northern blot analysis (Fig.
6). In the wild type strain (DTY1), basal levels of
CTR1, CTR3, and FRE1 mRNA are
clearly visible. However, in the presence of 1 or 10 µM
copper the steady-state levels of CTR1, CTR3, and
FRE1 mRNA were strongly repressed. Conversely, in the
presence of 100 µM BCS, CTR1, CTR3, and
FRE1 mRNA levels were induced 3-, 14-, and 6-fold over
basal levels, respectively. No mRNA was detected for
CTR1 and CTR3 in the mac1
mutant
strain (SLY2) even under conditions of copper starvation, although a low level of FRE1 mRNA was still observed, perhaps due
to the action of Aft1p (30). In the MAC1up1 strain
(DTY205), all three genes were highly expressed (8-fold for
CTR1, 37-fold for CTR3, and 8-fold for
FRE1 with respect to the basal level detected in DTY1), and
were virtually unregulated by copper or copper-starvation. Taken
together, these data demonstrate that Mac1p is an essential
trans-acting component of the copper signaling pathway for
appropriate expression and regulation of genes involved in high
affinity copper transport.
The CuRE Is Differentially Occupied under Copper Deprivation and Repletion
Although Mac1p exhibits homology to the Ace1p and Amt1p
copper-activated DNA-binding proteins, we have been unable to
demonstrate CuRE DNA binding activity for Mac1p expressed in E. coli or by in vitro transcription translation. To begin
to understand the molecular responses to copper mediated by the CuREs,
and the function of Mac1p in this pathway, we have examined the
CTR3 promoter in cells starved for copper or grown in the
presence of copper by in vivo dimethyl sulfate footprinting.
In the wild type strain (DTY1) under copper deprivation conditions that
parallel induced CTR3 gene expression, we observed that a
region encompassing and flanking the two CuREs located in the
CTR3 promoter is strongly protected from methylation by
dimethyl sulfate relative to copper-treated cells in which
CTR3 gene expression is repressed (Fig. 7).
Upon copper treatment of the wild type strain with 10 nM
CuSO4, we observed that all of the G residues encompassing
and immediately flanking the CuREs were strongly modified by dimethyl
sulfate. Specifically, the G residues corresponding to positions 182
and
184 inside of the downstream TTTGCTC
element (oriented in the opposite direction relative to the direction
of the transcription), and the G located at position
228 inside of
the upstream TTTGCTC element were strongly protected from
dimethyl sulfate modification. The protection detected for the G
residue at position
184 correlates with the critical requirement for
this G for copper-responsive gene regulation in
CTR3-CYC1-lacZ fusion gene (see construct M4, Fig. 4). Furthermore, each G residue between the two CuREs, plus two
additional G residues (at positions
172 and
165) located downstream
of the repeat were found to be protected from dimethyl sulfate under
conditions in which CTR3 is expressed, but not when CTR3 expression is repressed, suggesting the presence of one
or more proteins occupying this region under condition of expression. Furthermore, a similar pattern as the one described for the wild type
strain under copper deprivation conditions was observed when this
strain was grown in the presence of BCS (data not shown). Consistent
with the constitutive high level CTR3 gene expression observed in the MAC1up1 strain, a constitutive
protected pattern in the CTR3 promoter, comparable to that
found for the wild type strain (MAC1) under copper
deprivation conditions, was revealed by dimethyl sulfate footprinting.
Conversely, the CTR3 promoter region in the isogenic mac1
strain revealed no copper-dependent
changes in dimethyl sulfate reactivity and strongly resembled the
copper-repressed promoter configuration in the wild type strain (Fig.
7). Therefore, a functional MAC1 gene is required for
copper-dependent changes in the CTR3 promoter
region encompassing and flanking the CuREs.
Because of its essential yet toxic nature, all cells must maintain
tight homeostatic regulation of the levels of bioavailable copper.
Regulatory responses to copper include the activation or repression of
gene transcription, copper-modulated turnover of proteins that
transport or utilize copper, and alterations in the intracellular
trafficking of copper transporting ATPases in response to fluctuations
in copper concentrations (9-13, 27). In this report, we have
characterized the molecular mechanisms responsible for the
copper-responsive regulation of CTR3, CTR1, and
FRE1 genes that play a critical role in high affinity copper transport in yeast. Detailed studies of CTR3 gene regulation
have revealed that half-maximal repression of CTR3 mRNA
levels occurs in the presence of as little as 2.0 × 1011 M CuSO4 or 1.4 × 10
11 M AgNO3 (Fig. 1). This
represents an exquisitely sensitive and selective metal responsive
system since at least 1000-fold higher concentrations of cadmium or
mercury were required to achieve the same level of repression and no
other metal tested was able to repress CTR3 mRNA levels.
Since repression is observed with cadmium and mercury, however, we
cannot at present eliminate the possibilities that either trace levels
of copper, undetectable by atomic absorption, were present in our stock
solutions or that there are significant differences in the efficiency
with which copper and silver, versus cadmium and mercury,
are transported or distributed in yeast cells. The similarity in the
potency of silver in fostering the repression of CTR3
mRNA levels resembles previous results on the Ace1 and Amt1 copper
MRTFs, which are known to form tetragonal coordinates with Cu(I) via
cysteine thiolates (23). The electronic similarity of Ag(I) to Cu(I),
but not Cu(II), suggests that the copper sensing machinery involved in
the repression of yeast copper transport genes may sense Cu(I), rather
than Cu(II).
Our studies have identified strictly conserved repeated CuREs present
in two copies in each of the CTR1, CTR3, and
FRE1 promoters. Inspection of the CTR1 promoter
suggests the presence of a third CuRE, located between positions 529
and
523, however, the contribution of this element to
copper-responsive regulation of CTR1 has not yet been
determined. Since single or multiple point mutations within the CuREs
abolish both copper-dependent repression and copper-starvation-induced expression of CTR1,
CTR3, and FRE1, this suggests that yeast cells
utilize a common mechanism to coordinately regulate the expression of
the copper transport machinery. Examination of the CuRE sequences in
CTR1, CTR3, and FRE1, suggest a
consensus sequence WWWTTTGCTCR (W = A or T; R = purine). The
CuRE sequence bears an interesting sequence similarity to the binding
sites for the Ace1 and Amt1 copper MRTFs (HTHXXGCTG, H = A, C or T; X = any residue), however, the inability
to convert a CuRE to an Ace1p activation site by substituting the
terminal C residue for a G (Fig. 4, plasmid derivative M4)
suggests that other nucleotides within or flanking the CuRE confer
specificity for copper repression and copper-starvation induction of
gene expression which is independent of Ace1p (10).2
Although the center-to-center distances between the CuREs, for each of
the CTR1, CTR3, and FRE1 promoters
predict that they lie on opposite faces of the DNA, it is currently
unknown whether this geometry plays a role in the regulation of copper
transporter gene expression via the CuREs. The FRE1 promoter
fragment, which has the shortest distance between the two CuREs, gave
rise to the poorest repression by copper and the strongest activation by copper-starvation. Since this promoter fragment lacks binding sites
for the Aft1 iron-responsive regulatory protein (31), this supports the
notion that de-repression of the FRE1-CYC1-lacZ fusion gene
represents a response to copper starvation rather than an indirect
response to iron starvation. However, whether other promoter elements
contribute to the magnitude of these regulatory responses must await a
comprehensive dissection of each of the promoters of these genes
encoding proteins that function in high affinity copper transport.
It is currently unknown how such exquisitely low extracellular copper
concentrations are capable of signaling the repression of
CTR1, CTR3, and FRE1 gene expression.
Based on previous observations that S. cerevisiae cells
harboring a deletion of the MAC1 gene display pleiotropic
defects that are corrected by the addition of copper, and are defective
in the regulation of FRE1 transcription (25, 32), we tested
the possibility that MAC1 plays a role in the regulation of
all the genes known to be involved in high affinity copper transport in
yeast. Indeed, we have demonstrated that cells bearing a disruption of
the MAC1 gene are severely defective in the expression and
regulation of mRNA levels from each of these genes (Fig. 6).
Furthermore, isogenic cells harboring a dominant allele of
MAC1, in which a His residue in the first Cys-rich
carboxyl-terminal cluster repeat has been altered to Gln, fail to
respond to copper administration to repress CTR1, CTR3, or FRE1 mRNA levels. Although Mac1p
exhibits significant sequence similarity to the amino-terminal 40 amino
acids of Ace1p and Amt1p, and a Mac1--galactosidase fusion protein
has been localized to the yeast cell nucleus (32), we have been unable to demonstrate specific CuRE binding activity for Mac1p either expressed in E. coli cells or produced by in
vitro transcription and translation (data not shown). Therefore,
Mac1p may function in the copper-signaling pathway to regulate copper
transport gene expression through a number of potential mechanisms.
First, Mac1p may require specific post-translational modifications or
partner proteins for sequence-specific DNA binding that would not be
present through expression in heterologous systems. Alternatively,
Mac1p could be required for the synthesis or activity of another
protein(s) which directly interacts with CuREs, or Mac1p may play a
critical nuclear signaling role for copper that is upstream of a direct DNA binding activity. Consistent with all of these models,
electrophoretic mobility shift experiments using extracts from yeast
cells expressing a functional epitope-tagged MAC1 allele
have revealed the presence of a specific CuRE-protein complex from
control or BCS-treated cells which is absent in extracts from cells
treated with 10 nM CuSO4 (data not shown).
Furthermore, the formation of this CuRE-protein specific complex was
dependent on the presence of a functional MAC1 gene, and the
complex was not abolished by the addition of copper to a strain bearing
a MAC1up1 allele. On the other hand, the inability
of the anti-epitope monoclonal antibody to supershift this complex
leaves open the possibility that Mac1p may not directly contact DNA
sequences within the CuRE. Our in vivo footprinting studies
clearly demonstrate that in the wild type MAC1 strains the
CTR3 CuREs are occupied under conditions of active
CTR3 gene expression (i.e. copper-starvation), and are highly accessible to dimethyl sulfate modification under conditions in which CTR3 gene expression is repressed
(i.e. addition of 10 nM CuSO4). In
contrast, mac1
mutants give rise to constitutive dimethyl
sulfate modifications in the CTR3 promoter region
encompassing the CuREs that resemble the repressed state in the wild
type strain. Furthermore, MAC1up1 mutants, which
constitutively express the copper transport genes and are unresponsive
to copper for repression, give rise to constitutive protection from
dimethyl sulfate modification within and flanking the CTR3
CuREs. Taken together these results demonstrate that Mac1p is an
essential component of the copper-signaling pathway that directly or
indirectly modulates coordinated copper-responsive gene expression of
yeast high affinity copper transport genes through the CuREs.
We are grateful to David Engelke, Marj Peña, and Keith Koch for critical reading of the manuscript, Marj Peña for atomic absorption spectroscopy measurements, Keith Koch for advice about RNase protection assays and computer analyses, and our colleagues for valuable discussions during the course of this work and preparation of the manuscript. We thank Robert Fuller, Charles Moehle, and Daniel Kosman for the p330, pCM64, and pmac::URA3 plasmids, respectively.