From the Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada
Received for publication, December 13, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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Transcriptional regulation of genes encoding
critical components of copper transport is essential for copper
homeostasis and growth in yeast. Analysis of regulatory regions in the
promoter of the ctr4+ copper transporter gene
in fission yeast Schizosaccharomyces pombe reveals the
identity of a conserved copper-signaling
element (CuSE), which is recognized by the transcription
factor Cuf1. We demonstrate that CuSE is necessary for transcriptional
activation in response to copper deprivation conditions. Interestingly,
the CuSE element bears a strong sequence similarity to the recognition site, denoted MRE (metal regulatory element), which is recognized by a
distinct class of copper sensors required for copper detoxification, including Ace1 from Saccharomyces cerevisiae and Amt1 from
Candida glabrata. When a consensus MRE from S. cerevisiae is introduced into S. pombe, transcription
is induced by copper deprivation in a Cuf1-dependent
manner, similar to regulation by Mac1, the nuclear sensor for
regulating the expression of genes encoding components involved in
copper transport in S. cerevisiae. UV-cross-linking experiments show that the Cuf1 protein directly binds the CuSE. These
results demonstrate that the Cuf1 nutritional copper-sensing factor
possesses a module that functions similarly to domains found in the
Ace1/Amt1 class of metalloregulatory factors, which allows the protein
to act through a closely related MRE-like sequence to regulate copper
transport gene expression in S. pombe.
Aerobic life requires trace amounts of transition elements like
copper, which have the property to reversibly gain and lose electrons,
thereby serving as catalytic centers of numerous proteins involved in a
variety of critical enzymatic processes (1-3). Owing to its proclivity
to engage in redox reactions, excess copper results in the production
of detrimental hydroxyl radicals, which are extremely toxic to cells,
causing lipid peroxidation, protein denaturation, and nucleic acid
cleavage (4). Therefore, specialized proteins have evolved for the
sensing, transport, and sequestration of copper within cells to
maintain the delicate balance between essential and toxic levels
(5-7).
Three genes in the bakers' yeast Saccharomyces cerevisiae
are induced in response to copper excess: CUP1- and
CRS5-encoded metallothioneins and SOD1-encoded
Cu/Zn superoxide dismutase (8-10). Metallothioneins are known to
counteract metal cytotoxicity by sequestration of cytosolic copper (11,
12). Increased synthesis of these three proteins in response to copper
is controlled at the transcriptional level by the Ace1 copper
metalloregulatory transcription factor
(MRTF)1 (13-15).
Furthermore, the promoter element necessary for copper-inducible transcription of CUP1, CRS5, and SOD1
is denoted metal regulatory element (MRE) and is defined by the
consensus sequence, 5'-HTHNNGCTGD-3' (D = A, G, or T; H = A,
C, or T; N = any residue). The GCTG region is termed the core
sequence, whereas the region 5' to the core is known as the T-rich
element (16, 55). The core sequence of the MRE is recognized by Ace1 in
the major groove, whereas the AT-rich 5' region is contacted in the
minor groove (16, 17). The first subdomain (residues 1-40) (denoted
Zn2+ module) of the DNA binding domain of Ace1 was found to
bind a single Zn2+ atom (18). The second subdomain of the
DNA binding domain (residues 41-110), named copper-regulatory domain
(17), harbors 9 Cys residues found as follows: one
Cys-X2-Cys motif, three Cys-X-Cys motifs, and a single Cys residue. All of these Cys residues are necessary for copper response, except for the last,
Cys105 (17). The arrangement of the Cys residues is
predicted to coordinate four Cu1+ atoms through cysteine
sulfur bonds (19). Upon copper activation, it appears that the
formation of a tetra-copper cluster within the copper regulatory domain
via cysteinyl thiolates is critical to make an active detoxifying
factor, thereby making the cupro-Ace1 able to interact to MREs in a
copper and conformational changes-dependent manner (11,
17-20). The Candida glabrata ACE1 gene ortholog, AMT1, binds MREs with similar motifs and is essential for
copper resistance (21, 22).
A copper biochemical pathway distinct from the detoxification system
exists to allow S. cerevisiae cells to acquire trace amounts
of copper from the environment (2, 5, 23). Yeast genetic studies have
led to the identification of the FRE1 (24), CTR1
(25), and CTR3 (26) genes, which encode components involved in the high affinity copper uptake process. When grown under copper starvation conditions, the Fre1 plasma membrane protein reduces Cu2+ to Cu1+ (27), allowing copper ions to be
recognized and transported into the cell by two separate, high affinity
transport proteins, Ctr1 (28) and Ctr3 (29). A hallmark of these genes
is the fact that they are transcriptionally expressed according to
copper need; the transcription of FRE1, CTR1, and
CTR3 is up-regulated under copper starvation conditions.
This process is under the control of Mac1, another copper-sensing
transcription factor harboring homology with the amino-terminal 40 amino acids found in the Ace1/Amt1 copper-activated metalloregulatory
proteins but little homology outside of this region (30-32). The
carboxyl-terminal region of Mac1 harbors two Cys-rich repeats, REP-I
and REP-II (also identified as C1 and C2) (39-41). REP-I has been
demonstrated to function in copper sensing (31, 39, 41). The
copper-specific transcriptional regulation by Mac1 is mediated through
cis-acting promoter elements, denoted CuREs
(Cu-response elements) with the
consensus sequence 5'-TTTGC(T/G)C(A/G)-3' (32, 33). Under low copper
conditions, Mac1 binds to CuREs as a dimer (34, 35) by making contacts in both the major (with 5'-GC(T/C)C(A/G)-3' sequence) and minor (with
5'-TTT-3' sequence) grooves (36). Interestingly, it has been shown that
Cu1+ ions can bind directly to Mac1 (37). Under elevated
copper concentrations, Mac1 is released from the CuREs in
vivo (32), and it is suggested that it undergoes intramolecular
conformational changes to inactivate its transactivation domain (35,
37, 38).
The fission yeast Schizosaccharomyces pombe exhibits several
features, such as genome organization, transcription initiation, signal
transduction, post-translational modification, and cell division
processing, which are more similar to those from mammalian cells than
those of bakers' yeast S. cerevisiae (44). Recent studies
of copper homeostasis in S. pombe have identified a high affinity copper transporter, denoted Ctr4, that exhibits more overall
sequence homology to human Ctr1 than to S. cerevisiae Ctr1
(6, 45, 46). Likewise, the S. pombe high affinity copper
transporter gene ctr4+ is transcriptionally
regulated by copper availability via the Cuf1 copper-sensing
transcription factor (46). Cuf1 activates ctr4+
gene expression under copper starvation conditions (46). Therefore, both the Ctr4 copper transporter and the Cuf1 nuclear copper-sensing transcription factor are essential for fission yeast cells to use
copper ions from the environment. Although the Cuf1 protein is required
for S. pombe high affinity copper transport, Cuf1 displays
at its amino terminus an extended homology (amino acid residues 1-61)
to the amino-terminal 63 and 62 amino acids of the S. cerevisiae Ace1 and C. glabrata Amt1 class of
transcription factors and much less similarity to Mac1, its functional
ortholog (46). Cuf1 possesses a cysteine-rich domain at its carboxyl terminus containing five Cys and one His residue that is absent in
Ace1/Amt1 but found duplicated in both Mac1 and Grisea of
Podospora anserina (43, 46). With respect to copper
detoxification in S. pombe, the only known molecules for
sequestering excess of copper ions are the phytochelatins, since the
fission yeast lacks metallothioneins (42). No precise molecular
mechanism of how copper sequestration is regulated in S. pombe has yet been identified.
In this study, we identify a cis-acting element found
upstream of the copper transport genes in S. pombe termed
copper-signaling element (CuSE)
that is necessary for activation in response to copper deprivation
conditions. The CuSE sequence bears a strong sequence similarity to the
MRE for the Ace1 and Amt1 copper metalloregulatory detoxifying
transcription factors from the bakers' yeast (S. cerevisiae) and the yeast C. glabrata. When MREs from
S. cerevisiae are expressed in the fission yeast S. pombe, transcriptional regulation occurs in the opposite direction
from that observed for the S. cerevisiae CUP1,
SOD1, and CRS5 genes, which are activated in an
MRE-dependent manner in response to copper. Moreover, the
transcriptional copper starvation-mediated activation that is observed
when MREs of S. cerevisiae are introduced in S. pombe depends on Cuf1. UV-cross-linking experiments using extracts
derived from a heterologous system expressing a functional
cuf1+ gene reveal that the Cuf1 protein binds
the CuSE element. Taken together, these results show that the S. pombe Cuf1 nutritional copper-sensing factor acts through a
closely related MRE-like element to regulate expression of fission
yeast genes encoding components of the high affinity copper transport machinery.
Strains and Growth Conditions--
The S. pombe
strains used in this study were the wild-type FY254
(h Promoter Deletions and Site-directed Mutagenesis--
The
plasmid pSP1ctr4+-737 carries a 1607-bp
XhoI-BamHI PCR-amplified fragment, which harbors
the ctr4+ promoter region up to RNA Analysis--
To conduct the RNase protection analyses as
described previously (16), three plasmids, denoted
pSKctr4+, pSKact1+ (46),
and pKSlacZ (32), were used for making antisense RNA probes,
allowing the detection of steady-state levels of
ctr4+, act1+, and
lacZ mRNAs, respectively. pSKCUP1 was created
by the insertion of a 149-bp EcoRI-BamHI PCR
fragment from the S. cerevisiae CUP1 gene into the same
sites of pBluescript II SK.
Protein Epitope Tagging and Detection--
Both the low and high
copy plasmid versions of URA3-based
GPDcuf1+FLAG2 were created using two
oligonucleotides, FLAG2up
(5'-GGCCGAGACTACAAGGACGACGATGACAAAGGCGACTACAAGGACGACGATGACAAAGAC-3') and FLAG2low
(5'-GGCCGTCTTTGTCATCGTCGTCCTTGTAGTCGCCTTTGTCATCGTCGTCCTTGTAGTCTC-3'), encoding two copies of the FLAG epitope. These purified
oligonucleotides were annealed pairwise to form double-stranded DNA
with EagI overhangs and then ligated into the
NotI site of pSKcuf1+NotI
in which a NotI restriction site was previously engineered by PCR and placed in-frame just before the stop codon of the
cuf1+ gene (46). Subsequently, the
cuf1+FLAG2 was isolated
from this pSK derivative with EcoRI and BamHI and
then cloned into the pGEM-7Zf(+) vector (Promega, WI). To release the
cuf1+FLAG2 gene from this
pGEM derivative, both XbaI and BamHI were used,
allowing unidirectional gene insertion into the SpeI (which produces compatible ends to that of XbaI) and
BamHI sites of the p416GPD/p426GPD
vector for ectopic expression in S. cerevisiae cells (51).
For immunoblot analysis, S. cerevisiae DTY59 cells were
transformed with p416GPD (as control) and
p416GPDcuf1+FLAG2. Yeast
cells were grown to A600 = 1.1 either
with or without 10 µM copper chelator BCS and with or
without 100 µM CuSO4. Total cell lysates were
prepared by glass bead disruption in lysis buffer (20 mM
HEPES (pH 7.9), 10% glycerol, 75 mM NaCl, 1 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 1 µg/ml pepstatin A, and 0.5 µg/ml leupeptin). The cells
were lysed by vortexing for 1 min at top speed 5 times with 1-2-min
intervals on ice. After centrifugation for 6 min at 4,800 rpm, the
protein concentration of the supernatant was determined by the Bradford
assay. Equal amounts of each sample were used to detect the
Cuf1FLAG2 protein using anti-FLAG M2 antibody (Eastman
Kodak Co.) as described by the manufacturer. To carry out
immunodetection of the 3-phosphoglycerate kinase, monoclonal
anti-PGK antibody 22C5-D8 (Molecular Probes, OR) was used. When
examined in S. pombe cuf1 UV Cross-linking--
Cultures of DTY59 (ace1 Identification of Copper-signaling cis-Acting Elements Necessary
for Copper Starvation Activation of the ctr4+ Transporter
Gene Expression--
We have demonstrated previously that in S. pombe, expression of the high affinity copper transporter
ctr4+ is dependent on the Cuf1 copper-sensing
transcription factor (46). To identify the cis-acting
elements necessary for copper starvation activation of copper transport
gene expression by Cuf1, we constructed nested 5' deletions of the
ctr4+ promoter (Fig.
1). As shown in Fig. 1, all constructs
were analyzed in a ctr4 The CuSEs Confer Copper Responsiveness to the Minimal Promoter CYC1
Fused to lacZ--
To examine whether the CuSEs could regulate a
heterologous reporter gene in a copper-dependent manner,
the ctr4+ promoter segment of 213-bp (positions
Cuf1 Acts through Metal Regulatory Elements--
Although we have
demonstrated previously that Cuf1 regulates the expression of essential
copper transport genes in S. pombe, the Cuf1 amino-terminal
61 amino acids more closely resemble amino-terminal 63 and 62 amino
acids of Ace1 (51%) and Amt1 (45%) copper-detoxifying factors than
the corresponding region of Mac1 (39%) (46). Based upon this
observation and because of our finding that the CuSE bears a strong
sequence similarity to the Ace1 and Amt1 binding sites (MREs), we
sought to assess whether MREs of the S. cerevisiae CUP1 gene
could serve in S. pombe for gene regulation as a function of
copper availability by Cuf1. In the cuf1+
strain, when a 129-bp CUP1 promoter DNA fragment (positions
Cuf1 Interacts Directly with CuSEs--
Based on the gene
expression data we obtained, we predicted that the Cuf1 factor directly
interacts with CuSEs to mediate copper regulation. To test this
hypothesis, we generated a Cuf1FLAG2 fusion protein,
allowing its detection in DNA-protein complexes using commercially
available anti-FLAG M2 antibody. Importantly, to test whether insertion
of the FLAG tags interfered with Cuf1 function, the
Cuf1FLAG2 fusion protein was tested for its ability to
complement the cuf1 The cuf1+ gene encodes a nuclear
copper-sensing transcription factor that occupies a central role in the
S. pombe high affinity copper transport system (46). Indeed,
we have shown previously that deletion of the
cuf1+ gene results in at least three phenotypes
associated with copper starvation in yeast cells: inability to use
respiratory carbon sources, impaired superoxide dismutase activity, and
defects in iron accumulation (46). All three phenotypes are restored by the addition of exogenous copper to the medium, consistent with cuf1 To identify the cis-acting elements necessary for copper
starvation activation of copper transport gene expression by Cuf1, we
have conducted detailed studies on the ctr4+
promoter. Although there are two copies of the Mac1 binding site, 5'-TTTGC(T/G)C(A/G)-3', denoted CuRE (32, 33), located between positions To investigate further the mechanism by which Cuf1 regulates target
gene expression through CuSEs in response to copper, we have used a
S. cerevisiae yeast system (51) in which the endogenous ACE1 gene was inactivated. By using this approach, we sought
to ensure the presence of Cuf1 as sole protein with the ability to recognize MRE-like elements. A heterologous test is essential, because
we have identified a second gene in S. pombe, denoted cuf2+ (SPCC584.02). This
putative MRTF exhibits 41% identity to the amino-terminal 61 amino
acids of Cuf1.3 When expressed in S. cerevisiae
ace1 How does Cuf1 function in copper deprivation activation through the
Ace1/Amt1-like recognition sequence? It is intriguing that Cuf1
displays DNA binding specificity similar to that of S. cerevisiae Ace1 and C. glabrata Amt1, yet clearly this
recognition sequence is used for copper transporter gene expression in
S. pombe. Although the first 61 residues of Cuf1 exhibit a
strong homology to this same region of Ace1 and Amt1, Cuf1 does not
possess the second half of the Ace1/Amt1 copper regulatory domain in
which two highly conserved Cys-X-Cys sequence motifs are
found (46). These two pairs of Cys that are missing in Cuf1 make
improbable the formation of Ace1/Amt1-like copper regulatory domain
that consists of two lobes separated by a cleft in which the
Cu4S6 center takes place in the presence of
copper ions (17). Interestingly, Cuf1 harbors a REP-like motif near its
carboxyl terminus, which shows a high percentage of identity (48%) to
a similar domain found in S. cerevisiae Mac1 (46). Because
in Mac1, the REP-I domain is able to sense copper ions, fostering
intramolecular conformational changes and thereby inactivating the Mac1
DNA binding and its transactivation function, the possibility exists
that the Cuf1 REP-like domain acts in a similar manner. Therefore, Cuf1
resembles two distinct types of copper MRTFs, sharing recognition sequence specificity to bind DNA of the Ace1 and Amt1 proteins while
playing an essential role in the regulation of the high affinity copper
transporter protein gene expression in S. pombe as found for
the Mac1 protein in S. cerevisiae.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can1-1 leu1-32 ade6-M210
ura4-D18), the cuf1
disruption strain (h
can1-1 leu1-32 ade6-M210 ura4-D18
cuf1
::ura4+), and the
ctr4
disruption strain (h
can1-1 leu1-32 ade6-M210 ura4-D18
ctr4
::ura4+), which are
described elsewhere in detail (46). For ectopic expression of the
cuf1+ gene in S. cerevisiae, the
ace1
strain, denoted DTY59 (MAT
his6
leu2-3,-112 ura3-52 ace1-
225 CUP1R-3) (47), was
utilized to ensure the presence of Cuf1 as sole protein with the
ability of interacting with MREs. Under nonselective conditions,
S. pombe cells were grown in yeast extract plus
supplements (225 mg/liter adenine, histidine, leucine, uracil, and
lysine). When plasmid maintenance was required, Edinburgh minimal
medium was used as selective medium (48). S. cerevisiae strains were grown either in yeast
extract/peptone/dextrose medium or in the appropriate drop-out
synthetic media (49). To assess the role of Cuf1 in S. cerevisiae, the cells were grown in a modified minimal medium containing 0.67% yeast nitrogen base minus copper and iron, 2% dextrose, 50 mM MES buffer (pH 6.1), and 10 µM NH4Fe(SO4)2 as described previously (32).
737 from the
start codon of the ctr4+ gene in addition to the
gene itself. This pSP1 (52) derivative was digested with
BamHI (accessible end) and SacI (protected end) to perform unidirectional nested 5' deletions of the
ctr4+ promoter. The plasmid
pSKctr4+213 containing nucleotides
from position
737 to position
524 with respect to the A of the ATG
codon of the ctr4+ ORF was created to
introduce mutations in the CuSEs by site-directed mutagenesis.
Precisely, the oligonucleotides
5'-
571CTGTCCCTAAAATTCATTCTCTTATCATGCACTAATACCTCGATTG
616-3'
and
5'-
702CAGTCGGAATCTCATATCCCAAACAGTGACATTTCC
737-3'
(letters that are underlined represent multiple point mutations in the CuSEs) were used either together or separately in conjunction with pSKctr4+213 and the Chameleon
mutagenesis kit (Stratagene, CA). The DNA sequence for each mutant was
confirmed by sequencing and then used to replace the equivalent
fragment from either pSP1ctr4+-737 or
pCF83ctr4+-CYC1-lacZ. This
latter plasmid consists of the ctr4+ promoter
region (position
737 to position
524) within which lie all three
CuSEs (positions
721 to
712; positions
603 to
594; positions
592 to
583) followed by the minimal promoter of the CYC1
gene fused to lacZ. To assess the ability of the CuSEs to
regulate a heterologous reporter gene in a copper-dependent fashion, two versions of the ctr4+ promoter were
tested, one containing 213 bp (positions
737 to
524) and the other
harboring a shorter segment of 175 bp (positions
737 to
562). Both
regions were PCR-amplified and inserted in their natural orientation
into the SmaI and XhoI sites of a
CYC1-lacZ fusion plasmid pCF83 (46). A similar approach was
also used to create both the wild type and mutant
pCF83ctr5+-CYC1-lacZ
fusions. Furthermore, a DNA segment (positions
292 to
163)
encompassing four MREs from the S. cerevisiae CUP1
regulatory region was also amplified by PCR to obtain both wild type
and MRE mutant elements. Both types of fragments were inserted into pCFLEU2 using the SmaI and XhoI sites.
Plasmid pCFLEU2 was constructed by insertion of the
HindIII LEU2 fragment isolated from
pINV1-spc1+-HA6His (50) into the
HindIII sites of pCF83 (46), replacing the
ura4+ cassette for the LEU2 marker.
cells, the
cuf1+FLAG2 fusion allele
complements the cuf1
glycerol growth defect (data not
shown). To accomplish this complementation assay, the NdeI-BamHI
cuf1+FLAG2 fragment was
isolated from the
p416GPDcuf1+FLAG2 plasmid
and then inserted into the NdeI and BamHI sites of pSP1cuf1+S-B/4.2 plasmid (46).
),
expressing either the p416/426GPD vector alone or
p416/426GPDcuf1+FLAG2,
were grown to early log phase under conditions of low copper availability. Cell extracts were prepared as described above in the
lysis buffer. This whole cell extract preparation was then separated
into aliquots in the presence of binding buffer containing 12.5 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 50 mM KCl, 2 mM MgCl2, 2.5 µM ZnSO4, 0.6 mM dithiothreitol,
0.05% Nonidet P-40, 3% Ficoll, and 0.5 µg of random double-stranded
DNA (pdN250) and incubated for 20 min at 25 °C in the presence of 1 ng of oligomer probe. Typically, the probe was prepared by hybridizing
two oligomers denoted CuSE1up (5'-GTCACTGTTTGTGATGCTGGATTCCGACT-3') and
CuSE1lo (5'-ATCAGTCGGAATCCAGCA-3') that are partially complementary to each other by 15 bp. Once annealed, the oligomers were made completely double-stranded by incubation with the Klenow fragment of DNA polymerase I in the presence of the 5-bromo-2'-deoxyuridine
triphosphate (BrdUrd) (7.7 mM, made up in 40 mM Tris-HCl (pH 9.0) and 280 mM NaOH) and
[
32P]dCTP for 90 min at 25 °C, and then the
reaction was chased with dATP, dGTP, and dTTP for an additional 90 min.
The gel-purified BrdUrd and [
32P]dCTP oligomer probe
that was used corresponds to the CuSE found at positions
721 to
712. After DNA protein binding, reaction mixtures were irradiated for
40 min at 4 °C with an average intensity of 600 microwatts/cm2. The reactions were stopped in Laemmli
buffer and heated before SDS-polyacrylamide gel electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain, ensuring that the only
ctr4+ transcripts detected were from the
recombinant plasmids. Removal of the ctr4+
upstream region between
737 and
524 completely abolished the BCS
up-regulation of the ctr4+ gene. Similarly,
further deletion to position
331 gave constitutive levels of gene
expression with complete failure to repress or activate gene expression
in response to copper ions. Owing to the observation that the integrity
of the region between positions
737 and
524 is essential to
down-regulate (~3-fold) and up-regulate (~4-fold)
ctr4+ mRNA expression in the presence of
copper or BCS, respectively, we sought to identify the
cis-acting elements responsible for copper-regulated gene
expression within this promoter region. Interestingly, the
ctr4+ promoter region between
737 and
524
harbors three copies of a repeated sequence, 5'-D(T/A)DDHGCTGD-3'
(D = A, G, or T; H = A, C, or T), that is similar to the
binding sites for Ace1 and Amt1 copper MRTFs from the yeasts S. cerevisiae and C. glabrata, respectively (16). To
examine whether these MRE-like ctr4+ promoter
sequences could mediate gene expression in response to copper, we
inserted multiple point mutations that mimic changes known to abolish
binding of Ace1 to MREs in either the first element (positions
721 to
712), the two other elements found downstream (positions
603 to
594 and
592 to
583), or in all three elements (Fig.
2). Although the overall magnitude of the
response to copper is optimal with the presence of all three elements,
the presence of only one of the three elements is sufficient to
confer regulation in response to copper levels (Fig. 2). As compared
with the wild type promoter segment, ~70 and 60% of the response was
still observed when either the first element or the two other elements
were unaltered, respectively, suggesting that the first element
(positions
721 to
712) is slightly favored for copper starvation
induction. Although a low basal level of expression was observed when
all three elements were mutated, there was a complete lack of either down or up-regulation of the ctr4+ gene.
Furthermore, deletion of 24 bp (from
737 to
713) of the ctr4+ promoter already harboring mutated
elements abolished even the low basal gene expression of
ctr4+ (Fig. 2). Taken together, these data show
that a conserved element in the ctr4+ promoter,
which we term CuSE, with the sequence 5'-D(T/A)DDHGCTGD-3' (D = A,
G, or T; H = A, C, or T) is required, at least in a single copy,
for both repression of basal expression in the presence of copper ions
and activation under copper starvation conditions.
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Fig. 1.
Mapping of ctr4+
promoter elements critical for up-regulation under conditions of low
copper availability. A, repression by CuSO4
(1 and 100 µM) and derepression by BCS (B) of
ctr4+ mRNA steady-state levels when the gene
is under the control of the promoter region containing 737 bp relative
to the A of the ATG codon of the ctr4+ ORF. The
ctr4+ and act1+ mRNA
steady-state levels are indicated with arrows. Results shown
are representative of three independent experiments. B,
schematic representation of nested 5' deletions of
ctr4+ promoter sequences. For each of these
constructs, values for fold repression by copper and derepression by
BCS are shown. The gray boxes indicate the location of the
conserved element 5'-D(T/A)DDHGCTGD-3' within the
ctr4+ promoter. M, reference
marker.
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[in a new window]
Fig. 2.
The CuSEs confer copper responsiveness to the
ctr4+ promoter. A, wild type
(WT) DNA promoter and mutants (M) of the
conserved element found in the ctr4+ promoter
were analyzed by RNase protection assay. Although the overall magnitude
of the response is optimal with the presence of all three CuSEs
(WT1-3), the presence of only one of the three elements
(WT1, M2-3) is sufficient to confer the
regulation in response to copper levels. Results illustrated are
representative of three independent experiments. B, the
gray boxes depict the wild type elements, and the
filled boxes represent the mutated versions of the CuSEs.
1 indicates a promoter deletion to position
713. For
each of these derivatives, values for fold repression by copper and
activation by BCS are shown.
737 to
524) was inserted into the minimal promoter of the
CYC1 gene fused to lacZ (Fig. 3). In the presence of copper ions, this
fusion promoter gene was repressed by ~9-12-fold. Conversely, under
copper starvation conditions, lacZ mRNA expression was
induced (~3-fold). Interestingly, CYC1-lacZ fusion genes
containing the previously described CuSE mutants (Fig. 3) derived from
the ctr4+ promoter repress lacZ
mRNA with an overall efficiency similar to that observed in the
endogenous ctr4+ gene, except for the magnitude
of the response, which is more pronounced when a heterologous system is
used. Importantly, the sequence 5'-D(T/A)DDHGCTGD-3' (D = A, G, or
T; H = A, C, or T) was also found in multiple copies in the
ctr5+ promoter, which drives the expression of a
novel gene encoding an indispensable partner of the Ctr4 copper
transporter involved in high affinity copper uptake in S. pombe.2 As illustrated
in Fig. 4, fusion of short regions
containing 175 bp from the ctr4+ or
ctr5+ promoters encompassing CuSE elements were
able to copper-regulate expression of lacZ mRNA.
Although the ctr4+ promoter harboring this
shorter fragment (positions
737 to
562) of 175 bp is regulated in a
copper-dependent manner, the overall magnitude of the
response decreases by ~70% as compared with the longer fragment
(positions
737 to
524) of 213 bp. For both promoter fusions,
ctr4+-CYC1-lacZ and
ctr5+-CYC1-lacZ, the integrity of the
CuSEs is essential for copper-responsive gene expression since CuSE
mutants abrogate any regulation by copper (Fig. 4). Therefore, these
results suggest that the S. pombe copper transport genes
share a common promoter element, denoted CuSE (5'-D(T/A)DDHGCTGD-3'),
which is necessary for activation in response to copper limitation.
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Fig. 3.
The ctr4+
promoter CuSEs confer copper-dependent regulation of the
minimal promoter CYC1-lacZ gene expression.
A, CYC1-lacZ fusion genes containing wild type
(WT) DNA fragment and CuSE mutants (M) derived
from the ctr4+ promoter were analyzed. One of
the three elements (WT1, M2-3) is sufficient to
regulate a heterologous reporter gene in a copper-dependent
fashion. The lacZ and act1+ mRNA
levels are shown with arrows. Data illustrated are
representative of three independent experiments. B,
schematic representation of the plasmid derivatives analyzed by RNase
protection assay and quantitation of fold copper repression and copper
starvation activation by BCS. The gray boxes represent the
wild type CuSEs, and the filled boxes indicate the mutant
elements. 1 indicates a promoter deletion to position
713.
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Fig. 4.
The CuSEs are also present in the
ctr5+ promoter. A, RNase
protection analysis of repression by CuSO4 (1 and 100 µM) and derepression by BCS (B) through CuSEs
from the ctr5+ promoter. Wild type
(WT) DNA fragments and CuSE mutants derived from both
ctr5+ and ctr4+ (as
control) promoters were inserted in their natural orientation into the
CYC1 minimal promoter fused to lacZ. Results
illustrated are representative of three independent experiments.
B, shown is a schematic representation of the plasmid
derivatives assayed and quantitation of fold copper repression and BCS
derepression. The nucleotide numbers refer to the position
relative to the A of the ATG codon of the ctr4+
and ctr5+ ORFs.
292 to
163) encompassing four MREs was inserted in its natural
orientation into the minimal promoter of the CYC1 gene fused
to lacZ, the expression of lacZ mRNA was
indeed copper-regulated, exhibiting induction in the presence of the
Cu1+ chelator BCS and repression upon the addition of
copper (Fig. 5). In the
cuf1
mutant strain, although a high basal level of lacZ mRNA was detected in the presence of the wild type
MREs, perhaps owing to the action of another transcription factor, the steady-state levels of lacZ mRNA were unregulated by
modulation of copper status (Fig. 5). Furthermore, for both the wild
type and mutant strains, no regulation by copper was observed when the
MRE elements were mutated (Fig. 5). Therefore, when MREs from S. cerevisiae are introduced in the fission yeast S. pombe, transcriptional regulation occurs in the opposite direction
from that observed for the S. cerevisiae CUP1,
SOD1, and CRS5 genes, in which the presence of
the MRE fosters the activation of gene expression in response to
copper.
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Fig. 5.
The bakers' yeast MRE elements serve to
down-regulate gene expression in a copper- and
Cuf1-dependent manner. A, The isogenic fission
yeast strains FY254 (cuf1+) and SPY1
(cuf1 ), transformed with pCFLEU2CUP1-CYC1-lacZ
either with wild type (WT) or mutant MREs, were incubated in
the absence (
) or presence of CuSO4 (1 and 100 µM) or BCS (B) (100 µM) for
1 h followed by RNA isolation and analysis. Results illustrated
are representative of three independent experiments. B,
schematic representation of the two plasmids assayed in the wild type
and cuf1
mutant strains. The open boxes
represent the wild type MREs, and the boxes marked with an × indicate the mutant versions. For each assay, values for fold
repression by copper and activation by BCS are shown. The nucleotide
numbers refer to the position relative to the A of the ATG
codon of the CUP1 ORF.
nonfermentable carbon source growth
defect in S. pombe. The tagged Cuf1 protein fully
complemented the respiratory deficiency (data not shown). Furthermore,
as assessed by RNase protection experiments when transformed into the
cuf1
fission yeast strain, the
cuf1+FLAG2 allele
accurately functions to regulate the ctr4+ gene
expression in response to copper ions in a manner indistinguishable from the cuf1+ wild type allele (Fig.
6). To examine whether the Cuf1 protein interacts with CuSE, we used the
cuf1+FLAG2 fusion gene in
a heterologous context as the only S. pombe gene expressed
in a bakers' yeast system (51) lacking the endogenous ACE1
gene. The ace1
strain was utilized to ensure that Cuf1
was the sole protein with the ability to recognize the DNA probe, which
contains a GCTG core with a T located four nucleotides upstream of the
first G in the GCTG core. When expressed, the
cuf1+FLAG2 gene gave rise
to full-length Cuf1 protein with an electrophoretic mobility on
SDS-polyacrylamide gel electrophoresis corresponding to ~54-kDa (Fig.
7A). No Cuf1 protein was
detected from bakers' yeast extract preparations containing the
expression plasmid alone (Fig. 7A). As observed previously
for S. pombe (46), no drastic change in the ability to
detect Cuf1 was observed when the bakers' yeast cells were grown
either with or without exogenous copper ions (Fig. 7A).
Interestingly, in the S. cerevisiae ace1
strain expressing Cuf1FLAG2, the CUP1-encoded
metallothionein is regulated by copper in the opposite direction,
down-regulated by copper instead of exhibiting a rapid induction after
exposure to copper as compared with the CUP1 gene expressed
in the wild type S. cerevisiae ACE1 strain (Fig.
7B). This observation is consistent with the manner by which
Cuf1 regulates MRE elements from S. cerevisiae when these
elements are introduced in S. pombe (Fig. 5). Importantly, the Cuf1FLAG2 protein appears to conserve its property of
copper-responsive DNA binding when ectopically expressed in S. cerevisiae. Using extracts prepared from S. cerevisiae
ace1
cells expressing the Cuf1FLAG2 protein,
UV-cross-linking experiments revealed a Cuf1-dependent complex of ~64-kDa, which is consistent with the predicted ~10-kDa that the covalently attached oligomer is anticipated to add to the mass
of the Cuf1 protein (Fig. 8A).
Furthermore, this DNA-protein complex of ~64-kDa was absent when
S. cerevisiae ace1
cells expressing the
Cuf1FLAG2 protein were grown under copper-replete
conditions (100 µM CuSO4) (Fig.
8A). Furthermore, no DNA-protein complex was observed when
the CuSE oligomer probe contained multiple point mutations
(5'-GTCACTGTTTGGGATATGAGATTCCGACTGAT-3' instead
of 5'-GTCACTGTTTGTGATGCTGGATTCCGACTGAT-3') (data not shown). To test further whether the DNA binding complex observed is due to the Cuf1FLAG2 protein, duplicates of
each of the irradiated samples shown in Fig. 8A were
resolved simultaneously on an SDS-polyacrylamide gel and then
transferred for analysis by immunoblotting using anti-FLAG M2 antibody.
As shown in Fig. 8B, when UV irradiation was omitted,
Cuf1FLAG2 migrates at an apparent molecular mass of 54-kDa
(unbound form (U)). When irradiated, in addition to the
unbound form (U) of Cuf1FLAG2 (~54-kDa), a higher molecular mass band (~64-kDa) of slower electrophoretic mobility (bound form (B)) was detected (owing to the covalently attached oligomer) with anti-FLAG M2 antibody. Importantly, this higher
molecular mass band of ~64-kDa was only observed when extracts were
derived from cells grown under copper-limiting conditions (Fig.
8B). No such band of slower electrophoretic mobility was observed when extracts were prepared from copper-replete cells expressing Cuf1FLAG2 (Fig. 8B). Because of the
identical molecular mass of ~64-kDa observed by both immunoblotting
and autoradiography of the Cuf1-DNA binding complex found by UV
cross-linking (Fig. 8A), the results strongly suggest the
presence of the Cuf1FLAG2 protein in this complex. Taken
together, these data reveal that the S. pombe Cuf1 protein
activates copper transport gene expression under copper deprivation
conditions by directly interacting with the conserved element,
5'-D(T/A)DDHGCTGD-3', denoted CuSE, which exhibits a striking
similarity with the S. cerevisiae MRE (Fig. 9).
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Fig. 6.
The Cuf1FLAG2 fusion protein
regulates in a proper manner the ctr4+
mRNA levels. Cultures of the cuf1 mutant strain
transformed with either pSP1 vector or pSP1cuf1+
or pSP1cuf1+FLAG2 were
incubated in the absence (
) or presence of CuSO4 (1 and
100 µM) or BCS (B) (100 µM) for
1 h. After treatment at 30 °C, total RNA was isolated. Shown is
a representative RNase protection assay of ctr4+
and act1+ mRNA steady-state levels,
indicated by arrows, respectively.
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Fig. 7.
The CUP1 gene expression is
down-regulated in response to copper in ace1
cells expressing S. pombe Cuf1.
A, Bakers' yeast ace1
cells transformed with
pGPD416Cuf1FLAG2 were grown to early log phase in a
modified SD media containing ~16 nM copper (32).
After no treatment (
) or incubation in the presence of either 100 µM CuSO4 or 100 µM BCS
(B), protein extracts were prepared from the cells and then
analyzed by immunoblotting using either anti-FLAG M2 or anti-PGK
(as an internal control) antibody. We note that the tagged Cuf1
migrates at ~54-kDa despite a predicted molecular mass of 45-kDa.
B, the ace1
strain, transformed with either
the low-copy plasmid expressing
cuf1+FLAG2 or the vector
alone, was grown to mid-log phase and treated with CuSO4
(100 µM) or BCS (B) (100 µM) for
the indicated time. After total RNA isolation, shown is a
representative RNase protection analysis of CUP1 and
ACT1 mRNA levels, which exhibits repression of
CUP1 mRNA expression in the presence of copper in a
Cuf1FLAG2-dependent manner. As a control
(right side), the S. cerevisiae isogenic wild
type strain (ACE1) displays high steady-state levels of
CUP1 mRNA in response to copper. M, reference
marker.
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Fig. 8.
Cuf1FLAG2 protein ectopically
expressed in bakers' yeast binds to CuSE. A, representative
UV-cross-linking gel of the wild type BrdUrd- and
32P-labeled oligomer CuSE used with whole cell extracts
prepared from S. cerevisiae ace1 cells expressing either
the plasmid alone or Cuf1FLAG2
(pGPDcuf1+FLAG2) as their
sole source of Cuf1. Only cells grown under copper-limiting conditions
expressing cuf1+FLAG2
allele exhibits the CuSE oligomer-protein complex (arrow),
which is absent in extract preparations from cells expressing
cuf1+FLAG2 allele but
treated with CuSO4 (100 µM). No
oligomer-protein complex is observed with extracts prepared from cells
expressing the plasmid alone. P, probe DNA irradiated in the
absence of protein. B, five UV-irradiated reactions of each
of the three binding reactions shown in A were pooled and
resolved by SDS-polyacrylamide gel electrophoresis. By immunoblotting
using anti-FLAG M2 antibody, a fraction of the Cuf1FLAG2
protein was found shifted,m presumably because of its irreversible
attachment to CuSE by UV irradiation. This slower electrophoretic form
of Cuf1FLAG2 (bound (B)) migrates at ~64 kDa,
as observed for the CuSE oligomer-protein complex shown in
A. Only one form (unbound (U)) of the
Cuf1FLAG2 protein was detected at ~54 kDa in extract
preparations from cells treated with CuSO4 (100 µM). As a control (right side), UV irradiation
was omitted. V, extracts from cells expressing the vector
alone. M, reference marker.
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Fig. 9.
Comparison of CuSE sequences from S. pombe copper transport genes with the Ace1 and Amt1
consensus MRE binding site. The conserved residues shown in
gray play a critical role in copper-regulated gene
expression of the S. pombe ctr4+ and
ctr5+ transport genes. As highlighted for MREs
(16), CuSEs possess four nucleotides perfectly conserved, GCTG (also
named core element) and a T (or A for one CuSE) located four
nucleotides upstream of the first G in the GCTG. The nucleotide
numbers refer to the position relative to the A of the start
codon of the indicated ORF.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strains being specifically defective in the
expression of genes required for high affinity copper transport.
Furthermore, the previous observation that in S. pombe,
expression of the high affinity copper transporter Ctr4 is dependent on
the Cuf1 protein defines a critical role for
trans-activation of copper transport gene by Cuf1 (46).
323 and
381 in the ctr4+ promoter,
no effect on copper-responsive regulation of the
ctr4+ promoter was observed when these two
elements were mutated.3 This
latter observation is also consistent with the inability for Cuf1 to
suppress a number of copper-remedial phenotypes associated with
mac1
disruption when the S. pombe
cuf1+ gene is ectopically expressed in a S. cerevisiae mac1
strain.3 Our studies have revealed
that the ctr4+ and ctr5+
promoters are regulated in response to fluctuations in copper concentrations by a consensus cis-acting sequence,
5'-D(T/A)DDHGCTGD-3' (D = A, G, or T; H = A, C, or T) termed
CuSE, which is highly similar to the binding sites for the Ace1 and
Amt1 copper MRTFs, MREs. This finding is consistent with the high
percentage of homology within residues 1-61 of Cuf1 amino terminus to
the amino-terminal 63 (51% identity) and 62 (45% identity) amino
acids of Ace1 and Amt1, respectively (46). This amino-terminal region
of Cuf1 harbors a Zn2+ module domain known to be involved
in minor groove DNA contacts with the AT-rich element (53, 55) and also
appears to share with Ace1 and Amt1 critical residues involved in
recognition of the core sequence. Interestingly, only a single CuSE is
sufficient for near-maximal copper deprivation activation of the
ctr4+ gene expression. Similarly, both the Ace1
and Amt1 copper MRTFs can mediate their action through a single MRE,
which is not the case for the Mac1 protein, which requires at least two
copies of the CuREs for copper-regulated gene expression (32, 38). Although few differences are observed between the CuSE and MRE consensus sequences (Fig. 9), the binding sites appear to be
interchangeable since when MREs from S. cerevisiae are
introduced in the fission yeast S. pombe, Cuf1 can
BCS-activate target gene expression through those MREs (Fig. 5). It is
anticipated that some nucleotides within the CuSE should optimize
copper starvation induction of gene expression. Which nucleotides
contribute to the magnitude of this regulatory response must await a
comprehensive dissection of the CuSE. With respect to the conserved
T-positioned four nucleotides upstream of the GCTG core element (Fig.
9), it has been shown previously (16) that its substitution by adenine
results in ~20% reduction in DNA binding by Amt1. Based on this
observation, we would predict that the CuSE between positions
596 and
581 has a weaker strength for copper transcriptional regulation.
cells under conditions in which copper is scarce, the Cuf1
protein fosters high basal levels of CUP1 mRNA.
Conversely, when S. cerevisiae ace1
cells were grown in
the presence of copper, the CUP1 mRNA levels were repressed by Cuf1, consistent with the manner by which Cuf1 regulates copper transport gene expression in S. pombe. To determine
whether Cuf1 binds to CuSE when ectopically expressed in S. cerevisiae ace1
cells, a BrdUrd-double-stranded CuSE oligomer
derived from the ctr4+ promoter (positions
700
to
731) was used for UV-cross-linking analysis (54). Precisely, the
BrdUrd was placed on the noncoding strand two nucleotides upstream of
the GCTG core element (position
718) since the Cuf1 protein is
predicted to interact with one face of the DNA double helix at adjacent
minor (encompassing that position
718) and major (containing the GCTG
core element) grooves. Indeed, when the reactive base (BrdUrd) was
positioned into the CuSE, a DNA-Cuf1-dependent complex was
observed (Fig. 8). Interestingly, this DNA-protein complex of ~64-kDa
was only observed when the extract preparations were obtained from
yeast cells grown under conditions of low copper availability. When
cells were grown in the presence of copper, no such DNA-protein complex
was detected.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Dennis J. Thiele for strains and plasmids used in this study and for generously sharing data before publication. We are grateful to Dr. Kevin A. Morano for critical reading and constructive comments of the manuscript. We thank Dr. Maria M. O. Peña for helpful discussions regarding site-directed mutagenesis of the ctr4+ promoter. We greatly appreciate advice from Dr. Benoit Coulombe about the UV-cross-linking approach.
![]() |
FOOTNOTES |
---|
* This study was supported by Medical Research Council of Canada Grant MOP-42406 (to S. L.). Infrastructure equipment essential for conducting this investigation was obtained through Canada Foundation for Innovation Grant NOF-3754 (to S. L.).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.
A Fonds de la Recherche en Santé du Québec Scholar. To
whom correspondence should be addressed: Tel.: 819-820-6868 (ext. 15460) or 819-564-5281; Fax: 819-564-5340; E-mail:
slabbe@courrier.usherb.ca.
Published, JBC Papers in Press, January 26, 2001, DOI 10.1074/jbc.M011256200
2 D. J. Thiele, personal communication.
3 S. Labbé, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MRTF, metalloregulatory transcription factor; BCS, bathocuproinedisulfonate; bp, base pair(s); BrdU, 5-bromo-2'-deoxyuridine triphosphate; Ctr, copper transporter; Cuf1, copper factor 1; CuRE, copper response element; CuSE, copper-signaling element; MRE, metal regulatory element; ORF, open reading frame; SOD, superoxide dismutase; MES, 2-N-morpholinoethanesulfonic acid; PCR, polymerase chain reaction.
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