From the Department of Environmental Toxicology, University of California, Santa Cruz, California 95064
Received for publication, September 7, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Copper ions are essential at a proper level yet
toxic when present in excess. To maintain a proper intracellular level,
cells must be able to sense the changes in copper ion concentrations. The yeast transcription factor Mac1p plays a critical role in the
transcriptional regulation of CTR1 and CTR3,
both encoding high affinity copper ion transporters. Here we report
that the Mac1p binding of the copper ion-responsive elements (CuREs) in the promoters of CTR1 and CTR3 is affected by
copper ions. On one hand, the Mac1p DNA binding is Cu+
coordination-dependent, and on the other hand, exogenous
Cu+ and isoelectronic Ag+ ions disrupt the DNA
binding of Mac1p. These results suggest that the Mac1p is able to sense
two different levels of copper ions. These two levels are probably the
physiological and toxic copper levels in yeast cells.
Furthermore, we found that Mac1p undergoes posttranslational
phosphorylation modification in yeast and that the phosphorylation is
required for the Mac1p to become DNA-binding active. Nonphosphorylated
Mac1p is unable to bind the CTR1 promoter DNA. The data
support the model of intradomain interactions and indicate further that
the phosphorylation probably prevents the inhibition of DNA-binding
domain activity by the activation domain of Mac1p. Taken together,
these findings demonstrate that Mac1p functions critically in
maintaining a proper intracellular concentration of copper ions.
Due to its essential yet toxic nature in biological systems
(1-3), the intracellular concentration of copper ions is tightly controlled in the yeast Saccharomyces cerevisiae, in part,
by regulating the expression of the genes CTR1 and
CTR3 that encode copper ion transporters at both
transcriptional and posttranslational levels (4-10). The
CTR1 and CTR3 transcription is enhanced by copper
starvation (addition of chelator bathocuproinedisulfonate (BCS)1 in the growth
medium) and inactivated by even a slight increase in the copper
ion concentrations (of picomolar range) (9). The transporter Ctr1p is
induced to undergo degradation by toxic copper levels (of 10 µM or higher), which is thought to further inhibit copper
uptake under toxic conditions (6). Interestingly, a recent study (33)
reports that the Ctr3p, unlike the Ctr1p, does not undergo degradation
in response to toxic copper levels. This latest finding may explain the
earlier report that CTR3 is disrupted by a transposon
element in most yeast laboratory strains (5). These
concentration-dependent responses noted above indicate that
cells are able to sense and differentiate the physiological from toxic
levels of copper ions. How the degradation of Ctr1p is controlled is
currently unknown. The transcription of CTR1 and
CTR3 is regulated by the copper-sensing factor Mac1p through the cis-acting CuREs in the promoters (9). However, how the copper ion
signal, particularly that of physiological and toxic levels, is
propagated to the promoters of CTR1 and CTR3 is
currently poorly understood.
The primary sequence indicates that the Mac1p may sense copper ions
through direct copper ion coordination (7), similar to the two other
known copper-sensing factors Ace1p and Amt1p (11-14). Biochemical
studies show that the separated DNA-binding domain and the activation
domain of Mac1p expressed in the Escherichia coli both bind
Cu+ ions (15). The bacteria-expressed DNA-binding domain
was also found to coordinate Zn2+; a zinc-module was then
postulated to also exist in the Mac1p DNA-binding domain (15). The
DNA-binding domain of Amt1p expressed in the bacteria was the first
copper-sensing factor found to be Zn2+-coordinated (16).
However, this Zn2+ coordination was not required for the
Amt1p to bind DNA (16). DNA binding activities at the CuREs in the
promoters of CTR1 and CTR3 have been detected in
the yeast extracts prepared from the cells expressing various forms of
the truncated Mac1 protein (17). The purified truncate of
Mac1p(1-159), which lacks the activation domain, was also found able
to bind the CuREs (15). In vitro translated Mac1p has also
been shown to be able to bind the CTR1 promoter (8). These
studies have demonstrated that Mac1p is able to bind the CuREs.
However, whether or not copper affects Mac1p DNA binding has yet to be
resolved. In vivo footprinting showed that upon
CuSO4 treatment, the CuREs in the CTR3 promoter became more accessible to the methylation reagent (9), indicative of
possible dissociation of Mac1p. The DNA binding activities of the yeast
extracts, prepared from the cells expressing the truncated
Mac1p(1-194) and grown in the medium containing either BCS or
CuSO4, exhibited no difference (15). There is no report on
the copper effect on Mac1p DNA binding by the addition of
CuSO4 to the binding reactions using yeast cell extracts or
purified proteins.
The previous study using Mac1 fusion proteins with Gal4p (either
DNA-binding or activation domain) and VP16 provided indirect evidence that there are interactions between the DNA-binding and activation domain of the Mac1p (17). It is postulated that
copper-induced intramolecular interactions inhibit both DNA binding and
transactivation activities of the Mac1p (17, 18). A more recent study
using one- and two-hybrid methods also suggests that the intramolecular interaction negatively modulates Mac1p activity (19).
In this report, we describe the finding that the Mac1p undergoes
posttranslational phosphorylation and that the phosphorylation is
required for the Mac1p to bind to the CuRE in the CTR1
promoter. The dephosphorylated Mac1p is unable to bind the DNA. We also found that the Mac1p DNA binding, on one hand, is dependent on Cu+ coordination and, on the other hand, is disrupted by
the addition of copper ions. These studies indicate that Mac1p is able
to sense two different copper levels and that the posttranslational
phosphorylation of Mac1p is probably a key event in the regulation of
copper transport.
Plasmid Construction, Cell Growth, and Protein Analysis--
A
high copy plasmid pGPD-Mac1HA(opt) was constructed by cloning a
BamHI-XhoI fragment of an artificial
MAC1 into the p423GPD vector at the same sites (20). The
artificial MAC1 has optimal codons and is tagged with a
single copy of HA epitope at the carboxyl terminus. The DNA was
synthesized in vitro (GeneMed).
A single copy plasmid pRSMac1(3HA) was constructed essentially the same
as pRSMac1(HA) as described previously (10). To introduce 3 × HA
epitopes in the MAC1 coding region, a NarI site was generated by silent mutations at the DNA encoding residues Gly-222
and Ala-223. Complementary oligonucleotides were synthesized for
a DNA fragment encoding three copies of the nine amino acids of the HA
epitope with flanking NarI sites. The oligonucleotides were
annealed and cloned into the NarI site of the
MAC1.
Plasmids were transformed into the SLY2(mac1 In vivo 32P-Labeling and Immunoprecipitation--
SLY2
cells transformed with pRSMac1(3HA) or control plasmid pRS313 were used
to carry out in vivo 32P-labeling (22). Cells
were pre-grown overnight in the copper-depleted selective medium
synthetic complete medium lacking histidine without phosphate
and reinoculated into 5 ml of synthetic complete medium lacking
histidine medium of 0.02 of the normal phosphate level with a starting
OD650 of 0.4. 32P-Inorganic phosphate (ICN) was
added to a final concentration of 200 µCi/ml. Cells were grown until
the OD650 reached ~1.5 and then harvested. Cell pellets
were washed with H2O, and whole cell extracts were prepared
using the method described previously (6). The proteins were dissolved
in buffer A (50 mM Na3PO4, pH 7.0, 25 mM MES, pH 7.0, 1% SDS, 3 M urea, 0.5%
DNA Binding Experiments--
Yeast extracts were prepared
from cells of SLY2 harboring pGPD-Mac1HA(opt) or vector p423GPD grown
in copper-depleted medium, using the glass bead disruption method (21),
in lysis buffer of 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM Mac1p Undergoes Posttranslational Phosphorylation in S. cerevisiae--
Probably because of the low physiological level of
Mac1p in the yeast cells, our repeated efforts to detect the CuRE
binding activity of Mac1p in the yeast extracts did not produce
conclusive results. An artificial MAC1 of optimized codons,
tagged with a single copy of HA epitope at the carboxyl terminus, was
then synthesized in vitro, and the Mac1-HA protein was
expressed in the SLY2 cells carrying the synthetic gene on a high copy
plasmid pGPG-Mac1HA(opt) as described under "Experimental
Procedures." As shown in Fig. 1A, three proteins (two
labeled with p-Mac1-HA and one with Mac1-HA) were detected
by immunoblotting using polyclonal anti-HA antibody Y11 and found only
in cells harboring the plasmid pGPD-Mac1HA(opt). Compared with the
E. coli-expressed Mac1-HA, the two proteins had higher
molecular masses. Upon protein phosphatase treatment, the two
proteins collapsed to the protein Mac-HAp, which migrated at 57 kDa.
The E. coli-expressed Mac1-HA also migrated at 57 kDa. Protein phosphatase treatment did not change the E. coli-expressed Mac1-HA (data not shown). This result indicates
that the Mac1-HAp when overexpressed undergoes phosphorylation
modification in the yeast cells but not in the bacteria.
We observed that the overexpression causes hyperphosphorylation of the
Mac1-HA protein (data not shown). In SLY2 cells transformed with the
single copy plasmid pRSMac1(HA) (10), the level of the phosphorylated
Mac1-HAp was found to be very low and hardly detectable (Fig.
1B). To enhance immunological detection, the plasmid
pRSMac1(3HA) was constructed as described under "Experimental Procedures" in which three copies of the HA epitope were inserted in
Mac1p between the Gly-222 and Ala-223 residues. The Mac1-3HAp was
functional in the activation of CTR1 and CTR3
transcription, judging from the growth on nonfermentable carbon source
yeast medium containing the non-fermentable carbon source
ethanol plate (Fig. 1D). As shown in Fig.
1B, in SLY2 cells harboring pRSMac1(3HA) plasmid, two
specific protein bands were detected with one band significantly more
abundant than the other, corresponding to nonphosphorylated and
phosphorylated Mac1-3HAp, respectively. We then carried out metabolic labeling with 32P-inorganic phosphate and
subsequent immunoprecipitation. In Fig. 1C, a
32P-radioactive protein of the Mac1-3HAp molecular weight
was detected from 32P metabolically labeled cells
expressing Mac1-3HAp but not from the 32P-labeled control
cells. These results together have demonstrated that phosphorylation of
Mac1p also occurs at a physiological level.
The Exogenous Copper Ions Disrupt the DNA Binding of
Mac1p--
Based on the findings described above, we speculate that
our prior failure to detect the DNA binding activity of Mac1p in the
yeast extracts is possibly due to the observed very low abundance of
the phosphorylated Mac1p. We then conducted electrophoretic mobility
shift assays to determine whether or not the Mac1p DNA binding was
affected by copper ions. To conduct this experiment, we used
yeast whole cell extracts, prepared from the SLY2 containing the
pGPD-Mac1HA(opt) plasmid and 32P-labeled wild type
CTR1 promoter DNA fragment, and a mutant fragment in which
both CuREs are mutated (9). The yeast cells were grown in
copper-deficient medium, and extracts were prepared as described above.
As shown in Fig. 2, a shifted complex was
detected in the reactions between 32P-labeled wild type
CTR1 promoter DNA and the extract prepared from cells
expressing Mac1-HA (lanes 3 and 4) but not from
control cells (lane 2). The addition of CuSO4 at
both 1 µM and 100 µM concentrations
(lane 5 and 6) disrupted the complex. In the
reaction containing the mutant CTR1 promoter DNA, no such
complex was detected (lane 7), confirming previous genetic
analysis and in vivo footprinting that the CuREs serve as
the copper-responsive elements in the CTR1 and
CTR3 promoters (9). Inclusion of the anti-HA antibody in the reaction (lane 10) caused a supershift of the
complex, indicating that Mac1-HAp is present in the complex.
Competition reactions (lanes 8 and 9) show that
Mac1-HA formed a specific complex with the wild type CTR1
promoter DNA. Our data then demonstrate that the Mac1p DNA binding is
copper ion-responsive, suggesting that the copper ion-triggered
disruption of Mac1p DNA binding probably results in the transcriptional
inactivation of CTR1 and CTR3.
The Phosphorylation and Cu+ Coordination of
Mac1p Are Required to Bind the CuRE in the CTR1 Promoter--
We next
characterized the effect of Mac1p phosphorylation on its DNA binding by
performing the electrophoretic mobility shift assays using the same
yeast extracts, both treated and not treated with protein phosphatase
(as shown in Fig. 3). Consistent with the
data in Fig. 2, the Mac1-HAp-containing complex was detected (lanes 3 and 4), and the complex was disrupted by
copper (lanes 5 and 6) and by the isoelectronic
Ag+ as well (lane 7). Ag+ represses
the CTR1 and CTR3 transcription in a manner
indistinguishable to Cu+ (9). These data further
substantiate our finding that Mac1p DNA binding is copper
ion-responsive. Also shown in the Fig. 3, the addition of BCS
(lane 8), the Cu+-specific chelator, disrupted
the complex, but EDTA (lane 9), a mainly divalent metal ion
chelator, had no effect. These results indicate that the Mac1p DNA
binding is also Cu+ coordination-dependent, much like the
Ace1p and Amt1p, suggesting that the DNA-binding active Mac1p is likely
to be Cu+-bound even under copper-limiting conditions. In
lane 10, the phosphatase treatment almost completely
abolished the DNA binding activity of the Mac1-HAp in the extract
sample showing that the phosphorylation of Mac1p is required to bind
the CTR1 promoter. Interestingly, the addition of the
phosphatase inhibitors (lane 11) did not prevent copper ions
from disrupting Mac1-HA DNA binding in vitro, implying that
copper disrupts the DNA binding of Mac1p probably through a mechanism
other than dephosphorylation. These results together show that the two,
phosphorylation and Cu+ coordination, are both required for
the Mac1p to bind the CuREs in the promoters of CTR1 and
CTR3.
In this report, we have demonstrated that Mac1p undergoes
phosphorylation modification in yeast cells but not in bacteria and
that the phosphorylation is required for Mac1p to bind to its target
gene, the CTR1 promoter (and presumably the CTR3
promoter as well). The nonphosphorylated Mac1p failed to bind DNA at
all. A previous report, however, has shown that the separated
DNA-binding domain is able to bind the CTR1 promoter (15).
Our lab has also found that the purified DNA-binding domain of Mac1p is
able to bind CTR1 promoter eventhough the DNA-binding domain
protein is not phosphorylated (data not shown). Taken together, these
findings suggest that the activity of the DNA-binding domain is
inhibited in the nonphosphorylated Mac1p and that the inhibition is
abolished upon phosphorylation of Mac1p. Consistent with this notion,
two earlier studies (17, 18) have reported that there are probably intermolecular interactions between the DNA-binding and activation domains of Mac1p and that the activation domain negatively may regulate
the activity of the DNA-binding domain (19). Therefore, in Mac1p, the
activation domain regulates the activity of the DNA-binding domain
possibly in a phosphorylation-dependent fashion. Supporting
this mechanism, our preliminary studies indicate that the
phosphorylation occurs within the activation domain of Mac1p, not in
the DNA-binding domain.2 The
activation domain of Mac1p clearly plays an active role in the
regulation of CTR1 and CTR3 transcription in
response to changes in copper ion concentrations. The phosphorylation
of proteins is a general mechanism for cells to respond to
extracellular signaling (25). This report indicates that regulation of
copper ion uptake most likely involves a phosphorylation-based signal
transduction mechanism. A recent study (26) has also indicated that
Aft1p, the iron-sensing factor, undergoes possible phosphorylation
modification. Therefore, the phosphorylation modification may represent
a new yet common mechanism by which cells sense metal ions.
Our finding that copper ions affect the DNA binding of Mac1p represents
important progress in the understanding of the mechanism by which Mac1p
functions in the regulation of CTR1 and CTR3
transcription. The sequence homology among Mac1p, Ace1p, and Amt1p
within their NH2-terminal DNA-binding domain suggests that
these factors may function similarly in sensing copper ions (7). A
recent study (27) has reported that Cuf1, a critical factor in the
regulation of copper and iron uptake in Schizosaccharomyces
pombe, also shares this homology. It is a known fact that for the
Ace1p and Amt1p to activate the transcription of MT genes, both
proteins have to bind copper ions first and then become DNA-binding
active (28, 29). This mechanism is clearly suited to assure that this
detoxifying response is only activated in cells exposed to toxic levels
of copper ions, not under physiological conditions because MTs inhibit the copper acquisition by other copper-dependent enzymes,
such as Cu,Zn-SOD (30). Therefore, the Ace1p and Amt1p are sensors for
the toxic level of copper ions. Our studies have shown that the Mac1p
DNA binding is also Cu+ coordination-dependent,
eventhough the deficiency of copper ions triggers the Mac1p to activate
the transcription of CTR1 and CTR3. Equally
important, we have also demonstrated that the exogenous copper ion and
isoelectronic Ag+ disrupt the Cu+
coordination-dependent DNA binding of Mac1p. Our results are seemingly
contradictory, yet they unmistakably indicate that the Mac1p is able to
sense two different copper levels, reflecting the very nature of copper
in living organisms, essential yet toxic depending on its
concentrations. We speculate that the dependence of Mac1p DNA binding
on Cu+ coordination probably represents the ability of
Mac1p to sense the physiological copper level, whereas the copper
disruption of Mac1p DNA binding may exemplify its ability to sense the
toxic level of copper ions. Mac1p contains two clustered metal ion
binding motifs of typical CXC and CXXC
nature located within the DNA-binding and the activation
domains, respectively (7). The DNA-binding and the activation domains
are the possible structural basis for the sensing of the two copper
levels by Mac1p. An important question is how Mac1p can detect these
two different copper levels? And equally important, under the
copper-limiting condition, why is the Mac1p able to bind copper ions
and activate the CTR1 and CTR3 transcription but
not the Ace1p, eventhough Mac1p and Ace1p share an almost identical
Cu+ binding motif within their DNA-binding domains? One
plausible model is that the copper ion binding motifs within the
DNA-binding and activation domains of Mac1p may have different
affinities to copper ions and that Mac1p DNA-binding domain has much
higher affinity to copper ions than does the Ace1p DNA-binding domain. Biochemical studies currently underway in our laboratory will shed
light on this possible mechanism.
The transcription of CTR1 and CTR3 has been shown
previously to be extremely sensitive to increases in exogenous
copper ion (9). Our data have provided a framework for understanding
how Mac1p may activate the transcription under copper-limiting
conditions. However, the mechanism by which the increase in copper ion
concentrations triggers the inactivation of CTR1 and
CTR3 transcription is still poorly understood. We have
reported here that copper triggers the repression of CTR1
and CTR3 transcription possibly by disrupting the
DNA-binding of Mac1p. Because the phosphatase inhibitors did not
prevent copper from disrupting Mac1p-binding DNA (Fig. 3), this
suggests that copper disrupts Mac1p DNA binding potentially by a means
other than triggering dephosphorylation of Mac1p (10).2 The
new features of how Mac1p functions as reported in this work, particularly the ability to sense the two different levels of copper
ions, are consistent with its regulatory role in copper ion transport.
Further studies are warranted to determine the precise molecular
mechanism by which cells differentiate the physiological from toxic
levels of copper ions. This is particularly true because Menkes' and
Wilson diseases are caused by copper ion deficiency and excess copper
ion toxicity, respectively (for review see Refs. 31 and 32).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) cells as
described previously (10). The cells were grown in the copper
ion-depleted medium treated with Chelex (Bio-Rad) (6). Yeast whole cell extracts were prepared using the previously described method (6) or by
using the glass bead disruption method (21). The protein concentrations
of the extracts were determined using the Bradford protein assay (21).
The lysis buffer contained 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 5 mM
dithiothreitol. The protein extracts were treated with
-protein phosphatase (New England Biolabs). After the treatment,
proteins were precipitated using trichloroacetic acid, fractionated on
8.5% SDS-polyacrylamide gels, and then transferred to a nitrocellulose
membrane as described previously (21). Western blotting analyses were
then carried out using a polyclonal anti-HA antibody, Y11 (Santa Cruz
Biotechnology Inc.) (10).
-mercaptoethanol, 50 mM NaF). Protein concentrations in
the extracts were measured as described above. For immunoprecipitation,
250 µg of total proteins in 100 µl of buffer A was mixed with 700 µl of buffer B (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% Tween 20, 0.1 mM EDTA), and 5 µl of anti-HA antibody 12CA5 was added. The reactions were incubated on a rotating wheel for 2 h at 4 °C. Protein A-Sepharose CL-4B beads were added with 10 µl of 100 mM bovine serum
albumin, and the reactions were incubated overnight at 4 °C on a
rotating wheel. Immunocomplexes were precipitated by microcentrifuge at
13,000 rpm and washed five times with the buffer B. The complexes were eluted in 40 µl of SDS-polyacrylamide gel electrophoresis sample buffer by heating at 80 °C for 10 min. Twenty microliters of elutes were analyzed on a 6% SDS-polyacrylamide gel. The gel was fixed and
dried and exposed to Kodak Biomax film at
80 °C with an
intensifying screen. The exposure time that was required for
autoradiographic detection of Mac1p phosphorylation was 2-4 days.
-mercaptoethanol, 10%
glycerol. 32P-Labeled CTR1 promoter DNA fragment
was generated by polymerase chain reactions using
[32P]dATP. The primers used for the wild type DNA were
5'-CATGTATTGATGCAA-3' and 5'-CTTGAAAAGTGCTC-3', and primers used for
the mutant probe were
5'-CATGTATTGATGCAAATCATGGGATAGGGATGAAAGACGACGGTAAAA-3' and 5'-CTTGAAAAGTGCTCTTTTCAGGATCGTGCCATTGGGATGAATTTTACCGTC-3'. The radioactive DNAs were purified by the agarose gel electrophoresis. Because of the observed interference of the Mn2+ on Mac1p
DNA binding, calf intestinal phosphatase (New England Biolabs) was used
to dephosphorylate the Mac1p in the protein extracts. The phosphatase
inhibitors, glycerol phosphate (40 mM), NaF (50 mM), and okadeic acid (2 µM), were used as
previously described (24). The binding reactions were carried out as
described previously (23).
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (46K):
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Fig. 1.
Mac1p undergoes posttranslational
phosphorylation modification in S. cerevisiae.
A, protein phosphatase treatment of Mac1p overexpressed in
yeast. Whole cell extracts were prepared using the glass beads
disruption method from SLY2 (mac1 ) harboring either
control vector pGPD423 or pGPD-Mac1HA(opt) plasmid. The extracts were
both treated and left untreated with
- protein phosphatase at
30 °C for 1 h and then precipitated using trichloroacetic acid
and analyzed by immunoblotting with anti-HA antibody Y11 (1:500
dilution) and goat anti-rabbit IgG conjugated to horse radish
peroxidase (1:1000 dilution). PPase,
-protein
phosphatase; Lane
, not treated with PPase;
Lane +, treated with PPase. For A, B, and
C, the positions of the molecular weight standards are
indicated to the left, and on the right, the
phosphorylated and nonphosphorylated Mac1 proteins are marked with an
arrowhead and Mac1-HA, Mac1-3HA, or p-Mac1-HA and
p-Mac1-3HA, respectively. Lane E, the Mac1-HAp expressed in
E. coli; lane C, control cells; Mac1,
cells expressing Mac1p. B, detection of phosphorylated Mac1p
at its physiological level by immunoblotting. SLY2 cells harboring
pRSMac1(3HA) or pRSMac1(HA) or control vector pRS313 plasmid were grown
in copper-depleted medium. Yeast whole cell extracts were prepared, and
Mac1-HAp and Mac1-3HA were detected by immunoblotting as described
above. C, in vivo 32P-labeling and
immunoprecipitation of Mac1p. SLY2 cells transformed with pRSMac1(3HA)
or control plasmid pRS313 were grown in copper-depleted medium in the
presence of 32P-inorganic phosphate, and whole cell protein
extracts were prepared as described under "Experimental
Procedures." Immunoprecipitation was carried out with anti-HA
antibody 12CA5 and protein A-Sepharose beads. Immunocomplexes were
analyzed on an 8.5% SDS-polyacrylamide gel. D, Mac1-3HA is
functional in the activation of CTR1 and CTR3
transcription. Transformants of SLY2 with the vector pRS313
(mac1
), pRSMac1(HA) (MAC1-HA), or pRSMac1(3HA)
(MAC1-HA) were streaked onto yeast medium containing a
nonfermentable carbon source (YPE). Cells were incubated for
48 h and photographed.
View larger version (81K):
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Fig. 2.
Exogenous copper ions disrupt the DNA binding
of Mac1p. Electrophoretic mobility shift assays using
32P-labeled CTR1 promoter fragments containing
the CuREs or the mutant are shown (9). Whole cell extracts were
prepared from either control cells (SLY2 transformed with the vector
plasmid pGPD423) or Mac1-HAp expressing cells (SLY2 containing
pGPD-Mac1(opt)HA) and used in binding reactions in the presence of
poly[dI-dC]·poly[dI-dC]. CuSO4 was added to final
concentrations of 1 µM (lane 4) and 100 µM (lane 5). The mAb 12CA5 was incorporated
into reactions (lanes 10 and 11), and the
reactions were incubated at 4 °C for 20 min. DNA binding reaction
mixtures were electrophoretically fractionated on a 5% native
polyacrylamide gel. Free, free probe DNA;
Mac1(HA)-DNA, the Mac1(HA)-CTR1 complex;
Mac1(HA)-DNA/12CA5, the Mac1(HA)-CTR1 supershifted complex
by the mAb 12CA5.
View larger version (61K):
[in a new window]
Fig. 3.
Phosphorylation and Cu+
coordination of Mac1p are required for binding CTR1
promoter DNA. Electrophoretic mobility shift assays using
32P-labeled CTR1 promoter fragments and
phosphatase-treated protein extracts are shown. The same whole cell
extracts were used, and the reactions were carried out as described in
the Fig. 2 in the presence of poly[dI-dC]·poly[dI-dC].
CuSO4 (1 µM), AgNO3 (1 µM), BCS (2 mM), and EDTA (2 mM)
were added to the final concentrations as shown at the
bottom of the figure. The extract was treated with calf
intestinal phosphatase for 30 min at 37 °C, both in the presence and
absence of the phosphatase inhibitors (40). DNA binding reaction
mixtures were electrophoretically fractionated on a 5% native
polyacrylamide gel. Free, free probe DNA;
Mac1-HA-DNA, the Mac1(HA)-CTR1 complex.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank other members in the Zhu lab for critically reading the manuscript and for valuable suggestions.
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FOOTNOTES |
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* This work was supported by National Science Foundation Grant MCB-9807786 and National Institutes of Health Grant GM58082-01 (to Z. Z.).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.
Supported in part by Minority Biomedical Research Support (MBRS) program.
§ To whom correspondence should be addressed. Tel.: 831-459-3987; Fax: 831-459-3524; E-mail: zhu@biology.ucsc.edu.
Published, JBC Papers in Press, December 28, 2000, DOI 10.1074/jbc.M008179200
2 Z. Zhu, M. Crooks, M. Depaz, and J. Heredia, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: BCS, bathocuproinedisulfonate; CuRE or CuREs, copper ion-responsive element(s); HA, hemagglutinin; MES, 4-morpholineethanesulfonic acid.
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