From the Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, Michigan 48109-0606
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Copper is an essential metal ion that is toxic when accumulated to high intracellular concentrations. The yeast Mac1 protein is a copper-sensing transcription factor that is essential for both the activation and inactivation of genes required for high affinity copper ion transport. Here we demonstrate that in response to low copper ion concentrations Mac1 protein is rendered inactive for copper transporter gene transcription. Under high copper ion concentrations Mac1 is degraded in a rapid, copper-specific manner. This degradation is critical to prevent copper toxicity that would otherwise result from sustained expression of the copper transport genes. These results demonstrate that nutritional and toxic copper concentrations elicit distinct fates for the Mac1 copper-sensing transcription factor and establish a new mechanism by which trace metals regulate gene expression.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Copper is an essential trace metal that serves as a critical co-factor for a number of enzymes, including cytochrome oxidase for respiration and Cu,Zn-superoxide dismutase for oxidative stress protection (1, 2). When accumulated to excessive levels, copper is highly toxic due to its ability to engage in redox chemistry resulting in the generation of the destructive hydroxyl radical (3). Therefore, a delicate balance must be established and maintained between the accumulation of sufficient copper ion levels for biochemical reactions and the elevation of copper ions to toxic levels. High affinity Cu(I) transport into yeast cells requires the action of the integral membrane Cu(I) transport proteins encoded by the CTR1 and CTR3 genes and the Fe(III)/Cu(II) reductase encoded by the FRE1 gene (4-6). Transcription of the CTR1, CTR3, and FRE1 genes is activated under copper starvation conditions, while expression of these genes is potently and rapidly extinguished by exogenous Cu ion concentrations in the picomolar to nanomolar range (8). The transcriptional activation and inactivation of the yeast Cu(I) transport genes requires conserved cis-acting copper-responsive (CuRE)1 promoter elements and the Mac1 nuclear protein. In vitro the CuRE elements are specifically bound by Mac1p, while in vivo the CTR3 CuREs are occupied under copper starvation conditions and not bound when CTR3 expression is extinguished by the addition of 10 nM copper (7-11). In this work, we have investigated the mechanisms by which copper ions regulate expression of the yeast Cu(I) transport genes through Mac1p. We demonstrate that Mac1 is a stable protein in cells grown in low copper ion concentrations; however, Mac1 is degraded in a rapid, metal-specific fashion when cells are grown under high copper ion concentrations. Although Mac1 degradation is not obligatorily coupled to target gene regulation, the rapid turnover of Mac1 is essential to prevent copper ion toxicity as a consequence of sustained Cu(I) transporter gene expression. Copper ion activated degradation of the Mac1 transcription factor represents a new mechanism by which trace metals play important cellular regulatory roles.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast cells were grown under low copper ion conditions as
described (8). All molecular biology manipulations were carried out as
described (13). A coding region for a nine-amino acid epitope from the
influenza hemagglutinin HA protein (YPYDVPDYA) was inserted in frame at
the carboxyl terminus of the MAC1 or MAC1up1 open reading
frame by polymerase chain reaction from genomic DNA of a wild type
(DTY1) or MAC1up1 (DTY205) strain, respectively. The
sequence of the gene was verified and the HA epitope-tagged allele,
under the control of the MAC1 promoter, was subcloned via
HindIII (Klenow) and BamHI sites into the
SalI (Klenow) and BamHI sites of the centromeric
yeast plasmid pRS313 to generate pRSMac1(HA). The epitope-tagged
MAC1 and MAC1up1 alleles accurately
reflected the function of the unadulterated MAC1 or
MAC1up1 alleles as ascertained by equivalent
growth rates on YPE medium (1% yeast extract, 2% bactopeptone, and
3% ethanol) and copper-responsive regulation of the CTR3
gene that was indistinguishable from the parental alleles, as assessed
by RNase protection experiments when transformed into the
mac1 strain SLY2 (MATa a gal1 trp1-1 his3 ade8
ura3::Knr mac1::URA3). Plasmids
pRSMac1-REP-I and pRSMac1-REP-II were constructed by site directed
mutagenesis to generate the corresponding allele, followed by the
exchange of a 544-base pair BspEI fragment encompassing the
mutations in the MAC1 REP-I and REP-II alleles, with the same fragment
from the wild type MAC1HA gene in plasmid pRSMac1(HA). All mutations
and fusion junctions were verified by DNA sequencing. Point mutations
in MAC1 were isolated based on the inability of cells to
repress the expression of a fusion between the CTR3 promoter to the URA3 gene in the presence of 20 µM
copper. One category of uracil prototrophs mapped to the
MAC1 gene and recombination mapping and sequence analysis
demonstrated the presence of single amino acid substitutions in REP-I
designated up3 and up4. The up3 and
up4 alleles regulate CTR3 mRNA levels in
response to copper ions in a manner indistinguishable from the
up1 allele.2
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mac1 Degradation Occurs Only in the Presence of High Copper Ion Concentrations-- To investigate the mechanisms by which Mac1p senses copper levels to control transcription of the high affinity Cu(I) transport genes, both Mac1 protein and mRNA levels were measured (12, 13). Mac1p-HA contains a single HA epitope at the Mac1p carboxyl terminus and completely functionally complements a mac1 deletion strain. The analysis of Mac1p-HA from cells grown under low copper ion concentrations, and treated with increasing copper concentrations for up to 90 min, demonstrated that Mac1p-HA is stable at low copper concentrations. However, Mac1p-HA steady state levels are rapidly and nearly completely extinguished at or above 10 µM CuSO4 (Fig. 1A). The steady state levels of another yeast nuclear transcription factor, heat shock transcription factor (HSF; Ref. 14), remained constant at all copper ion concentrations over the course of these experiments (Fig. 1A). In contrast to Mac1p-HA protein levels, MAC1-HA mRNA levels were not responsive to copper ions and were constant for all samples (Fig. 1B). To ascertain if the reduction in Mac1p-HA levels parallels the inactivation of high affinity Cu(I) transporter gene expression, RNA was prepared from aliquots of the same cell cultures, and CTR3 mRNA levels were measured by RNase protection assays. The data in Fig. 1C show that, as observed previously, CTR3 mRNA levels were rapidly reduced in response to nanomolar copper ion concentrations. These data demonstrate that the loss of Mac1p-HA occurs at copper ion concentrations that exceed those required for normal inactivation of CTR3 transcription. We observed the same copper ion-mediated extinction of Mac1p steady state levels in response to high copper ion concentrations by using a single or a double MYC epitope tag at the carboxyl terminus of Mac1 (data not shown). Taken together, these results suggest that loss of Mac1p in response to high copper ion concentrations can be uncoupled from low copper ion-mediated Mac1-dependent extinction of Cu(I) transport gene transcription.
|
Mac1p Degradation Is Uncoupled from Target Gene Regulation-- The inactivation of yeast Cu(I) transporter gene transcription is highly specific for the metal ions copper and Ag(I), with responses to Cd(II) and Hg(II) only at metal concentrations 1000 times that of copper or Ag(I) (8). To further test whether the dramatic reduction in Mac1p-HA levels observed with high copper ion concentrations is obligatorily coupled with Cu(I) transporter gene regulation, Mac1p-HA and CTR3 mRNA levels were measured from cells treated with 10 µM Ag(I) or Cd(II). The data in Fig. 2A clearly demonstrate that while CTR3 expression was inactivated by Ag(I) and Cd(II) as observed previously (8), steady state levels of Mac1p-HA were not affected by either metal. Therefore, although Cu(I) transporter gene expression is inactivated by Cu and Ag(I) ions with equal efficacy, and by Cd(II) at much higher concentrations, the dramatic reduction of Mac1p-HA levels is highly specific to copper ions and is not essential for regulation of Cu(I) transport gene expression at low copper ion concentrations. Since high Cu(I) ion levels are known to activate transcription of genes encoding proteins such as the yeast metallothioneins, which protect cells from copper toxicity and Cu,Zn-superoxide dismutase (15-17), an experiment was conducted to ascertain whether the reduction in Mac1p-HA steady state levels depends on the copper ion-dependent synthesis of new proteins. Yeast cells expressing the Mac1p-HA protein were pretreated with 100 µg/ml cycloheximide for 30 min to inhibit new protein synthesis, followed by either no addition or the addition of 100 µM CuSO4. As shown in Fig. 2B, Mac1p-HA levels were stable when cells were grown under low copper ion concentrations and in the presence of cycloheximide. However, Mac1p-HA levels were dramatically reduced when cells were incubated with copper ions under conditions where protein synthesis was inhibited. The levels of yeast HSF protein remained constant under both conditions over the course of the experiment (Fig. 2B). Therefore, the reduction in Mac1p-HA levels in response to high copper ion concentrations is not dependent on the synthesis of new proteins. Furthermore, these data demonstrate that high, but not low, copper ion concentrations rapidly and specifically trigger the degradation of Mac1p-HA.
|
Mac1p Degradation Is Critical for Copper Resistance--
A
dominant allele, MAC1up1, encodes a Mac1 protein
that is only partially responsive to copper and therefore drives robust
transcription of the CTR1, CTR3, and
FRE1 genes even in the presence of high copper ion
concentrations (7-11). The MAC1up1 mutation
resides in one of two repeats that are located in the carboxyl-terminal
half of the protein, designated REP-I and REP-II, each containing 5 cysteines and 1 histidine residue (Fig.
3A, WT). To ascertain the role
of the REP-I and REP-II elements in Cu(I) transporter gene regulation
and to determine whether they are involved in copper
ion-dependent degradation of Mac1p, all of the Cys and His
residues were mutated in each independent REP element (Fig. 3A,
REP-I and REP-II). The analysis of CTR3
mRNA levels in a strain expressing the wild type Mac1p (Fig.
3B, WT) gave typical rapid and complete inactivation of
CTR3 expression. In contrast, isogenic cells with the
MAC1up1 allele (up1) exhibited
elevated basal CTR3 mRNA levels that were only
transiently repressed by copper ions. Longer exposure of cells to
copper ions resulted in sustained CTR3 expression as
compared with wild type cells (Fig. 3B, up1). The
comparison of cells expressing either the REP-I or REP-II MAC1 allele demonstrated strikingly distinct phenotypes with
respect to CTR3 gene expression. REP-I cells displayed the
transient but largely refractile response to copper ion levels similar
to that observed with MAC1up1 cells (Fig.
3B) or in Mac1up2 (11) and
Mac1up3 and Mac1up4
cells.2 Although we have ascertained that the copper
ion-dependent turnover of Mac1p is not obligatory for
CTR3 mRNA regulation, we observed in fact increased
CTR3 mRNA in strains with the Mac1up1 and
REP-I MAC1 alleles. The fact that CTR3 expression
in REP-I cells was slightly lower than in
MAC1up1 cells may be due to the multiple
mutations in REP-I rather than a single amino acid substitution in the
Mac1up1 protein. In yeast cells expressing the Mac1 REP-II
protein, CTR3 mRNA levels were undetectable (Fig.
3B, compare REP-I and REP-II). These
data demonstrate that, although identical mutations were made in the
Mac1 REP-I and REP-II elements, these elements are not functionally
equivalent with respect to their role in expression of the high
affinity Cu(I) transport genes. This conclusion is strongly supported
by additional experimental observations. First, the in vivo
selection of yeast mutations linked to the MAC1 gene that
exhibit high constitutive expression of the Cu(I) transport genes gave
rise to single amino acid substitutions in REP-I, but not REP-II (Fig.
3A and Refs. 7, 8, and 11). Second, the kinetics of copper
ion-dependent degradation of the protein expressed from the
MAC1 REP-I allele were much slower than the wild type, with
Mac1p-HA REP-I levels still detectable 90 min after copper ion
treatment (Fig. 3C). In contrast, Mac1p expressed from the MAC1 REP-II allele exhibited copper
ion-dependent degradation kinetics indistinguishable from
that of the wild type Mac1p (compare Figs. 1A and
3C). Therefore, although the Mac1p-HA expressed from the
REP-II allele is stable under low copper ion concentrations, it is
incapable of activating CTR3 expression and is similar to a
strain harboring a disrupted mac1::URA3 allele (8,
10, 11). The observation that Mac1 REP-II protein, which is defective in activation of copper transporter gene expression, is degraded as
well as the wild type protein, is likely due to the fact that copper
ions (100 µM) are transported via the low affinity copper transport system. It should be noted that single or clustered point
mutations in REP-II, in the context of a Gal4-Mac1 fusion protein,
failed to exhibit a defect in regulating a GAL UAS-driven lacZ gene in response to 100 µM copper (9). We
suggest two possible explanations. First, the fusion of Mac1 to the
Gal4 DNA binding domain would mask any requirement for REP-II in Mac1
sequence-specific DNA binding to the CuREs (11). Second, since the Mac1
REP-II mutant is subject to degradation when cells are incubated in the presence of high copper ion concentrations (Fig. 3C), it is
possible that regulation of the GAL UAS-lacZ fusion gene was
due to copper ion-dependent degradation of the Gal4-Mac1
fusion protein. Although the REP-I and MAC1up1 cells are
capable of high affinity Cu(I) transport and respiration, REP-II
mutants are incapable of respiration, presumably because they are
completely defective in expression of the high affinity Cu transport
genes (Fig. 3D). Finally, both the MAC1up1 and
REP-I mutants are sensitive to copper in a manner that parallels the
magnitude of sustained expression of the CTR3 gene, while the REP-II mutant is as resistant to copper as the parental wild type
strain. Under the conditions used (Fig. 3D) the high
affinity Cu(I) transport genes contributes only modestly to the
copper ion sensitivity of the Mac1up1 and REP-I
strains. Nonetheless, when the copper transport genes are deleted
(ctr1 ctr3
) in the Mac1up1 and REP-I
strains, significant copper resistance is restored (Fig.
3D).
|
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Robert Fuller, Randal Kaufman, and members of the Thiele laboratory for comments on this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant RO1 GM41840 (to D. J. T.) from the National Institutes of Health, Postdoctoral Fellowship-National Research Service Awards GM17067 and GM18089 (to Z. Z. and M. M. O. P.), and a Postdoctoral Fellowship from the Fonds de la Recherche en Santé du Québec (F.R.S.Q) (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.
These authors share first authorship.
§ Present address: Dept. of Biology, University of California, Santa Cruz, CA 95064.
¶ Burroughs Wellcome Toxicology Scholar. To whom correspondence should be addressed. Tel.: 313-763-5717; Fax: 313-763-4581; E-mail: dthiele{at}umich.edu.
1 The abbreviations used are: CuRE, copper-response element; HSF, heat shock factor.
2 S. Labbé and D. J. Thiele, unpublished data.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|