NORMAL GENOMIC

• Tohoyama H, Kadota H, Shiraishi E, Inouhe M, Joho M
• Induction for the expression of yeast metallothionein gene, CUP1, by cobalt.
• Microbios. 2001; 104: 99-104
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• Induction for the expression of the metallothionein gene, CUP1, in the yeast Saccharomyces cerevisiae by cobalt was examined using a reporter gene with the promoter of this gene fused to the coding region of lacZ. The expression of the gene was induced by cobalt as well as by copper and silver ions. The activity of beta-galactosidase showed high levels after treatment with 1.0 mM cobalt chloride. It has been reported that the induction for the transcription of CUP1 by copper and silver is mediated by the Ace1 transcription factor. However, the expression of the gene by cobalt occurred in yeast cells lacking the Ace1 factor. These results suggest the presence of a novel cobalt-specific transcription factor for the CUP1 gene.

• Winge DR
• Copper-regulatory domain involved in gene expression.
• Prog Nucleic Acid Res Mol Biol. 1998; 58: 165-95
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• Copper ion homeostasis in yeast is maintained through regulated expression of genes involved in copper ion uptake, Cu(I) sequestration, and defense against reactive oxygen intermediates. Positive and negative copper ion regulation is observed, and both effects are mediated by Cu(I)-sensing transcription factors. The mechanism of Cu(I) regulation is distinct for transcriptional activation versus transcriptional repression. Cu(I) activation of gene expression in S. cerevisiae and C. glabrata occurs through Cu-regulated DNA binding. The activation process involves Cu(I) cluster formation within the regulatory domain in Ace1 and Amt1. Cu(I) binding stabilizes a specific conformation capable of high-affinity interaction with specific DNA promoter sequences. Cu(I)-activated transcription factors are modular proteins in which the DNA-binding domain is distinct from the domain that mediates transcriptional activation. The all-or-nothing formation of the polycopper cluster permits a graded response of the cell to environmental copper. Cu(I) triggering may involve a metal exchange reaction converting Ace1 from a Zn(II)-specific conformer to a clustered Cu(I) conformer. The Cu(I) regulatory domain occurs in transcription factors from S. cerevisiae and C. glabrata. Sequence homologs are also known in Y. lipolytica and S. pombe, although no functional information is available for these candidate regulatory molecules. The presence of the Cu(I) regulatory domain in four distinct yeast strains suggests that this Cu-responsive domain may occur in other eukaryotes. Cu-mediated repression of gene expression in S. cerevisiae occurs through Cu(I) regulation of Mac1. Cu(I) binding to Mac1 appears to inhibit the transactivation domain. The Cu(I) specificity of this repression is likely to arise from formation of a polycopper thiolate cluster.

• Pena MM, Koch KA, Thiele DJ
• Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae.
• Mol Cell Biol. 1998; 18: 2514-23
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• The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity. These studies (i) demonstrate that the nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

• Koch KA, Thiele DJ
• Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.
• Mol Cell Biol. 1996; 16: 724-34
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• Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed mutagenesis, and quantitative in vitro DNA binding studies. The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.

• Mascorro-Gallardo JO, Covarrubias AA, Gaxiola R
• Construction of a CUP1 promoter-based vector to modulate gene expression in Saccharomyces cerevisiae.
• Gene. 1996; 172: 169-70
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• We report the construction and characterization of a yeast shuttle-expression vector that can be used to express genes in yeast in a modulated form. This vector is centromeric, with a polylinker that optionally can provide an ATG start of translation. Expression features are based on the CUP1 yeast metallothionein gene promoter, which can be tightly modulated by copper.

• Strain J, Culotta VC
• Copper ions and the regulation of Saccharomyces cerevisiae metallothionein genes under aerobic and anaerobic conditions.
• Mol Gen Genet. 1996; 251: 139-45
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• We have previously reported that the Saccharomyces cerevisiae CRS5 metallothionein gene is negatively regulated by oxygen. The mechanism of this repression was the focus of the current study. We observed that the aerobic repression of CRS5 is rapid and occurs within minutes of exposing anaerobic cultures to air. Furthermore, the CUP1 metallothionein gene of S. cerevisiae was found to be subject to a similar downregulation of gene expression. We provide evidence that the aerobic repression of yeast metallothioneins involves copper ions and Ace1, the copper trans-activator of CUP1 and CRS5 gene expression. A functional Ace1 binding site was found to be necessary for the aerobic repression of CRS5. Moreover, the aerobic down-regulation of the metallothioneins was abolished when cells were treated with elevated levels of copper. Our studies show that anaerobic cultures accumulate higher levels of copper than do aerobic cells and that this copper is rapidly lost when cells are exposed to air. In fact, the kinetics of this copper loss closely parallels the kinetics of CUP1 and CRS5 gene repression. The yeast metallothionein genes, therefore, serve as excellent markers for variations in copper accumulation and homeostasis that occur in response to changes in the oxidative status of the cell.

• Culotta VC, Howard WR, Liu XF
• CRS5 encodes a metallothionein-like protein in Saccharomyces cerevisiae.
• J Biol Chem. 1994; 269: 25295-302
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• Protection from copper toxicity in the bakers' yeast Saccharomyces cerevisiae involves the action of a copper binding metallothionein encoded by the CUP1 locus. To identify additional factors contributing to copper ion homeostasis and detoxification, we screened a genomic library for genes that confer high levels of copper resistance to yeast strains lacking CUP1. This screen led to the identification of the CRS5 (copper-resistant suppressor) gene. By sequence analyses, CRS5 encodes a small molecular weight cysteine-rich protein with an amino acid sequence bearing all the features of a eukaryotic metallothionein. The CRS5 polypeptide exhibits a striking similarity to a number of mammalian and invertebrate metallothioneins, yet shares surprisingly little homology with CUP1. In yeast, CRS5 is expressed as a 0.5-kilobase mRNA that is regulated both by copper ions and by oxidative stress, and expression is dependent upon ACE1, a copper and DNA binding transcription factor also known to regulate CUP1. Deletion of the chromosomal CRS5 locus was found to increase cellular sensitivity to copper, but not cadmium, toxicity. These studies support an important role for the CRS5 metallothionein-like protein in copper homeostasis and detoxification.

• Zhou P, Thiele DJ
• Rapid transcriptional autoregulation of a yeast metalloregulatory transcription factor is essential for high-level copper detoxification.
• Genes Dev. 1993; 7: 1824-35
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• Copper detoxification in the yeast Candida glabrata is carried out in large part by a family of metallothionein (MT) genes: a unique MT-I gene, a tandemly amplified MT-IIa gene, and a single unlinked MT-IIb gene. In response to elevated environmental copper levels, members of this MT gene family are transcriptionally activated by a copper-dependent, sequence-specific DNA-binding transcription factor, AMT1. AMT1 shares several structural and functional features with the Saccharomyces cerevisiae copper metalloregulatory transcription factor ACE1, which is constitutively expressed and poised for rapid transcriptional responses to the toxic metal copper. In this paper, we demonstrate that AMT1 is subject to positive transcriptional autoregulation, which is exerted through binding of copper-activated AMT1 to a single copper responsive element in the AMT1 promoter. A nonautoregulatory amt1 mutant displayed a marked decrease in both copper tolerance and expression of the MT-II genes, which are critical for high-level copper detoxification in Candida glabrata. Kinetic analysis demonstrated the remarkably rapid AMT1 mRNA accumulation in the presence of copper, which is followed by increased expression of the metallothionein gene products. These results demonstrate that AMT1-positive autoregulation plays a critical role in metal detoxification and suggest that the rapid autoactivation of the AMT1 metalloregulatory transcription factor biosynthesis is essential for C. glabrata to quickly build up a cellular defense line to protect cells upon exposure to high environmental copper levels.

• Zhou P, Szczypka MS, Sosinowski T, Thiele DJ
• Expression of a yeast metallothionein gene family is activated by a single metalloregulatory transcription factor.
• Mol Cell Biol. 1992; 12: 3766-75
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• The opportunistic pathogenic yeast Candida glabrata elicits at least two major responses in the presence of high environmental metal levels: transcriptional induction of the metallothionein gene family by copper and the appearance of small (gamma-Glu-Cys)nGly peptides in the presence of cadmium. On the basis of a trans-activation selection scheme in the baker's yeast Saccharomyces cerevisiae, we previously isolated a C. glabrata gene which encodes a copper-activated DNA-binding protein designated AMT1. AMT1 forms multiple specific DNA-protein complexes with both C. glabrata MT-I and MT-IIa promoter DNA fragments. In this report, we localize and define the AMT1-binding sites in the MT-I and MT-IIa promoters and characterize the mode of AMT1 binding. Furthermore, we demonstrate that the AMT1 protein trans activates both the MT-I and MT-IIa genes in vivo in response to copper and that this activation is essential for high-level copper resistance in C. glabrata. Although AMT1-mediated trans activation of the C. glabrata metallothionein genes is essential for copper resistance, AMT1 is completely dispensable for cadmium tolerance. The distinct function that metallothionein genes have in copper but not cadmium detoxification in C. glabrata is in contrast to the role that metallothionein genes play in tolerance to multiple metals in higher organisms.

• Carri MT, Galiazzo F, Ciriolo MR, Rotilio G
• Evidence for co-regulation of Cu,Zn superoxide dismutase and metallothionein gene expression in yeast through transcriptional control by copper via the ACE 1 factor.
• FEBS Lett. 1991; 278: 263-6
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• Saccharomyces cerevisiae mutant strain DTY26, lacking ACE1, the protein mediator for the induction of metallothionein gene expression, is unable to increase Cu,Zn superoxide dismutase mRNA in response to copper. In the wild-type strain DTY22 transcription of both Cu,Zn superoxide dismutase and metallothionein genes is induced by copper and silver, as expected on the basis of previous results indicating that ACE1 binds only Ag(I) besides Cu(I). We conclude that at the transcriptional level Cu,ZnSOD is co-regulated with metallothionein. Furthermore, structural similarities between the two promoters were found, which could explain the co-regulation effect and the quantitative differences in the response of the two genes to copper.

• Gralla EB, Thiele DJ, Silar P, Valentine JS
• ACE1, a copper-dependent transcription factor, activates expression of the yeast copper, zinc superoxide dismutase gene.
• Proc Natl Acad Sci U S A. 1991; 88: 8558-62
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• Copper, zinc superoxide dismutase (SOD1 gene product) (superoxide:superoxide oxidoreductase, EC 1.15.1.1) is a copper-containing enzyme that functions to prevent oxygen toxicity. In the yeast Saccharomyces cerevisiae, copper levels exert some control over the level of SOD1 expression. We show that the ACE1 transcriptional activator protein, which is responsible for the induction of yeast metallothionein (CUP1) in response to copper, also controls the SOD1 response to copper. A single binding site for ACE1 is present in the SOD1 promoter region, as demonstrated by DNase I protection and methylation interference experiments, and is highly homologous to a high-affinity ACE1 binding site in the CUP1 promoter. The functional importance of this DNA-protein interaction is demonstrated by the facts that (i) copper induction of SOD1 mRNA does not occur in a strain lacking ACE1 and (ii) it does not occur in a strain containing a genetically engineered SOD1 promoter that lacks a functional ACE1 binding site.

• Culotta VC, Hsu T, Hu S, Furst P, Hamer D
• Copper and the ACE1 regulatory protein reversibly induce yeast metallothionein gene transcription in a mouse extract.
• Proc Natl Acad Sci U S A. 1989; 86: 8377-81
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• We describe a cell-free system in which the transcription of the yeast metallothionein gene is inducible by the addition of metal ions plus a specific regulatory protein. Efficient transcription requires the complete yeast ACE1 metalloregulatory protein, including both its DNA-binding and transactivation domains; a mouse nuclear extract providing RNA polymerase and general transcription factors; a template containing the ACE1 binding site; and Cu(I). Because the binding of ACE1 to DNA is dependent on Cu, it is possible to inhibit transcription by the use of Cu-complexing agents such as CN-. We have used this specific inhibition to show that the ACE1 regulatory protein is required for the maintenance as well as the formation of a functional preinitiation complex. The ability to reversibly induce yeast metallothionein gene transcription in vitro provides a powerful system for determining the molecular mechanism of a simple eukaryotic regulatory circuit.

• Huibregtse JM, Engelke DR, Thiele DJ
• Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences.
• Proc Natl Acad Sci U S A. 1989; 86: 65-9
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• Copper-inducible transcription of the yeast metallothionein gene (CUP1) occurs by means of cis-acting upstream activation sequences (UAS) and trans-acting cellular factors. We have used a high-resolution chromosomal footprinting technique to detect the interaction of cellular factors with UASCUP1. Our results demonstrate that one or more cellular factors bind to UASCUP1 in a copper-inducible fashion. This copper-inducible binding is enhanced in a yeast strain that harbors several copies of the positive regulatory gene ACE1 and is not detectable in yeast cells that contain a nonfunctional (ace1-delta 1) locus. The correlation between yeast metallothionein gene activation and copper-inducible occupation of UASCUP1 sequences suggests that the binding of metallothionein transcriptional regulatory factors to cis-acting control sequences is copper-inducible.

• Welch J, Fogel S, Buchman C, Karin M
• The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.
• EMBO J. 1989; 8: 255-60
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• The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.

• Furst P, Hamer D
• Cooperative activation of a eukaryotic transcription factor: interaction between Cu(I) and yeast ACE1 protein.
• Proc Natl Acad Sci U S A. 1989; 86: 5267-71
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• Cu ions activate yeast metallothionein gene transcription by altering the conformation and DNA-binding activity of the ACE1 transcription factor. We show that this conformational switch occurs in an all-or-none highly cooperative fashion (Hill coefficient = 4). Analysis of the subunit composition of ACE1 bound to DNA indicates that cooperativity results from the binding of multiple Cu(I) ions to the cysteine-rich DNA-binding domain. Surprisingly, DNA has little effect on the interaction between Cu(I) and ACE1 as assayed by partial proteolysis; this suggests that the effect of the metal on DNA binding is primarily kinetic rather than thermodynamic. Although Ag(I) also activates ACE1, it acts less cooperatively than the smaller Cu(I) ion and the resulting metalloprotein has a reduced affinity for DNA. The cooperative interaction between Cu and ACE1 allows the cell to respond to a small change in metal concentration by a large change in gene expression.

• Wright CF, Hamer DH, McKenney K
• Autoregulation of the yeast copper metallothionein gene depends on metal binding.
• J Biol Chem. 1988; 263: 1570-4
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• The yeast CUP1 gene product, copper metallothionein, acts to repress the basal transcription of its own structural gene. By creating a series of truncation and amino acid substitutions in CUP1, we show that the ability of the protein to autoregulate is directly correlated to its ability to bind and detoxify copper. These results support a model in which metallothionein controls the level of free intracellular copper available to interact with positive transcription factors. In addition, mutations in chemically equivalent cysteine residues were functionally dissimilar, suggesting that partial sites in the molecule are critical for the formation of the sulfur-metal cluster.

• Thiele DJ
• ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene.
• Mol Cell Biol. 1988; 8: 2745-52
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• Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein. Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels. This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper. The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor. The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant. Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus. ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5. The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

• Butt TR, Sternberg E, Herd J, Crooke ST
• Cloning and expression of a yeast copper metallothionein gene.
• Gene. 1984; 27: 23-33
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• The induction of a copper-binding metallothionein (Cu-MT) was studied in yeast, Saccharomyces cerevisiae, and a relationship between copper resistance and intracellular levels of Cu-MT in these eukaryotes was established. Poly(A)-containing RNA from a copper-resistant (Cur) yeast strain, which synthesized abundant quantities of Cu-MT and in which Cu-MT gene transcription was enhanced 50-fold upon exposure to CuSO4, was used to screen yeast genomic DNA clones. Restriction analysis revealed common XbaI and KpnI sites in five genomic clones isolated. The transcription of these clones was regulated by copper. Transformation of a copper-sensitive (Cus) yeast strain by one of these clones confers copper resistance in yeast. The results suggest that the expression of the Cu-MT gene is, in part, responsible for mediating copper resistance in yeast.