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PubMed2002

Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals.

Cisplatin is a chemotherapeutic drug used to treat a variety of cancers. Both intrinsic and acquired resistance to cisplatin, as well as toxicity, limit its effectiveness. Molecular mechanisms that underlie cisplatin resistance are poorly understood. Here we demonstrate that deletion of the yeast CTR1 gene, which encodes a high-affinity copper transporter, results in increased cisplatin resistance and reduced intracellular accumulation of cisplatin. Copper, which causes degradation and internalization of Ctr1 protein (Ctr1p), enhances survival of wild-type yeast cells exposed to cisplatin and reduces cellular accumulation of the drug. Cisplatin also causes degradation and delocalization of Ctr1p and interferes with copper uptake in wild-type yeast cells. Mouse cell lines lacking one or both mouse Ctr1 (mCtr1) alleles exhibit increased cisplatin resistance and decreased cisplatin accumulation in parallel with mCtr1 gene dosage. We propose that cisplatin uptake is mediated by the copper transporter Ctr1p in yeast and mammals. The link between Ctr1p and cisplatin transport may explain some cases of cisplatin resistance in humans and suggests ways of modulating sensitivity and toxicity to this important anticancer drug.

Animals↗
PubMed1997

hCTR1: a human gene for copper uptake identified by complementation in yeast.

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

Amino Acid Sequence↗
PubMed1995

Molecular characterization of a putative Arabidopsis thaliana copper transporter and its yeast homologue.

At the molecular level, little is known about the transport of copper across plant membranes. We have isolated an Arabidopsis thaliana cDNA by complementation of a mutant (ctr1-3) of Saccharomyces cerevisiae defective in high affinity copper uptake. This cDNA codes for a highly hydrophobic protein (COPT1) of 169 amino acid residues and with three putative transmembrane domains. Most noteworthy, the first 44 residues display significant homology to the methionine- and histidine-rich copper binding domain of three bacterial copper binding proteins, among these a copper transporting ATPase. Mutant yeast cells expressing COPT1 exhibit nearly wild type behavior with regard to growth on a nonfermentable carbon source and resistance to copper and iron starvation. Expression of COPT1 is also associated with an increased sensitivity to copper toxicity. Additionally, COPT1 shows significant homology to an open reading frame of 189 amino acid residues on yeast chromosome VIII. This gene (CTR2) may encode an additional yeast metal transporter able to mediate the uptake of copper. A mutation in CTR2 displays a higher level of resistance to toxic copper concentrations. Overexpression of CTR2 provides increased resistance to copper starvation and is also associated with an increased sensitivity to copper toxicity. The amino acid sequence of CTR2, like Arabidopsis COPT1, contains three potential transmembrane domains. Taken together, the data suggest that a plant metal transporter, which is most likely involved in the transport of copper, has been identified.

Amino Acid Sequence↗
PubMed1997

The yeast Fre1p/Fre2p cupric reductases facilitate copper uptake and are regulated by the copper-modulated Mac1p activator.

Fre1p and Fre2p are ferric reductases which account for the total plasma membrane associated activity, a prerequisite for iron uptake, in Saccharomyces cerevisiae. The two genes are transcriptionally induced by iron depletion. In this communication, we provide evidence that Fre2p has also cupric reductase activity, as has been previously shown for Fre1p (Hassett, R., and Kosman, D.J. (1995) J. Biol. Chem. 270, 128-134). Both Fre1p and Fre2p enzymes are functionally significant for copper uptake, as monitored by the accumulation of the copper-regulated CUP1 and CTR1 mRNAs in fre1Delta, fre2Delta, and fre1Deltafre2Delta mutant strains. However, only Fre1p activity is induced by copper depletion, even in the presence of iron. This differential copper-dependent regulation of Fre1p and Fre2p is exerted at the transcriptional level of the two genes. We have shown that Mac1p, known to affect the basal levels of FRE1 gene expression (Jungmann, J., Reins, H.-A., Lee, J., Romeo, A., Hassett, R., Kosman, D., and Jentsch, S. (1993) EMBO J. 12, 5051-5056), accounts for both the copper-dependent induction of FRE1 and down-regulation of FRE2 gene. Finally, Mac1p transcriptional activation function is itself modulated by the availability of copper.

Carrier Proteins↗
PubMed1997

Copper-specific transcriptional repression of yeast genes encoding critical components in the copper transport pathway.

Copper is an essential micronutrient that is toxic in excess. To maintain an adequate yet non-toxic concentration of copper, cells possess several modes of control. One involves copper uptake mediated by genes encoding proteins that play key roles in high affinity copper transport. These include the FRE1-encoded Cu2+/Fe3+ reductase and the CTR1 and CTR3-encoded membrane-associated copper transport proteins. Each of these genes is transcriptionally regulated as a function of copper availability: repressed when cells are grown in the presence of copper and highly activated during copper starvation. Our data demonstrate that repression of CTR3 transcription is exquisitely copper-sensitive and specific. Although copper represses CTR3 gene expression at picomolar metal concentrations, cadmium and mercury down-regulate CTR3 expression only at concentrations 3 orders magnitude greater. Furthermore, copper-starvation rapidly and potently induces CTR3 gene expression. We demonstrate that the CTR1, CTR3, and FRE1 genes involved in high affinity copper uptake share a common promoter element, TTTGCTC, which is necessary for both copper repression and copper-starvation activation of gene expression. Furthermore, the Mac1p is essential for down- or up-regulation of the copper-transport genes. In vivo footprinting studies reveal that the cis-acting element, termed CuRE (copper-response element), is occupied under copper-starvation and accessible to DNA modifying agents in response to copper repression, and that this regulated occupancy requires a functional MAC1 gene. Therefore, yeast cells coordinately express genes involved in high affinity copper transport through the action of a common signaling pathway.

Antiporters↗
PubMed1997

Homeostatic regulation of copper uptake in yeast via direct binding of MAC1 protein to upstream regulatory sequences of FRE1 and CTR1.

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 and CTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1 is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1(up2), is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to the MAC1(up1) allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA ... TGAGCAAA, appearing within the 5'-flanking region of the CTR1 promoter, is necessary and sufficient for the copper- and MAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in the FRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264-279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

Alleles↗
PubMed1997

Restriction of copper export in Saccharomyces cerevisiae to a late Golgi or post-Golgi compartment in the secretory pathway.

The CCC2 gene in the yeast Saccharomyces cerevisiae encodes a P-type ATPase (Ccc2p) required for the export of cytosolic copper to the extracytosolic domain of a copper-dependent oxidase, Fet3p. Ccc2p appears to be both a structural and functional homolog of ATPases impaired in two human disorders of intracellular copper transport, Menkes disease and Wilson disease. In the present work, three approaches were used to determine the locus of Ccc2p-dependent copper export within the secretory pathway. First, like ccc2 mutants, sec mutants blocked in the secretory pathway at steps prior to and including the Golgi complex failed to deliver radioactive copper to Fet3p. Second, also like ccc2 mutants, vps33 and certain other mutants with defects in post-Golgi sorting exhibited phenotypes traceable to deficient copper delivery to Fet3p. These findings were sufficient to explain the respiratory deficiency of these mutants. Third, immunofluorescence microscopy revealed that Ccc2p was distributed among several punctate foci within wild-type cells, consistent with late Golgi or post-Golgi localization. Thus, copper export by Ccc2p appears to be restricted to a late or post-Golgi compartment in the secretory pathway.

Adenosine Triphosphatases↗
PubMed1999

Evidence for (Mac1p)2.DNA ternary complex formation in Mac1p-dependent transactivation at the CTR1 promoter.

The Mac1 protein in Saccharomyces cerevisiae is required for the expression CTR1 and FRE1, which, respectively, encode the copper permease and metal reductase that participate in copper uptake. Mac1p binds to a core GCTC sequence present as a repeated unit in the promoters of both genes. We show here that Mac1p DNA binding required an intact N-terminal protein domain that includes a likely zinc finger motif. This binding was enhanced by the presence of a TATTT sequence immediately 5' to the core GCTC, in contrast to a TTTTT one. This increased binding was demonstrated clearly in vitro in electrophoretic mobility shift assays that showed Mac1p.DNA complex formation to a single TATTTGCTC element but not to a TTTTTGCTC one. Furthermore, the fraction of Mac1p in a ternary (Mac1p)2.DNA complex in comparison to a binary Mac1p.DNA complex increased when the DNA included two TATTTGCTC elements. A similar increase in ternary complex formation was demonstrated upon homologous mutation of the FRE1 Mac1p-dependent promoter element. The in vivo importance of this ternary complex formation at the CTR1 promoter was indicated by the stronger trans-activity of this promoter mutated to contain two TATTT elements and the attenuated activity of a mutant promoter containing two TTTTT elements that in vitro supported only a weak ternary complex signal in the shift assay. The stronger binding to TATTT appeared due to a more favorable protein contact with adenine in comparison to thymine at this position. An in vivo two-hybrid analysis demonstrated a Mac1p-Mac1p protein-protein interaction. This Mac1p-Mac1p interaction may promote (Mac1p)2.DNA ternary complex formation at Mac1p-responsive upstream activating sequences.

Base Sequence↗
PubMed1999

The yeast transcription factor Mac1 binds to DNA in a modular fashion.

Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

Amino Acid Sequence↗
PubMed1999

Structure-function analysis of the protein-binding domains of Mac1p, a copper-dependent transcriptional activator of copper uptake in Saccharomyces cerevisiae.

The Mac1 protein in Saccharomyces cerevisiae is essential for the expression of yeast high affinity copper uptake. A positive transcription factor, Mac1p binds via its N-terminal domain to GCTC elements in the promoters of CTR1 and FRE1, encoding a copper permease and metal reductase, respectively. Mac1p-dependent transcriptional activation is negatively regulated by copper. We have mapped the domains in Mac1p responsible for its nuclear localization and for the protein-protein interactions that underlie its transcriptional activity. Immunofluorescence studies indicate that Mac1p contains two nuclear localization signals, one each in the N- and C-terminal halves of the protein. Yeast one-hybrid analysis demonstrates that the copper-dependent transcriptional activity in Mac1p resides primarily in a cysteine-rich element encompassing residues 264-279. Two-hybrid analysis indicates that a copper-independent Mac1p-Mac1p interaction linked to DNA binding is due primarily to a predicted helix in the C-terminal region of the protein encompassing residues 388-406. Point mutations within this putative helix abrogate the Mac1-Mac1 interaction in vivo and formation of a ternary (Mac1p)(2).DNA complex in vitro. When produced in normal abundance, Mac1pI396D and Mac1pF400D helix mutants do not support transcriptional activation in vivo consistent with an essential Mac1p dimerization in transcriptional activation. Lastly, the one- and two-hybrid data indicate that an intramolecular interaction between the DNA-binding and transactivation domains negatively modulates Mac1p activity.

Binding Sites↗
PubMed1999

A copper-sensing transcription factor regulates iron uptake genes in Schizosaccharomyces pombe.

Copper and iron serve essential functions as catalytic co-factors in a wide variety of critical cellular enzymes. Studies in yeast have demonstrated an absolute dependence upon copper acquisition for proper assembly and function of the iron transport machinery. We have cloned genes for a high affinity copper transporter (Ctr4) and copper-sensing transcription factor (Cuf1) from Schizosaccharomyces pombe. Interestingly, the primary structure of Ctr4 and a putative human high affinity copper transport protein, hCtr1, suggests that they are derived from a fusion of the functionally redundant but structurally distinct Ctr1 and Ctr3 copper transporters from Saccharomyces cerevisiae. Furthermore, although Cuf1 activates ctr4(+) gene expression under copper starvation conditions, under these same conditions Cuf1 directly represses expression of genes encoding components of the iron transport machinery. These studies have identified an evolutionary step in which copper transport modules have been fused, and describe a mechanism by which a copper-sensing factor directly represses expression of the iron uptake genes under conditions in which the essential copper co-factor is scarce.

Antiporters↗
PubMed2002

Copper ion-sensing transcription factor Mac1p post-translationally controls the degradation of its target gene product Ctr1p.

Copper ion uptake must be regulated to avoid both deficiency and excess because its essential yet toxic biological nature depends on the concentration. Yeast copper uptake is controlled at both the transcriptional and post-translational levels. The transcription of CTR1 and CTR3, encoding high affinity copper ion transporters, is regulated by the copper ion-sensing transcription factor Mac1p through the cis-acting copper ion-responsive elements in CTR1 and CTR3 promoters. Ctr1p is known to undergo degradation in cells exposed to high copper levels. We report that Mac1p is also required for copper-dependent Ctr1p degradation. Both mutations within a conserved copper ion binding motif, the "Cu-fist" in the Mac1p DNA-binding domain, and within a metal ion binding motif, REP-III located in the cytosolic domain of Ctr1p, cause defects in Ctr1p turnover. Furthermore, we show that the Mac1p limits intracellular copper accumulation likely by controlling Ctr1p degradation. The findings have uncovered an unprecedented mechanism by which a transcription factor not only regulates its target gene transcription but also controls the degradation of its target gene product.

Base Sequence↗
PubMed2000

Phosphorylation and Cu+ coordination-dependent DNA binding of the transcription factor Mac1p in the regulation of copper transport.

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.

Base Sequence↗
PubMed2001

Biochemical characterization of the human copper transporter Ctr1.

The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated (64)Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.

Biopolymers↗
PubMed2002

Biochemical and genetic analyses of yeast and human high affinity copper transporters suggest a conserved mechanism for copper uptake.

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.

Amino Acid Sequence↗
PubMed2002

Molecular characterization of hCTR1, the human copper uptake protein.

We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.

Amidohydrolases↗
PubMed2002

Characterization of mouse embryonic cells deficient in the ctr1 high affinity copper transporter. Identification of a Ctr1-independent copper transport system.

The trace metal copper is an essential cofactor for a number of enzymes that have critical roles in biological processes, but it is highly toxic when allowed to accumulate in excess of cellular needs. Consequently, homeostatic copper metabolism is maintained by molecules involved in copper uptake, distribution, excretion, and incorporation into copper-requiring enzymes. Previously, we reported that overexpression of the human or mouse Ctr1 copper transporter stimulates copper uptake in mammalian cells, and deletion of one Ctr1 allele in mice gives rise to tissue-specific defects in copper accumulation and in the activities of copper-dependent enzymes. To investigate the physiological roles for mammalian Ctr1 protein in cellular copper metabolism, we characterized wild type, Ctr1 heterozygous, and Ctr1 homozygous knock-out cells isolated from embryos obtained by the inter-cross of Ctr1 heterozygous mice. Ctr1-deficient mouse embryonic cells are viable but exhibit significant defects in copper uptake and accumulation and in copper-dependent enzyme activities. Interestingly, Ctr1-deficient cells exhibit approximately 30% residual copper transport activity that is saturable, with a K(m) of approximately 10 microm, with biochemical features distinct from that of Ctr1. These observations demonstrate that, although Ctr1 is critical for both cellular copper uptake and embryonic development, mammals possess additional biochemically distinct functional copper transport activities.

Animals↗