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We present the design, synthesis, spectroscopy, and biological applications of Mitochondrial Coppersensor-1 (Mito-CS1), a new type of targetable fluorescent sensor for imaging exchangeable mitochondrial copper pools in living cells. Mito-CS1 is a bifunctional reporter that combines a Cu+-responsive fluorescent platform with a mitochondrial-targeting triphenylphosphonium moiety for localizing the probe to this organelle. Molecular imaging with Mito-CS1 establishes that this new chemical tool can detect changes in labile mitochondrial Cu+in a model HEK 293T cell line as well as in human fibroblasts. Moreover, we utilized Mito-CS1 in a combined imaging and biochemical study in fibroblasts derived from patients with mutations in the two synthesis of cytochrome coxidase 1 and 2 proteins (SCO1and SCO2), each of which is required for assembly and metalation of functionally active cytochrome coxidase (COX). Interestingly, we observe that although defects in these mitochondrial metallochaperones lead to a global copper deficiency at the whole cell level, total copper and exchangeable mitochondrial Cu+pools in SCO1and SCO2patient fibroblasts are largely unaltered relative to wild-type controls. Our findings reveal that the cell maintains copper homeostasis in mitochondria even in situations of copper deficiency and mitochondrial metallochaperone malfunction, illustrating the importance of regulating copper stores in this energy-producing organelle.

The study of patient tissues and cell lines shows that SCO1 and SCO2 function collaboratively to generate a redox-dependent signal that is transduced from mitochondria to the cytosol by COX19, where it is relayed to ATP7A to regulate the rate of copper efflux from the cell.

Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.

The twin-Cx9C motif protein Pet191 is essential for cytochrome c oxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx9C motif class of proteins in being independent of the Mia40 pathway.

ABSTRACTThe twin-Cx9C motif protein Pet191 is essential for cytochrome coxidase maturation. The motif Cys residues are functionally important and appear to be present in disulfide linkages within a large oligomeric complex associated with the mitochondrial inner membrane. The import of Pet191 differs from that of other twin-Cx9C motif class of proteins in being independent of the Mia40 pathway.

ABSTRACTAce1 and Mac1 undergo reciprocal copper metalloregulation in yeast cells. Mac1 is functional as a transcriptional activator in copper-deficient cells, whereas Ace1 is a transcriptional activator in copper-replete cells. Cells undergoing a transition from copper-deficient to copper-sufficient conditions through a switch in the growth medium show a rapid inactivation of Mac1 and a corresponding rise in Ace1 activation. Cells analyzed after the transition show a massive accumulation of cellular copper. Under these copper shock conditions we show, using two epitope-tagged variants of Mac1, that copper-mediated inhibition of Mac function is independent of induced protein turnover. The transcription activity of Mac1 is rapidly inhibited in the copper-replete cells, whereas chromatin immunoprecipitation studies showed only partial copper-induced loss of DNA binding. Thus, the initial event in copper inhibition of Mac1 function is likely copper inhibition of the transactivation activity. Copper inhibition of Mac1 in transition experiments is largely unaffected in cells overexpressing copper-binding proteins within the nucleus. Likewise, high expression of a copper-binding, non-DNA-binding Mac1 mutant is without effect on the copper activation of Ace1. Thus, metalloregulation of Ace1 and Mac1 occurs independently.

The third isoform of mammalian metallothioneins (MT-3), mainly expressed in brain and down-regulated in Alzheimer's disease, exhibits neuroinhibitory activity in vitroand a highly flexible structure that distinguishes it from the widely expressed MT-1/-2 isoforms. Previously, we showed that two conserved prolyl residues of MT-3 are crucial for both the bioactivity and cluster dynamics of this isoform. We have now used genetic engineering to introduce these residues into mouse MT-1. The S6P,S8P MT-1 mutant is inactive in neuronal survival assays. However, the additional introduction of the unique Thr5 insert of MT-3 resulted in a bioactive MT-1 form. Temperature-dependent and saturation transfer113Cd NMR experiments performed on the113Cd-reconstituted wild-type and mutant Cd7-MT-1 forms revealed that the gain of MT-3-like neuronal inhibitory activity is paralleled by an increase in conformational flexibility and intersite metal exchange in the N-terminal Cd3-thiolate cluster. The observed correlation suggests that structure/cluster dynamics are critical for the biological activity of MT-3. We propose that the interplay between the specific Pro-induced conformational requirements and those of the metal-thiolate bonds gives rise to an alternate and highly fluctuating cluster ensemble kinetically trapped by the presence of the5TCPCP9motif. The functional significance of such heterogeneous cluster ensemble is discussed.

Cox11 is a protein essential for respiratory growth and has been implicated in the assembly of the Cu(B) site of cytochrome c oxidase. In the present study, we demonstrate that Cox11 is a copper-binding protein. The soluble C-terminal domain of Cox11 forms a dimer that coordinates one Cu(I) per monomer via three thiolate ligands. The two Cu(I) ions in the dimer exist in a binuclear cluster and appear to be ligated by three conserved Cys residues. Mutation of any of these Cys residues reduces Cu(I) binding and confers respiratory incompetence. Cytochrome c oxidase activity is reduced in these mutants. Thus, the residues important for Cu(I) binding correlate with in vivo function, suggesting that Cu(I) binding is important in Cox11 function.

Sco1 is a conserved essential protein, which has been implicated in the delivery of copper to cytochrome coxidase, the last enzyme of the electron transport chain. In this study, we show for the first time that the purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer. X-ray absorption spectroscopy suggests that the Cu(I) is ligated via three ligands, and we show that two cysteines, present in a conserved motif C XXXC, and a conserved histidine are involved in Cu(I) ligation. The mutation of any one of the conserved residues in Sco1 expressed in yeast abrogates the function of Sco1 resulting in a non-functional cytochrome coxidase complex. Thus, the function of Sco1 correlates with Cu(I) binding. Data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.

The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and theYRO2homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c andYRO2appears to be direct. Induction of HAA1using a GAL1/HAA1fusion gene resulted in rapid galactose-induced expression of both HAA1and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochromecoxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome coxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys → Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys → Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.

In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates copper-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiaegenes, including CTR1,CTR3, FRE1, FRE7, YFR055w, and YJL217w. Two of the last three newly identified Mac1 target genes have no known function; the third, YFR055w, is homologous to cystathionine γ-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1up1, are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells because of constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1andCRS5and two genes, FET3and FTR1, in the iron uptake system. Copper-induced FET3andFTR1expression arises from an indirect copper effect on cellular iron pools.

Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the264CXC(X)4CXC(X)2C(X)2H279C1 motif. The lone exception was a L260S substitution. Two additionalMAC1mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.

The Zap1 transcriptional activator ofSaccharomyces cerevisiaeplays a major role in zinc homeostasis by inducing the expression of several genes under zinc-limited growth conditions. This activation of gene expression is mediated by binding of the protein to one or more zinc-responsive elements present in the promoters of its target genes. To better understand how Zap1 functions, we mapped its DNA binding domain using a combined in vivoand in vitroapproach. Our results show that the Zap1 DNA binding domain maps to the carboxyl-terminal 194 amino acids of the protein; this region contains five of its seven potential zinc finger domains. Fusing this region to the Gal4 activation domain complemented a zap1Δ mutation for low zinc growth and also conferred high level expression on a zinc-responsive element-lacZreporter. In vitro, the purified 194-residue fragment bound to DNA with a high affinity (dissociation constant in the low nanomolar range) similar to that of longer fragments of Zap1. Furthermore, by deletion and site-directed mutagenesis, we demonstrated that each of the five carboxyl-terminal zinc fingers are required for high affinity DNA binding.

Abstract:  Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu12MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu4MT, Cu6MT, and Cu12MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu4 to Cu12MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs.

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 Mac1t) with the two CuRE sites found in the yeast CTR1promoter. We conclude from these experiments that Mac1tbinds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5′ end of the CTR1CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

A metallothionein cDNA was generated from pea ( Pisum sativumL.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of ∼6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

A metallothionein cDNA was generated from pea (Pisum sativumL.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium‐containing material after DEAE‐cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of ∼6 g.atoms per mole of protein was estimated. N‐terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine‐rich (putative) metal‐binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Cadmium-resistant Saccharomyces cerevisiaestrain 301N exhibits high basal as well as cadmium-induced expression of the CUP1metallothionein gene. Since regulation of CUP1is usually restricted to copper ions, our goal was to identify the factor responsible for the high metallothionein levels in strain 301N. The gene responsible for the observed phenotype is a spontaneously mutated heat shock transcription factor gene (HSF1). A double, semidominant HSF1mutant with substitutions at codons 206 and 256 within the DNA-binding domain of the heat shock factor (HSF) confers two phenotypes. The first phenotype is elevated transcriptional activity of the HSF mutant (HSF301), which results in constitutive thermotolerance. A second HSF301 phenotype is enhanced binding affinity for the heat shock element (HSE) within the CUP15′-sequences, resulting in high basal transcription of metallothionein. The CUP1HSE is a minimal heat shock element containing only two perfectly spaced inverted repeats of the basic nGAAn block. Cells containing HSF301 are resistant to cadmium salts. The single R206S mutation is responsible for the high affinity binding to the CUP1HSE. In addition, the R206S HSF substitution exhibits constitutive transcriptional activation from a consensus HSE (HSE2). The F256Y substitution in HSF attenuates the effects of R206S on the consensus HSE2, but not on the CUP1HSE.

The three dimensional structure of the N-terminal domain (residues 1–42) of the copper-responsive transcription factor Amt1 from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel ß-sheet with two short helical segments that project from one end of the ß-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.

LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and¹H-¹⁵N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of ≤0.03 Å and penalties (squared sum of distance violations) of ≤0.06 Ųwere generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(±0.14) Å. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel β-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an α-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module–CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the α-helix appears to be packed more tightly against the preceding antiparallel β-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.

Mac1 from Saccharomyces cerevisiaeactivates transcription of genes, including CTR1in copper-deficient cells. N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1–159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1–159) purified from Escherichia colicontains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1–159) to a DNA duplex containing the copper-responsive element TTTGCTCA. The DNA binding affinity of Mac1-(1–159) for a duplex containing a single promoter element or an inverted repeat was 5 nmfor the 1:1 complex. The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1mutation yielding a Cys11→ Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivofunction. Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1expression.

The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiaeinvolve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in theS. cerevisiaegenome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae,and their expression is metalloregulated as is true with FRE1and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6is elevated in AFT1–1cells and attenuated in aft1null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7is specifically copper-metalloregulated. FRE7is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7in MAC1cells and the lack of expression in mac1–1cells are consistent with Mac1 being the critical transcriptional activator of FRE7expression. The 5′ promoter sequence of FRE7contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7and CTR1when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).

The bakers' yeast Saccharomyces cerevisiaecontains a metallothionein (MT) gene family comprised of the amplified CUP1locus and the single copy CRS5gene. We demonstrate that CUP1plays the dominant role in copper detoxification. A single copy of CUP1was far more effective in conferring copper resistance than was CRS5.The CUP1promoter contributes to this resistance; in a promoter exchange experiment, the Crs5 MT conferred strong copper resistance when its expression was driven by the CUP1promoter, and conversely, the CRS5promoter reduced the effectiveness of Cup1 MT. Unlike CUP1, the CRS5promoter appears to be refractory to high concentrations of copper. The CUP1coding sequences also contribute to copper tolerance, presumably reflecting the enhanced binding avidity of Cup1 MT for Cu(I) ions. In studies with the bathocuproine Cu(I) chelator, the Cu(I) ions bound to Crs5 were kinetically more labile than the Cu(I) binding to Cup1. Our findings are consistent with the assembly of Crs5 into two metal-binding clusters, similar to mammalian MTs, but unlike Cup1. Overall, the striking differences in gene structure, regulation, and function of CUP1and CRS5are remarkably reminiscent of the MTI and MTII genes of the pathogenic yeast Candida glabrata.

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