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J. Biol. Chem., Vol. 279, Issue 34, 35334-35340, August 20, 2004
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From the University of Utah Health Sciences Center, Salt Lake City, Utah 84132
Received for publication, April 28, 2004 , and in revised form, June 1, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Four proteins (Cox11, Cox17, Cox19, and Sco1) have been implicated in copper ion delivery and insertion into CcO (reviewed in Ref. 5). The first protein implicated in copper ion delivery to newly synthesized CcO was Cox17 (6). Yeast harboring a mutant cox17–1 were shown to be deficient in respiratory growth and CcO activity, but these phenotypes were reversed by the addition of 0.4% copper salts to the growth medium (6). Among a collection of yeast respiratory-deficient mutants defective in cytochrome c oxidase activity, only cox17–1 cells were suppressed by exogenous copper, suggesting a role for Cox17 in copper ion metallation of CcO. Sco1 was first implicated in the copper delivery pathway to CcO by the observation that the respiratory-deficient phenotype of a cox17–1 strain was suppressed by overexpression of SCO1 (7). Sco1 is an integral inner membrane (IM) protein that is tethered to the IM by a single transmembrane helix and contains a globular domain, projecting into the intermembrane space (IMS) (8, 9). Null sco1 cells, like cox17
cells, are respiratory-deficient and have diminished CcO activity. Unlike cox17 cells, these phenotypes are not reversed in yeast by the addition of exogenous copper. The role of Cox11 in CuB site formation was suggested by the observation that CcO isolated from Rhodobacter sphaeroides cox11 cells lacked CuB and Mg(II) but contained the other cofactors (10). Yeast lacking Cox11 are devoid of CcO activity (11). Cox11 is also an intrinsic IM protein tethered by a transmembrane helix. A fourth protein, Cox19, is implicated in copper metallation of CcO by virtue of its limited sequence similarity with Cox17 (12). Yeast cells lacking Cox19 are respiratory-deficient, but this phenotype is not reversed by the addition of exogenous copper salts.
The roles of Cox17, Sco1, and Cox11 in copper metallation of CcO were substantiated by the observation that each protein is a Cu(I)-binding protein and that mutations that abrogate in vivo function attenuate Cu(I) binding (11, 13–15). Cox17 is a soluble protein that localizes within the IMS and also within the cytoplasm (8). The dual localization of Cox17 suggested that it served as a copper ion shuttle to the mitochondrion. However, we recently demonstrated that Cox17 is fully functional if tethered to the IM by a heterologous transmembrane domain (16).
The pathway of copper ion delivery to CcO has not been resolved. Sco1 was proposed to mediate the transfer of Cu(I) ions from Cox17 to the CuA site in Cox2 (17). The observation that Sco1 and Cox2 interact supports this model (18). Cox17 has not been shown to interact with any of these proteins, so its role as a copper donor to Sco1 and/or Cox11 remains unsubstantiated. Cox19, like Cox17, is a small soluble protein within the IMS, so it may also have a copper transfer function. However, nothing is known of which step in the CcO assembly is affected by Cox19.
Three of the seven cysteine residues in the 69-residue Cox17 are in a CCXC motif that is important for Cu(I) binding (13). Multiple Cu(I) ions bind to Cox17 within a polycuprous-thiolate cluster that stabilizes a mixture of dimers and tetramers (14). A second functional domain in Cox17 lies near the C terminus. The original cox17–1 mutation creates a C57Y substitution that abrogates in vivo function without compromising either Cu(I) binding or oligomerization. Studies with C-terminal domain Cox17 mutants suggest a role in mitochondrial retention, perhaps docking with another molecule (16).
The two IM proteins Sco1 and Cox11 are postulated to be the direct copper donors to Cox2 and Cox1, respectively. Both Sco1 and Cox11 have soluble domains that project into the IMS. Sco1 has a conserved CXXXC sequence motif that together with a conserved His residue forms the Cu(I) binding site (15). The structure of the Bacillus subtilis Sco1 reveals that these two essential Cys residues lie within an exposed 
-hairpin loop (19). We predict that the single Cu(I) ion coordinated to Sco1 is solvent-exposed and poised for a ligand exchange transfer reaction. Cox11 coordinates Cu(I) by three cysteinyl residues (11). Mutation of any of these Cys residues reduces Cu(I) binding and abrogates CcO activity.
The postulate that Sco1 and Cox11 mediate copper ion insertion into Cox2 and Cox1, respectively, is consistent with protein-mediated copper metallation of other yeast proteins. Copper insertion into Cu,Zn-superoxide dismutase (Sod1) in yeast requires the function of the Ccs1 metallochaperone (20, 21). Ccc2, a P-type ATPase copper ion transporter, receives copper ions from the Atx1 metallochaperone (22–24). With both Sod1 and Ccc2, copper transfer occurs via ligand exchange reactions in protein-protein complexes of the target protein and its specific metallochaperone. Thus, the prediction is that putative copper transfer from Sco1 to Cox2 and Cox11 to Cox1 is mediated through protein-protein interactions. Likewise, copper ion metallation of Sco1 and Cox11 is expected to occur in protein-mediated reactions. At least two major questions remain: the identity of the copper donor(s) to Sco1 and Cox11 and the mechanism of copper routing to the mitochondrial IMS. In this report we have addressed the first major question and demonstrate for the first time that Cox17 is the specific copper donor to both Sco1 and Cox11.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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variant (6) were used for all experiments. Yeast strains were cultured on plates or in liquid in complete medium lacking uracil for pYEF2 selection or lacking leucine for pRS315 selection. Cells were cultured with glucose, raffinose, or galactose as carbon sources as specified under "Results." DNA transformations were performed using a lithium acetate protocol. Plasmids—SCO1 corresponding to codons 95–296 fused to a 5' sequence encoding a His purification tag was amplified from pTNDW4 (15) by PCR with NotI and EcoRI restriction sites added to the 5'- and 3'-ends, respectively. Likewise, mutant SCO1 encoding the same segment with the mutation (C148A,C152A) was amplified from pTNDW4–148,152 with NotI and EcoRI terminal sites. The truncated SCO1 genes were subcloned into pYEF2 containing the GAL1 promoter and CYC1 terminator for transformation of W303 cells (25). The plasmid is designated YEp-GAL1-SCO1. A fusion of thioredoxin and COX11 lacking the first 300 base pairs of the open reading frame was PCR-amplified from the pET32a vector pHCDW4 (11) and subcloned into pYEF2, creating plasmid YEp-GAL1-COX11. DNA encoding a Cox11 mutant with a triple alanine substitution of Cys-111, Cys-208, and Cys-210 was amplified as a thioredoxin fusion from an existing mutant plasmid for subcloning into pYEF2. The construction of YCp-MET25-COX17 and YCp-MET25-SCO2/COX17 was described previously (16). Maximum expression with the MET25 promoter occurs in medium lacking methionine, whereas minimal expression occurs in medium containing 0.67 mM methionine (5x the normal Met level). The Sco2-Cox17 fusion contains the 5'104 residues of Sco2 containing the N-terminal mitochondrial import sequence and transmembrane domain of Sco2 fused to Cox17. YEp-MET25-COX17–1 was constructed by insertion of COX17–1 encoding the C57Y variant of Cox17 in pRS426 (26). The correct sequences of all plasmids generated in this study were confirmed by sequencing.
Protein Purification—Recombinant Cox17 was purified as described previously by Heaton et al. (14). E. coli BL21 (DE3) transformants with COX17 on a pAED3 vector were used for the Cox17 purification using conventional chromatography on an AKTA fast protein liquid chromatography unit (Amersham Biosciences). The C57Y mutant variant of Cox17 was purified from cells harboring the COX17–1 mutant. Recombinant Sco1 and mutant Sco1 (C148A,C152A double substitution) were purified from BL21 (DE3) transformants harboring pTNDW2 (His-tagged SCO1 or mutant SCO1) as described previously by Nittis et al. (15). The Sco1 molecules purified were soluble truncates lacking the N-terminal 95 residues that form the mitochondrial target sequence and transmembrane domain. Nickel-NTA Superflow (Qiagen) was used for the purification of the His-tagged Sco1 proteins. ApoSco1 was isolated from E. coli transformants induced in the absence of added copper to the growth medium. Under these conditions, Sco1 is recovered with less than 0.1 mole equivalent of bound copper. E. coli strain BL21 (DE3) harboring pHCDW4, a pET32a (Novagen) vector containing COX11 lacking the first 300 base pairs, was used for the purification of thioredoxin-Cox11 as a His-tagged fusion. Ni-NTA (Qiagen) column chromatography was used as described previously (11). ApoCox11 was isolated from bacterial transformants induced in the absence of added copper to the growth medium. Yeast transformants with YEp-GAL1-SCO1 or YEp-GAL1-COX11 were cultured in raffinose medium to an A600 nm of 0.6. Galactose was then added to induce expression of the His-tagged Sco1 or Cox11. Cells were harvested after 5 h for preparation of lysates by use of a French press and subsequent Ni-NTA purification from clarified samples. The overexpression of the soluble domain of Sco1 or Cox11 did not cause impaired mitochondrial respiration.
Copper Transfer Assay—For in vitro studies, CuCox17 and mutants were purified from E. coli, concentrated, and stored anaerobically in 50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM dithiothreitol. ApoSco1 or apoCox11 purified from E. coli was reduced in 50 mM dithiothreitol and subsequently desalted into transfer buffer (20 mM Tris-HCl, pH 7.0, 100 mM NaCl). The transfer was initiated by dilution of the Cox17 into the transfer buffer followed by the addition of the apoprotein. Emissions were monitored (with a 350-nm bandwidth filter) between 350 and 700 nm after excitation at 300 nm. Excitation and emission slits were set at 5 and 20 nm, respectively. After analysis transfer, mixtures were separated by either affinity purification by Ni-NTA chromatography or by size exclusion on a Superdex 75 equilibrated in 20 mM Tris-HCl, pH 7.0, 100 mM NaCl. Ni-NTA chromatography was carried out by the addition of Ni-NTA resin to the protein mixture followed by a 10-min incubation at 4 °C with gentle agitation. Resin was removed by centrifugation, and the resulting supernatant was designated unbound. The resin was washed once in 1 volume of transfer buffer before elution in 1 volume of 20 mM Tris, pH 7.0, 250 mM imidazole (designated as bound). Protein content was monitored by absorbance at 280 nm with an elution buffer blank. Copper content was measured by atomic absorption spectroscopy.
Assays—The copper concentration of the protein samples was measured using a PerkinElmer (AAnalyst 100) atomic absorption spectrophotometer or a PerkinElmer Optima (3100XL) ICP spectrometer. Protein was quantified by amino acid analysis after hydrolysis in 5.7 N HCl at 110 °C in vacuo on a Beckman 6300 analyzer. Optical absorption spectroscopy was carried out on a Beckman DU640. Luminescence was measured on a PerkinElmer LS55 fluorimeter with excitation set at 300 nm.
Westerns Analysis—Protein (10–50 µg) from the mitochondrial or postmitochondrial (cytosolic) fraction was electrophoresed on a 15% SDS-PAGE gel system and transferred to nitrocellulose (Bio-Rad Laboratories). Membranes were blocked in 1x phosphate-buffered saline (50 mM Na2PO4, 100 mM NaCl, pH 7.0), 0.01% Tween 20, and 10% milk solution prior to detection with appropriate antibodies and visualization with Pierce chemiluminescence reagents using a horseradish peroxidase-conjugated secondary antibody. Antisera to phosphoglycerate kinase was from Molecular Probes. Chicken anti-yeast Cox11 antisera and rabbit anti-Sco1 antisera were generated as described previously (11, 15).
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RESULTS |
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The concentration dependence of the copper transfer reactions was determined. Titrations using a fixed CuCox17 concentration and increasing the level of the apoproteins to excess conditions revealed that only a subset of the Cox17-bound Cu is transferred to Sco1 as monitored by the CuCox17 emission (Fig. 3, A and B). Even after the addition of excess apoSco1, residual Cu(I) luminescence persisted in Cox17. Only 
40% of the Cox17-bound Cu(I) is transferred regardless of the excess of Sco1 used. The incomplete copper transfer from Cox17 arises from an inherent property within Cox17 and not Sco1. Doubling the Cox17 concentration doubles the amount of Sco1 that can be metallated. Concerning the metallation of Sco1, the nature of the titration curve suggests that copper transfer to Sco1 appears maximal near 1 mole equivalent (Fig. 3A). This observation is corroborated by recovery of Sco1, using the affinity tag, after a transfer reaction with limiting Sco1 levels. The resulting Sco1-bound copper content was near 1 mole equivalent. In a second series of titrations using excess Sco1 and varying the CuCox17 concentration, we observed a concentration dependence for copper transfer from Cox17 at limiting Cox17 levels (Fig. 3B). At concentrations below 10 µM Cox17, transfer to Sco1 appears impaired. One possibility is the known concentration dependence in Cox17 oligomerization from dimers to tetramers (KD near 20 µM) (14). Dimeric Cox17 species may be less efficient than tetrameric species in the copper transfer reaction. Cox17 incubated in the presence and absence of apoSco1 was fractionated by gel filtration, and the distribution of Cox17 oligomers was assessed by Western analysis of the column fractions (Fig. 4A). In the absence of Sco1, Cox17 was distributed in species representing tetramers, dimers, and monomers as was shown previously (14) (Fig. 4B). After incubation with apoSco1, less tetrameric Cox17 species were observed (Fig. 4B).
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These in vitro studies suggested that Cox17 is a specific copper donor to both Sco1 and Cox11. To further corroborate these studies, we expressed the soluble domains of each protein in a nonnative cellular compartment, the yeast cytoplasm. DNA encoding the soluble domains of SCO1 and COX11 was subcloned in a yeast episomal expression plasmid under the GAL1 promoter, permitting galactose-induced expression of each truncated gene. The soluble domains lack the mitochondrial import sequence and transmembrane domain. A His tag was added to each soluble domain to allow for affinity purification of each molecule. Galactose was added to the medium with W303 cells harboring the YEp-GAL1-SCO1 construct for 5 h prior to harvest and preparation of lysates. Fractionation of clarified lysates by Ni-NTA chromatography yielded single step purification of His-tagged Sco1. Analysis of the recovered protein by polyacrylamide gel electrophoresis revealed Sco1 as the predominant species. Quantitation of the bound copper by atomic absorption spectroscopy and protein by amino acid analysis revealed that Sco1 contained 0.2 Cu/mol (Fig. 5A). The copper content in Sco1 was increased to 0.6 mole equivalent if the cells were cultured in the presence of 0.8 mM CuSO4. These results suggested that copper metallation of Sco1 in the yeast cytoplasm was inefficient but could be achieved by high exogenous levels of copper. This situation is related to the inefficient copper metallation of Sod1 in cells lacking the Ccs1 metallochaperone (28).
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The COX17-mediated metallation of Sco1 likely arises from direct copper transfer from Cox17. To confirm that cytoplasmic Cox17 contains bound copper, we expressed a His-tagged Cox17 gene under the GAL1 promoter on a YEp vector. Induction of COX17 expression by transferring the cultures to galactose medium for 5 h resulted in appreciable accumulation of Cox17 in the cytoplasm as shown by Western analysis (Fig. 5C). Recovery of the protein by Ni-NTA chromatography revealed mean copper stoichiometries between 0.3 and 0.5 in multiple experiments. Thus, a fraction of the Cox17 molecules are copper-loaded in cells cultured in standard growth medium.
As mentioned, in vitro copper transfer studies with apoSco1 revealed no apparent copper transfer from the C57Y mutant Cox17. To test whether this mutant was capable of copper transfer in the yeast cytoplasm, double transformants containing both YEp-GAL1-SCO1 and YEp-MET25-COX17–1 (encoding C57Y Cox17) vectors were cultured in raffinose followed by 5 h in galactose as mentioned above. Sco1 purified from lysates showed a copper content near 0.2 mole equivalent, a value similar to cells not overexpressing Cox17 (Fig. 5A). The expression level of C57Y Cox17 was lower than the overexpressed Cox17 used in the above experiments (Fig. 5C). As mentioned above, reduction of the wild-type Cox17 levels by culturing cells in medium containing excess methionine did not appreciably lower copper metallation of Sco1, but the Cox17 protein levels were now comparable with the level of the C57Y mutant protein (data not shown). Thus, the C57Y Cox17 is an ineffective copper donor in both the in vitro and in vivo systems.
In the absence of Cox17 overexpression, the copper content of the recovered Sco1 is 0.2 as mentioned, potentially because of low levels of endogenous, cytoplasmic Cox17. We did not test the metallation of Sco1 in cox17 cells because they grew poorly in raffinose medium. To test whether the limited Sco1-bound copper arose from endogenous cytoplasmic Cox17, we used cox17 cells containing a SCO2-COX17 chimeric protein. This chimera is functional and resides solely within the mitochondrial IM (16). YEp-GAL1-SCO1 and YCp-ME25-SCO2-COX17 cotransformants were cultured in the usual conditions (raffinose followed by 5 h in galactose). Sco1 recovered from these cells had a copper content of <0.1 mole equivalent, suggesting that copper ion metallation of Sco1 is dependent on the Cox17 protein under normal physiological growth conditions (Fig. 5A).
The cytosolic assay was repeated using YEp-GAL1-COX11 transformants in the presence and absence of YCp-MET25-COX17. In the absence of co-expression of Cox17, Cox11 was recovered from yeast cytosol using Ni-NTA chromatography. The purity of the recovered Cox11 was insufficient for determination of the binding stoichiometry, so the samples were further purified by gel filtration on a S200 matrix. Purified Cox11 was recovered with 0.1 mole equivalent of bound copper from cells cultured in synthetic medium without any supplemental copper (Fig. 5B). Recovery of Cox11 from cells cultured in medium supplemented with 0.8 mM CuSO4 resulted in 0.4 mole equivalent of copper bound. The protein eluted from the S200 gel filtration column in a volume consistent with a dimeric species. We reported previously that Cox11 purified from E. coli forms a stable dimer (11).
Co-expression of GAL1-COX11 and MET25-COX17 chimeric genes in cells cultured without supplemental copper results in enhanced copper metallation of Cox11 (Fig. 5B). The copper content increased from 0.1 mole equivalent in the absence of Cox17 to nearly 0.5 in the presence of Cox17. This stoichiometry is 2-fold less than that observed when COX11 is expressed in E. coli cells cultured in copper-supplemented medium (11). The copper ions in the dimeric Cox11 purified from E. coli are closely spaced (11), so the repeated recovery of Cox11 from the yeast cytosol with only 0.5 mole equivalent of bound copper may suggest a species exists with a single copper ion bound at the dimer interface. When a mutant Cox11 was induced in which Cys-111, Cys-208, and Cys-210 were substituted by alanines, no appreciable copper binding was observed in the recovered protein (Fig. 5B). Thus, copper binding by Cox11 is dependent on this Cys motif. Consistent with this result, we showed previously that recombinant Cox11 purified from E. coli as a thioredoxin fusion revealed Cu(I) binding to Cox11 and not the thioredoxin (11).
Whereas the C57Y Cox17 was an ineffective copper donor to Sco1 either in vitro or in vivo, C57Y Cox17 was nearly as efficient as wild-type Cox17 as a copper donor to Cox11 in the yeast cytosol assay system (Fig. 5B). Cox11 purified from YEp-GAL1-COX11 transformants containing YEp-MET25-COX17–1 (C57Y variant) contained 0.4 mole equivalent of bound copper, confirming that C57Y Cox17 is an efficient copper donor to Cox11 but not Sco1.
The efficient copper transfer by the C57Y mutant to Cox11, but not Sco1, implies that the substitution perturbs transfer to only one target. The specificity of the mutant copper transfer is consistent with Cox17 docking with each target molecule in distinct interfaces. Cox17 did not form stable interactions with either Sco1 or Cox11 in the studies. To determine whether a weak interaction of Cox17 with either Sco1 or Cox11 existed, the purified Sco1 and Cox11 samples were tested by Western analysis for co-purification of low levels of Cox17. Cox17 was not detected in either purified Sco1 or Cox11 samples. Cox17 may only transiently interact with these target molecules. In an attempt to stabilize a putative interaction between Cox17 and Sco1, we co-expressed COX17 and a mutant SCO1 encoding a C148A,C152A double mutation of the two Cu(I) binding cysteine residues (15). No co-purification was observed of Cox17 and the Sco1 mutant lacking the ability to bind Cu(I) (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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strain by overexpression of SCO1 (7) and on the fact that Sco1 is a copper-binding protein (15, 17). In the present studies we directly confirm this postulate and extend the functional significance of Cox17 to show for the first time that it is also a copper donor to Cox11. Like Sco1, Cox11 is a copper-binding protein, suggesting that copper metallation of Sco1 and Cox11 is an intermediate step in the transfer of copper to the CuA site in Cox2 and CuB site in Cox1, respectively (11). Two assays were used to confirm the specificity of the Cox17-mediated copper transfer to both Sco1 and Cox11. First, in vitro studies with purified proteins showed physical copper transfer from CuCox17 to either Sco1 or Cox11. Specificity of this copper transfer reaction was indicated by the absence of transfer to heterologous proteins, including bovine serum albumin and carbonic anhydrase. In addition, the C57Y mutant of Cox17 failed to transfer copper to Sco1 but was competent for copper transfer to Cox11. Second, the in vitro transfer studies were corroborated by a yeast cytoplasm assay. Soluble domains of Sco1 and Cox11 lacking the mitochondrial targeting sequence and transmembrane domains were expressed in the yeast cytoplasm in the presence and absence of Cox17 co-expression. Recovery of either Sco1 or Cox11 revealed that copper metallation of either protein was dependent on either growth of cells in medium containing high exogenous copper ions or co-expression of Cox17. The Cox17-mediated metallation of Sco1 or Cox11 occurred in yeast cultured in non-copper-supplemented medium. Residual copper binding was observed in Sco1 recovered from wild-type yeast containing chromosomally encoded Cox17. This residual copper metallation of Sco1 arises from the low, endogenous levels of Cox17 within the cytoplasm (8). Copper metallation of Sco1 was nearly abolished when purified from cells containing Cox17 tethered to the mitochondrial inner membrane. Specificity in the yeast cytoplasm assay was further demonstrated by a lack of copper transfer to Sco1 by the C57Y Cox17 mutant, yet copper transfer by the mutant to Cox11 in agreement with the in vitro assay.
Yeast harboring C57Y Cox17 are respiratory-deficient and lack appreciable cytochrome c oxidase activity (6). The mutant protein fails to accumulate to a significant level within the mitochondrial IMS in the presence or absence of a heterologous IMS targeting sequence (13, 16). The recombinant C57Y Cox17 protein binds Cu(I) normally and forms similar tetrameric conformers as the wild-type protein (14, 16). Thus, the non-functionality of the protein does not arise from defective copper ion binding.
The present data show that C57Y Cox17 is defective only in copper transfer to Sco1. The ability of the mutant protein to transfer copper to Cox11 suggests that Cox17 may have different interaction interfaces for Sco1 and Cox11. We reported recently that the C-terminal segment containing Cys-57 may adopt an amphipathic helix (16). This segment of the protein may be an important interface for Sco1, but not Cox11. We were unable to detect any stable interactions between Cox17 and Sco1 or Cox11 with in vitro or yeast cytoplasm assays by evaluating co-purification, implying that its interactions with Sco1 and Cox11 may be transient. A similar situation exists with the other two known copper metallochaperones. Stable interactions of the Atx1 and Ccs1 metallochaperones and their target proteins has not been observed.
The observation that copper transfer did not deplete Cox17 of bound Cu(I) in the in vitro assay was surprising. Despite incubation with excess Sco1 or Cox11, residual copper remained associated with Cox17. Because Cox17 contains a polycopper cluster (14), the possibility remains that only a subset of the bound Cu(I) ions is available for transfer. The prediction is that the copper ions bound within the polycopper cluster are not equivalent. We have failed to obtain clear binding stoichiometries by electrospray mass spectrometry to address the number of residual bound coppers after transfer reactions.
These studies suggest the following model of Cox17-mediated metallation of CcO. Cox17 may be metallated within the IMS by transfer of copper from the matrix copper pool (30). CuCox17 transfers Cu(I) ions to both Sco1 and Cox11 for subsequent donation to Cox2 and Cox1, respectively. The postulated Sco1-mediated metallation of Cox2 is likely to occur after extrusion of the Cox2 soluble domain into the IMS by Cox18 (31). This transfer may occur upon physical interaction between Sco1 and Cox2 as shown previously (18). It is unclear at this time whether both copper ions in the CuA site are derived from CuCox17. Studies are underway to extend this yeast cytoplasm assay system as a nonnative cellular environment to evaluate copper transfer from Sco1 to Cox2 and to test the function of Cox19.
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FOOTNOTES |
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Supported by Grant T32 DK07115 from the National Institutes of Health.
To whom correspondence should be addressed. Tel.: 801-585-5103; Fax: 801-585-5469; E-mail: dennis.winge{at}hsc.utah.edu.
1 The abbreviations used are: CcO, cytochrome c oxidase; IM, inner membrane; IMS, intermembrane space; Ni-NTA, nickel-nitrilotriacetic acid; Sod1, superoxide dismutase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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