SCO1 and SCO2 act as high copy suppressors of a mitochondrial copper recruitment defect in Saccharomyces cerevisiae.

C129/U1 is a respiratory defective mutant of Saccharomyces cerevisiae arrested in cytochrome oxidase assembly due to a mutation in COX17, a nuclear gene encoding a low molecular weight cytoplasmic protein proposed to function in mitochondrial copper recruitment. In the present study we show that the respiratory defect of C129/U1 is rescuable by two multicopy suppressors, SCO1 and SCO2. SCO1 was earlier reported to code for a mitochondrial inner membrane protein with an essential function in cytochrome oxidase assembly (Buchwald, P., Krummeck, G., and Rodel, G. (1991) Mol. Gen. Genet. 229, 413-420). SCO2 is a homologue of SCO1, whose product is also localized in the mitochondrial membrane but is not required for respiration. SCO1 also suppresses a cox17 null mutant, indicating that overexpression of Sco1p can compensate for the absence of Cox17p. In contrast, neither copper, COX17 on a multicopy plasmid, or a combination of the two is able to restore respiration in sco1 mutants. Rescue of cox17 mutants by Sco1p suggests that this mitochondrial protein plays a role either in mitochondrial copper transport or insertion of copper into the active site of cytochrome oxidase. Although SCO2 can also partially restore respiratory growth in the cox17 null mutant, rescue in this case requires addition of copper to the growth medium. SCO2 does not suppress a sco1 null mutant, although it is able to partially rescue a sco1 point mutant. We interpret the ability of SCO2 to restore respiration in cox17, but not in sco1 mutants, to indicate that Sco1p and Sco2p have overlapping but not identical functions.

C129/U1 is a respiratory defective mutant of Saccharomyces cerevisiae arrested in cytochrome oxidase assembly due to a mutation in COX17, a nuclear gene encoding a low molecular weight cytoplasmic protein proposed to function in mitochondrial copper recruitment. In the present study we show that the respiratory defect of C129/U1 is rescuable by two multicopy suppressors, SCO1 and SCO2. SCO1 was earlier reported to code for a mitochondrial inner membrane protein with an essential function in cytochrome oxidase assembly (Buchwald, P., Krummeck, G., and Rodel, G. (1991) Mol. Gen. Genet. 229, 413-420). SCO2 is a homologue of SCO1, whose product is also localized in the mitochondrial membrane but is not required for respiration.
SCO1 also suppresses a cox17 null mutant, indicating that overexpression of Sco1p can compensate for the absence of Cox17p. In contrast, neither copper, COX17 on a multicopy plasmid, or a combination of the two is able to restore respiration in sco1 mutants. Rescue of cox17 mutants by Sco1p suggests that this mitochondrial protein plays a role either in mitochondrial copper transport or insertion of copper into the active site of cytochrome oxidase. Although SCO2 can also partially restore respiratory growth in the cox17 null mutant, rescue in this case requires addition of copper to the growth medium. SCO2 does not suppress a sco1 null mutant, although it is able to partially rescue a sco1 point mutant. We interpret the ability of SCO2 to restore respiration in cox17, but not in sco1 mutants, to indicate that Sco1p and Sco2p have overlapping but not identical functions.
The COX17 gene of Saccharomyces cerevisiae has been shown to code for a cytoplasmic protein that is essential for assembly of cytochrome oxidase (1). The respiratory defect of cox17 mutants is correctable by exogenous copper, indicating an insufficiency of mitochondrial copper as the basis for the assembly arrest. Cox17p is a low molecular mass protein (8 kDa) with 7 cysteine residues, of which at least one is functionally important (1). The copper deficiency in cox17 mutants appears to be confined to cytochrome oxidase. The presence in a cox17 null mutant of cytoplasmic superoxide dismutase (2) and the iron transporter encoded by FET3 (3), both of which use copper as cofactors, provides strong evidence that Cox17p targets copper to mitochondria (1).
The COX17 gene was cloned by complementation of the cytochrome oxidase deficiency of the cox17 mutant, C129/U1, with a yeast genomic plasmid library (1). Transformations of this mutant yielded two other plasmids that did not contain COX17. In this communication, we show that these high copy suppressors of C129/U1 are SCO1 (4) and SCO2 (5). SCO1 has been reported to code for a cytochrome oxidase assembly factor (4). SCO2 was identified through the yeast genome sequencing project (5). Although the two genes code for homologous proteins, a null mutation in SCO2 does not elicit a discernable phenotype. Sco1p and Sco2p are both membrane constituents of mitochondria, but do not appear to be associated in a stable complex. Suppression of cox17 mutants by SCO1 indicates that the product of this gene also plays a role in providing copper for cytochrome oxidase. The failure of sco1 mutants to form the mature complex is proposed to be caused either by a deficiency in mitochondrial copper uptake or by failure to insert copper during assembly of cytochrome oxidase.

MATERIALS AND METHODS
Yeast Strains and Media-The genotypes and sources of the strains of S. cerevisiae used in this study are listed in Table I. The media used for growth of yeast have been described elsewhere (1).
Cloning of SCO1 and SCO2-The SCO1 and SCO2 genes were cloned by transformation of the cox17 mutant, C129/U1, with a recombinant library of yeast nuclear DNA, by the method of Schiestl and Gietz (7). The library used for the transformation was constructed from partial Sau3A fragments of nuclear DNA (averaging 7-10 kb) 1 cloned into the BamHI site of the shuttle vector YEp24 (8). This library was kindly provided by Dr. Marian Carlson, Department of Genetics and Development, Columbia University. Approximately 5 ϫ 10 8 cells were transformed with 100 g of plasmid DNA. The transformation mixtures were plated on minimal glucose medium to select for plasmid-bearing clones (approximately 10 5 uracil-positive clones). The minimal glucose plates were replicated on rich glycerol medium, and growth was scored after 2-5 days of incubation at 30°C. SCO1 was also cloned independently by transformation of the sco1 mutant E428/U1 with the same genomic library.
Preparation of Yeast Mitochondria and Enzyme Assays-Wild-type and mutant yeast were grown to stationary phase in YPGal (2% galactose, 1% yeast extract, and 2% peptone) and mitochondria were prepared by the procedure of Faye et al. (9), except that Glusulase was replaced by Zymolyase 20,000 (ICN Biomedicals, Inc.) to prepare spheroplasts. Cytochrome oxidase activity was measured by following oxidation of ferrocytochrome c at 550 nm (10).
Construction of a SCO1-BIO Fusion Gene-To create a gene expressing Sco1p with covalently attached biotin at its carboxyl terminus (11), the termination codon of SCO1 was destroyed and replaced by a BamHI site. A HindIII site was created 296 bp upstream of the start codon, and the amplified 1.2-kb fragment was digested with HindIII and BamHI and cloned into YEp352-Bio5. The resultant construct consisted of the entire SCO1 coding sequence fused in-frame to the 270-nucleotide fragment coding for the biotinylation signal sequence of bacterial transcarboxylase (12). The fusion gene was transferred to the multicopy shuttle vector YEp351 and to the integrative plasmid YIp351 (13). § To whom all correspondence should be addressed. Tel.: 212-854-2920; Fax: 212-865-8246. 1 The abbreviations used are: kb, kilobase pair; bp, base pair.
Construction of W303⌬SCO1 and W303⌬SCO2-A null allele of SCO1 was made by cloning the 1.7-kb EcoRI fragment containing SCO1 into YEp352E. YEp352E is identical to YEp352 (13), except that the multiple cloning region of the latter plasmid is replaced with a unique EcoRI site. This plasmid was linearized at the SphI site inside SCOI and ligated to a 1.2-kb linear SphI fragment with the yeast URA3 gene. Respiratory deficient and uracil-independent transformants (W303⌬SCO1) were obtained and verified to have the disrupted allele by backcrosses to a sco1 mutant and by Southern analysis of genomic DNA.
The sco2::URA3 allele was created by replacement of the 450-bp AflII fragment internal to the coding sequence with the URA3 gene. pSG74/ ST4 was digested with AflII, and the linear plasmid was ligated to an SphI linker. The yeast URA3 gene (as a 1.1-kb SphI fragment) was ligated to the SphI site in the gapped gene. The disrupted sco2::URA3 allele was isolated on a linear fragment and used to transform the respiratory competent haploid strains W303-1A and W303-1B by the one-step gene replacement procedure (14). Uracil-independent transformants were selected and verified to have the disrupted alleles by Southern analysis of their chromosomal DNA (see Fig. 2).
Preparation of Antibodies to Sco2p-In order to generate antibodies to the SCO2 gene product, the gene was amplified by PCR, with primers which created a BamHI site at amino acid residue 11 and a HindIII site 120-bp downstream of the stop codon. This fragment was ligated into the expression vector pATH20 (15), creating an in-frame fusion with the Escherichia coli trpE gene. The fusion protein expressed from the trpE fusion constituted most of the insoluble protein fraction of the E. coli cells. This fraction was dissolved in a 10 mM Tris-HCl, 1 mM EDTA, pH 7.5 buffer, containing 2% SDS, 5 mM ␤-mercaptoethanol, and 20 g/ml phenylmethylsulfonyl fluoride, and the Sco2-fusion protein was further purified on a Bio-Gel A0.5 column developed with a buffer containing 10 mM Tris-HCl, 0.1 mM EDTA, and 5 mM ␤-mercaptoethanol. Fractions containing primarily the fusion protein were pooled, concentrated by acetone precipitation, and used to raise antibodies in rabbits.
Miscellaneous Procedures-Standard procedures were used for the preparation and ligation of DNA fragments and for transformation and recovery of plasmid DNA from E. coli (16). The preparation of yeast nuclear DNA and the conditions for the Southern hybridizations were as described by Myers et al. (17). DNA probes were labeled by random priming (18), and DNA was sequenced by the method of Maxam and Gilbert (19). Proteins were separated by polyacrylamide gel electrophoresis in the buffer system of Laemmli (20), and Western blots were treated with antibodies against Sco2p followed by a second reaction with 125 I-protein A (21). Alternatively, biotin-containing proteins were visualized with peroxidase conjugated to avidin (1). Protein concentrations were determined by the method of Lowry et al. (22).

Isolation of Suppressors of a cox17
Mutant-C129 is a cytochrome oxidase defective mutant of S. cerevisiae with a single point mutation in COX17 (1). The COX17 gene was cloned by complementation of C129/U1 with a yeast genomic library (1). Some of the respiratory competent clones obtained from the transformations, however, were found to have plasmids with genomic fragments unrelated to one another or to COX17. The physical maps of two such plasmids indicated the presence of SCO1, a gene coding for a cytochrome oxidase assembly factor (4,23). The identity of SCO1 as the suppressor was corroborated by the ability of pG41/T2 and pG41/ST8 to confer respiration to C129/U1. pG41/T2 was cloned independently by transformation of a sco1 mutant. pG41/ST8 is a subclone of pG41/T2 containing only SCO1 (Fig. 1).
SCO1 codes for a constituent of the yeast mitochondrial inner membrane and is essential for the expression of cytochrome oxidase (4,23). Mutations in SCO1 induce a specific deficiency in cytochrome oxidase but do not substantially affect other enzymes of the respiratory chain or of the ATPase (4, 23). The ability of sco1 mutants to synthesize the mitochondrial encoded subunits of cytochrome oxidase (23) and to accumulate the nuclear gene products has led to the suggestion that Sco1p promotes some late post-translational step in the assembly pathway (4,23), although its precise function has not been clarified.
Two other plasmids (pSG74/T1 and T2) obtained by transformation of C129/U1 had overlapping inserts unrelated to either COX17 or SCO1 (Fig. 1). Partial sequencing of the insert in pSG74/T1 revealed that it contained a fragment of yeast chromosome II with SCO2, a homologue of SCO1 (5). Restoration of respiration in C129/U1 by pSG74/ST4, but not by pSG74/ST5 (with a 450-bp deletion in the SCO2-coding region), confirmed SCO2 to be a second high copy suppressor. The function of the SCO2 product is not known. The phenotype of a mutant with a null allele of the gene (see below) precludes a  requirement for the protein in respiration.
Phenotype of a sco2 Mutant and Localization of the Product-To facilitate further studies on the relationship of Cox17p to Sco1p and Sco2p, null alleles of SCO1 or SCO2 were constructed as described under "Materials and Methods." Mutants with a disrupted chromosomal copy of SCO1 (aW303⌬SCO1) are unable to grow on nonfermentable carbon sources and exhibit a deficiency in cytochrome oxidase, as reported previously (4). In contrast, W303⌬SCO2, carrying a null mutation in SCO2, had a respiratory competent phenotype and normal levels of cytochrome oxidase. The replacement of the wild-type gene by the sco2::URA3 allele in W303⌬SCO2 was confirmed by a genomic Southern (Fig. 2). Additional evidence for the absence of Sco2p in W303⌬SCO2 was obtained by immunological assays of Sco2p in mitochondria from the mutant, the parental wild-type strain, and a transformant harboring SCO2 on a multicopy plasmid. Western analysis of total mitochondrial proteins, with an antibody against the protein expressed from the SCO2/trpE fusion gene, indicates the presence of a protein of approximately 30 kDa in wild-type mitochondria (Fig. 3A). This mass is consistent with the molecular mass of Sco2p based on the derived protein sequence, assuming that some 4 kDa are lost during processing of the precursor. The 30-kDa protein detected by the antibody is much more abundant in mitochondria from a transformant harboring SCO2 on a high copy plasmid and is absent in W303⌬SCO2. The absence of a signal in the null mutant confirms that this mitochondrial constituent is not required for respiration. Mitochondria from two other strains were also probed with the same antibody. W303⌬SCO1 has a disrupted chromosomal copy of SCO1, and W303⌬SCO1/ST12 is the identical mutant transformed with a fusion gene expressing biotinylated Sco1p-Bio, approximately 7 kDa larger than the native protein. The absence of a crossreacting protein of 37 kDa in mitochondria from W303⌬SCO1/ ST12 and the lack of an effect of the sco1 mutation on the strength of the signal indicates that the antibody specifically recognizes Sco2p.
The Sco2p antibody was used to determine whether this protein is a mitochondrial membrane protein like Sco1p. Mitochondria prepared from the respiratory competent strain W303-1B were disrupted by sonication and the membrane and soluble fractions were probed for Sco2p. The Western blot, shown in Fig. 3B, indicates Sco2p to be present exclusively in the submitochondrial membrane fraction. Solubilization of Sco2p requires extraction of mitochondria with 0.2-0.5% deoxycholate and 0.5 M NaCl, further confirming the hydrophobic nature of this protein (Fig. 3C).
Suppression of a cox17 Null Mutant by SCO1 and SCO2-C129/U1 has a point mutation in COX17 resulting in the substitution of a tyrosine for the carboxyl-proximal cysteine in the protein (1). To test if overexpression of SCO1 and SCO2 can also suppress a cox17 null mutant, W303⌬COX17, harboring a disrupted copy of COX17, was transformed with pG41/ST8 (SCO1), pSG74/ST4 (SCO2), and as a control, also with pG74/ ST8, a multicopy plasmid containing COX17. The respiratory competent parental strain, W303-1B, and the different transformants were replicated on rich glycerol/ethanol medium (YEPG) and scored for growth after 3 days of incubation at 30°C (Fig. 4, top plate). The null mutant was partially suppressed by SCO1, but not by SCO2 (Fig. 4, top plate). The generation time of the mutant transformed with SCO1 was approximately double that of the wild-type strain.
Growth of C129/U1 and W303⌬COX17 on mitochondrial substrates is rescued by addition of exogenous copper to the growth medium (1). The effect of copper supplementation was also tested on the cox17 mutants transformed with SCO1 and SCO2. The somewhat leaky growth phenotype of C129/U1 made it difficult to score differences in the growth of this strain as a function of the different plasmids and the copper supplement. In the case of W303⌬COX17, copper supplementation clearly enhanced the suppressor activity of SCO1 with the null mutant. Addition of copper to the medium also allowed SCO2 to partially suppress the respiratory defect in the null mutant (Fig. 4, middle plate), at a concentration lower than that required to rescue the null mutant itself (Fig. 4, bottom plate).
sco1 Mutants Are Not Rescued by Copper Supplementation and by Overexpression of COX17 and CTR1-Suppression by SCO1, and to a lesser degree by SCO2, of the cytochrome oxidase deficiency of cox17 mutations suggested that the products of the two genes might be involved in mitochondrial copper metabolism. Unlike cox17 mutants, neither the sco1 null mutant, nor seven independent sco1 point mutants were rescued by inclusion of 0.01-0.8% copper in the medium. Neither were the mutants rescued with high copy numbers of either COX17 or CTR1 (the structural gene for the plasma membrane copper pump) (25), either in the presence or absence of added copper. In previous studies, the concentration of copper needed to restore respiratory growth of the cox17 null mutant could be significantly lowered (Ͼ10-fold) when it was transformed with CTR1 on a high copy plasmid (1). Overexpression of the plasma  (28), Pseudomonas (stutzeri) (29), and Cowdria (ruminantium) (30). Identical and conserved residues in Cox2p and the Sco homologues are marked by the asterisks. Four of the residues in Cox2p that make contact with the two coppers at the Cu A site of subunit 2 are indicated by the arrows. The contact with glutamic acid is at the carbonyl of the peptide backbone (31).
FIG. 6. Possible roles of Sco1p in cytochrome oxidase assembly. Copper imported by the plasma membrane pump (Ctr1p) is transferred to mitochondria via the Cox17p. In mechanism A, Sco1p can function by transporting copper into the mitochondrial matrix compartment where it is used to make the active sites of cytochrome oxidase subunits 1 and 2 (Cox1 and Cox2). Alternatively (mechanism B), copper is transferred from Cox17p to Sco1p in the intermembrane space where it is then used to mature subunits 1 and 2. membrane copper pump was presumed to increase internal copper pools, thereby allowing rescue to occur in the presence of lower exogenous copper (1).
Allele-specific Suppression of a sco1 Mutation by SCO2-The primary sequence homology of Sco1p and Sco2p, and their activity as suppressors of the cox17 allele in C129/U1, suggested that they might have similar or overlapping functions. Transformation of W303⌬SCO1 with SCO2 (pSG74/ST4), however, failed to restore respiratory growth in the sco1 null mutant, indicating that the two proteins are not exchangeable and therefore cannot be functionally equivalent. When tested for suppression of point mutations, SCO2 was found to partially rescue one of the seven sco1 mutants tested (W30).
The allele-specific suppression of a sco1 mutant by SCO2 could indicate that a physical interaction of Sco1p and Sco2p is necessary for function. This is contradicted by the observation that the sco2 null mutant has no phenotype on nonfermentable carbon sources. Attempts to detect a physical complex of the two proteins also were unsuccessful. Extraction of Sco1p-Bio and purification of the biotinylated protein by affinity chromatography on a monomeric avidin column failed to disclose copurification of Sco2p. Secondly, although both Sco1p and Sco2p sediment with apparent molecular masses of approximately 60 kDa, the sedimentation behavior of Sco2p was the same in a strain with a sco1 null mutation (data not shown). Finally, suppression by overexpression of Sco1p or Sco2p alone is difficult to rationalize if the two proteins are subunits of a stoichiometric complex. DISCUSSION The cytochrome oxidase deficiency of cox17 mutants was previously shown to be corrected by exogenous copper (1). This observation together with the cytoplasmic localization of Cox17p led us to propose a role for this low molecular weight protein in targeting copper to mitochondria (1). In the present study we demonstrate rescue of cox17 mutants, including a strain with a null allele of the gene, by overexpression of Sco1p, a constituent of the mitochondrial inner membrane, encoded by SCO1 (23). The suppressor activity of SCO1 suggests an involvement of the product, Sco1p, in mitochondrial copper metabolism. Since sco1 mutants are not rescued by COX17, Sco1p is likely to act downstream of Cox17p, either prior to or during the copper maturation step. An alignment of yeast Sco1p with several homologues from eucaryotic and procaryotic sources reveals a potential copper binding domain characterized by the presence of two conserved cysteines whose spacing and proximity to a short conserved hydrophobic domain is reminiscent of the copper binding domain of subunit 2 of cytochrome oxidase (Fig. 5).
Sco1p could be a mitochondrial copper carrier, accepting copper from Cox17p and translocating it to the mitochondrial matrix. This model implies that copper addition to subunits 1 and 2 of the enzyme occurs on the matrix side of the inner membrane (Fig. 6, scheme A), perhaps before subunits 1 and 2 of cytochrome oxidase are fully integrated in the lipid bilayer. Excess Sco1p may help to correct lesions in Cox17p by increasing the efficiency of copper uptake from alternate cellular pools. Sco1p could also be a mitochondrial copper storage and/or transfer protein more directly involved in addition of copper to the cytochrome oxidase precursor (Fig. 6, scheme B). Copper need not be transferred across the inner membrane if addition occurs in the intermembrane space where the active site of the mature enzyme resides (31). According to this model also, overexpression of Sco1p could compensate for the absence of the mitochondrial copper targeting protein by allowing for a more efficient uptake or transfer of copper during maturation of the enzyme.
Overexpression of Sco2p can partially suppress the respiratory defect of a cox17 point mutant and a strain with a null allele, the latter in the presence of added copper. The activity of SCO2 on a high copy plasmid as an allele-specific suppressor of sco1 mutations could mean that Sco1p and Sco2p exist and function as a complex. Even though the molecular weight of Sco1p and Sco2p is two times the monomer size, no evidence could be obtained for a complex of the two proteins. The dependence of function on such a complex, even if it exists, is also unlikely in view of the absence of a discernable phenotype in the sco2 deletion mutant. An alternative explanation for allelespecific suppression of a sco1 mutation by SCO2 is that the product of this gene is able to provide one of the Sco1p functions lost in the mutant. A redundancy in one of the activities of these two proteins would explain the lack of a phenotype in the sco2-disrupted strain.