Purification and Characterization of Yeast Sco1p, a Mitochondrial Copper Protein*

The present studies were undertaken to further characterize the properties of Sco1p, a constituent of the mitochondrial inner membrane implicated in copper transfer to cytochrome oxidase. We report a procedure capable of yielding Sco1p of >95% purity. Sco1p has been purified from strains of Saccharomyces cerevisiae that overexpress the protein. The amino-terminal sequence of purified Sco1p indicates that the first 40 amino acids of the primary translation product constitute a mitochondrial targeting sequence that is proteolytically cleaved during import. We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification. Sco1p contains 1 mol of copper/mol protein. The copper is not removed by the treatment of Sco1p with EDTA, indicating that it is bound with high affinity. Purified Sco1p sediments identical to Sco1p in crude extracts of mitochondria from wild type yeast or from a strain transformed with SCO1 on a high copy plasmid. Native Sco1p has an estimated mass of 88 kDa, suggesting that it is a homotrimer. Sco1p expressed as a soluble protein lacking the internal 17 amino acids of the membrane-anchoring domain has been localized in the matrix. The protein has also been targeted to the intermembrane space. Neither soluble matrix nor intermembrane-localized Sco1p is able to complement a sco1 mutant, suggesting that only the membrane form with the carboxyl-terminal domain facing the intermembrane space is able to exert its normal function.

Cytochrome oxidase (COX) 1 contains two distinct copper centers. The first center, CuA, is associated with Cox2p and acts as the first acceptor of electrons from reduced cytochrome c. The second center, CuB, consists of a single copper atom bound to Cox1p where together with heme A of cytochrome a 3 it functions in reduction of molecular oxygen. Two nuclear genes of Saccharomyces cerevisiae have been proposed to function in mitochondrial copper homeostasis and COX assembly (1)(2)(3). COX17 codes for a low molecular weight protein located in the cytoplasm and the mitochondrial intermembrane space (3). This protein was proposed to deliver copper to mitochondria (1,3) and is an example of a larger group of cytoplasmic proteins that target copper to different cellular compartments (4). The ability of Cox17p to bind up to three copper atoms supports its proposed role as a copper carrier (3,5). SCO1 is a mitochondrial inner membrane protein (2,6). Mutations in SCO1 elicit a COX deficiency as a result of a block in some late steps of the assembly process (2). A third protein encoded by COX11 has been proposed to be required for the maturation of the CuB center in Rhodobacter spheroids (7). This gene is also required for the expression of COX in yeast (8) where it is presumed to have the same function.
The proposed function of Sco1p as a copper transferase was based on the ability of SCO1 to act as a high copy suppressor of cox17 mutants (6), the presence in Sco1p of a domain with sequence similarity to the copper binding site of Cox2p (6), and the physical interaction of Sco1p with Cox2p (9). The involvement of Sco1p in mitochondrial copper metabolism is more directly supported by recent studies showing that a soluble fragment of Sco1p, expressed in Escherichia coli, binds 1 copper/molecule of protein (10).
To learn more about the properties of Sco1p, we have purified the native protein from yeast and characterized its copperbinding property. We have also determined the site at which the Sco1p precursor is processed by the matrix protease and the size of the native protein. The evidence obtained with constructs expressing Sco1p lacking the membrane-spanning domain or having its normal import signal substituted with the leader of cytochrome c 1 shows that the localization and orientation of the protein are essential for its function.

MATERIALS AND METHODS
Yeast Strains and Media-Sco1p was purified from two different strains of S. cerevisiae. E428/U1/ST5 is a sco1 mutant (6) transformed with pG41/ST5, a high copy plasmid containing the wild type SCO1 gene on a 1.9-kilobase pair EcoRI fragment (Fig. 1). E428/U1/ST28 was obtained by transformation of the same mutants with pG41/ST28, which contains both SCO1 and COX17 on a 1.2-kilobase pair HindIII fragment (Fig. 1). For large-scale purifications, cells maintained on minimal galactose (yeast nitrogen base plus 2% galactose) were inoculated and grown to stationary phase in liquid galactose medium (YPGal) containing 4% galactose, 1% yeast extract, and 1% peptone with or without 50 M copper sulfate.
Construction of Genes Expressing Modified Sco1p-A gene lacking the sequence for the internal membrane-spanning domain of Sco1p was constructed by PCR amplification of the gene in pG41/ST5 (6) with the bidirectional primers described by Buchwald et al. (11). The resultant plasmid, pG41/ST23, was identical to pG41/ST5 with the exception that it lacked the internal 51 nucleotides coding for amino acid residues 75-90 of Sco1p (Fig. 1).
The sequence of CYT1 (12) coding for the 5Ј-untranslated region and amino-terminal import and intermembrane targeting signal was obtained by PCR amplification of yeast nuclear DNA with the forward PCR primer (Primer 1) 5Ј-AGACTATCTGAGCTCTTAGTAGAG-GCC-3Ј and the reverse primer (Primer 2) 5Ј-TGCAATCCGGGATC-CGCTGCGGTC-3Ј.
The PCR product lacking the sequence coding for the amino-terminal 40 residues of the import signal was digested with BclI and SalI and cloned into pG101/ST10. The gene in the resultant plasmid (pG41/ ST24) codes for the following sequence at the junction of cytochrome c 1 and Sco1p: glu-ala 2 met-thr-ala-ala-Asp-gln-ser-asn-gly where the arrow demarcates the processing site in cytochrome c 1 , the lowercase residues are part of the cytochrome c 1 leader, the capitalized residues are created by the new restriction site at the junction, and the italicized residues correspond to the amino-terminal end of mature Sco1p. This gene was further modified by removing the sequence coding for the transmembrane segment by PCR amplification of pG41/ST24 with the bidirectional primers described by Buchwald et al. (11). The resultant plasmids were designated as pG41/ST44 (Fig. 1). All the high copy plasmids were used to transform the sco1 null strain W303⌬SCO1 (6).
Purification of Sco1p-An overnight culture of E428/U1/ST5 or E428/ U1/ST28 (15 ml) grown in YPGal was inoculated into 800 ml fresh YPGal medium and incubated with shaking at 30°C for 17-18 h. In a typica1 purification, 40 flasks each containing 800 ml of YPGal were used yielding ϳ500 -600 g of wet weight cells. All steps are carried out at 4°C.
The materials obtained after steps 3 and 5-10 can be stored frozen at Ϫ80°C.
In step 2, the spheroplasts were centrifuged at 2,600 ϫ g for 20 min, washed twice with 3 liters of 1.2 M sorbitol, and lysed in 1.2 liters of STE buffer (0.5 M sorbitol, 50 mM Tris-HCl, pH 7.5, and 1 mM phenylmethylsulfonyl fluoride). The lysed spheroplasts were homogenized in a Waring blender for 40 s and centrifuged at 640 ϫ g for 10 min. The supernatant was collected, and the pellet was washed with 600 ml of STE buffer (0.5 M sorbitol, 50 mM Tris-HCl, pH 7.5, and 1 mM phenylmethylsulfonyl fluoride). The first supernatant and wash were combined and centrifuged at 640 ϫ g to remove remaining cell debris.
In step 3, the mitochondria obtained by centrifugation of the supernatant from step 2 at 14,700 ϫ g av for 30 min were washed three times in 0.5 M sorbitol and 50 mM Tris-Cl, pH 7.5, suspended in the same buffer at a protein concentration of 20 -30 mg/ml, and sonically irradiated for 45 s in a 100-ml beaker with a Branson sonifier using a microtip probe at a power output of 60 watts. The submitochondrial particles (SMP) were sedimented in a Beckman ultracentrifuge at 79,000 ϫ g av for 45 min and suspended in Tris-HCl, pH 7.5 at a final protein concentration of 20 mg/ml.
In step 4, to the SMP suspension were added solid KCl to a final concentration of 1 M, 0.01 volumes of 20 mg/ml phenylmethylsulfonyl fluoride, and 0.1 volumes of 10% potassium deoxycholate. After centrifugation at 79,000 ϫ g for 10 min, the supernatant containing Sco1p was collected. The materials obtained after steps 3 and 5-10 can be stored frozen at Ϫ80°C.
In step 5, to the deoxycholate extract from step 4 was added an equal volume of cold water and 20% potassium cholate to a final concentration of 0.5%. Saturated ammonium sulfate (4°C) was added to a final concentration of 26% saturation, and the precipitate was removed by centrifugation at 79,000 ϫ g for 10 min. The clear reddish supernatant was adjusted to 42% ammonium sulfate saturation with cold saturated ammonium sulfate. The greenish pellet obtained after centrifugation at 79,000 ϫ g for 10 min was dissolved in 15 ml of TT buffer (20 mM Tris, pH 7.5, and 0.05% Triton X-100) and was desalted on a 120-ml column of Sephadex G-50 equilibrated in TT buffer. The desalted proteins elute as a green-colored band in ϳ30 ml.
In step 6, the fraction from step 5 was diluted to 200 ml with TT buffer and applied to a 5 ϫ 17-cm column of Cibacron Blue 3GA cross-linked to agarose (Sigma). The column was washed sequentially with 1) 400 ml of TT buffer, 2) 200 ml of TT buffer containing 1.0 M KCL, 3) 200 ml of TT buffer, 4) 500 ml of a 0 -0.5% linear gradient of potassium deoxycholate in TT buffer, and 5) 700 ml of 0.5% potassium deoxycholate in TT buffer. Fractions (15 ml) were collected, separated on a 12% polyacrylamide gel, and stained with silver. Most of Sco1p elutes in the potassium deoxycholate gradient and subsequent 0.5% deoxycholate wash. Fractions containing Sco1p were pooled (ϳ1 liter). When frozen, this material may develop a white precipitate upon thawing. The precipitate can be removed on a 0.45filter without loss of Sco1p.
In step 7, the pool from the Cibacron Blue column was applied to a 50-ml column of hydroxyapatite (Bio-Gel HTP, Bio-Rad) equilibrated with TT buffer. Following loading of the sample, the column was    Table I. Proteins were separated on a 12% polyacrylamide gel and silver-stained. Mature Sco1p migrates slightly faster than the carbonic anhydrase (31 kDa) standard. The arrowhead in the margin identifies Sco1p. Most of the extra stain above the 66-kDa marker in lanes 5, 6, and 7 is because of a staining artifact. B, preparation of Sco1p purified from E428/U1/ST5 used for copper analysis. C, preparation of Sco1p purified from E428/U1/ST28 used for copper analysis.
FIG. 3. Concentration of Sco1p in wild type and E428/U1/ST5 mitochondria. The indicated amounts of E428/U1/ST5 (ST5) and W303-1A (WT) mitochondria and purified Sco1p were separated on a 12% polyacrylamide gel. The proteins were transferred to nitrocellulose paper, and the Western blot was treated with antibody against Sco1p. The antibody-antigen complex was visualized by treatment of the blot with 125 I-protein A and exposure to x-ray film. The density of the signals obtained with purified Sco1p was determined with a Visage 110 Bioimager (Millipore) and was used to estimate the concentration of Sco1p in the two different samples of mitochondria. Each sample was loaded on 4.8 ml of a 12-30% linear sucrose gradient containing 20 mM Tris-Cl, pH 7.5, 0.05% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. The gradients were centrifuged at 4°C for 44 h at 60,000 rpm in a Beckman SW 65Ti rotor. Fractions were collected and assayed for hemoglobin by absorption at 410 nm and for Sco1p by Western blot analysis. The distribution of hemoglobin is plotted, and the distribution of Sco1p is shown by the photographs in the inserts. The size of Sco1p in each gradient was calculated from its sedimentation relative to hemoglobin (18). a Sco1p was reduced in the presence of 0.1 M DTT for 10 min on ice. The DTT was removed on a Sephadex G-25 column that had been treated with DTT and then equilibrated with a degassed/nitrogen-saturated buffer containing 100 mM NaCl in TT buffer (TTNaCl). The reduced Sco1p eluted from the Sephadex column was incubated at 23°C with an equal volume of saturated CuCl (approximately 0.2 mM). EDTA was added to a concentration of 0.5 mM and the mixture further incubated for 45 min at 23°C. Copper and EDTA were removed on a Sephadex G-100 column equilibrated with nitrogen-saturated TTNaCl buffer. Fractions containing protein were pooled, concentrated on a Centricon filter (Amicon), and analyzed for copper.
b Sco1p was treated in the same way as above with the exception that a mole equivalent to purified Cox17p was present during the initial incubation in the presence of 0.1 M DTT. Cox17 did not separate from Sco1p during the desalting step on Sephadex G-25 and therefore was present during the subsequent treatment with the CuCl. Cox17p was separated from Sco1p on the Sephadex G-100 column.
c Sco1p was incubated on ice for 30 min in the presence of 5 mM EDTA and was desalted by passage over Sephadex G-25 equilibrated with 5 mM Tris-Cl, pH 7.5, and 0.05% Triton X-100. The protein was concentrated on a Centricon filter.
washed with 75 ml of TT buffer. The protein is eluted with 50 ml of 0.3 M potassium phosphate, pH 7.5, and 0.05% Triton X-100. This fraction is desalted on a 350-ml column of Sephadex G-25 superfine (Amersham Biosciences) equilibrated with TT buffer. The desalted protein eluting as a single 280-nm absorbing peak is collected in ϳ50 ml. Because of the weak adsorption of Sco1p to mono-S, in the next step it is important to remove all the salt on the Sephadex column.
In step 8, a preparative high pressure liquid chromatography mono-S column (8 ml) (Amersham Biosciences) was washed with 25 ml of TT buffer and 1.0 M NaCl followed by 50 ml of TT buffer. After application of the desalted fraction from step 7, the column was washed with 1) 20 mM NaCl in TT buffer, 2) 80 ml of a 0 -0.1 M linear gradient of NaCl in TT buffer, and 3) 80 ml of 0.1 M NaCl in TT buffer. Fractions of 8 ml were collected and analyzed for Sco1p on a 12% polyacrylamide gel. Sco1p elutes in ϳ70 ml of TT buffer peaking at 0.1 M NaCl.
In step 9, the pool from step 8 was diluted to 150 ml with TT buffer and applied to a 1 ml of fast protein liquid chromatography mono-Q column (Amersham Biosciences). The column was washed with 5 ml of TT buffer followed by 5 ml of 0.35 M NaCl in TT buffer. Sco1p elutes as a single 280-nm absorbing band and is recovered in ϳ2 ml.
In step 10, purified Sco1p was desalted on a 10-ml Sephadex G-25 superfine column equilibrated in TT buffer.
Miscellaneous Procedures-Standard procedures were used for the preparation and ligation of DNA fragments and for the transformation and recovery of plasmid DNA from E. coli (14). Proteins were analyzed on 12% polyacrylamide gel by SDS-PAGE (15). Western blots were treated with antibodies against the Sco1p (6). Antibody-antigen complexes were visualized by a secondary reaction with 125 I-protein A (16). Protein concentrations were determined by the method of Lowry et al. (17). Copper was determined by atomic absorption in a graphite furnace (Galbraith Laboratories, Knoxville, TN). The amino terminus of purified Sco1p was sequenced with an AP Biosystems Procise Model 494 microsequencer (Protein Chemistry Core Facility, Columbia University).

RESULTS AND DISCUSSION
Purification of Sco1p-The concentration of Sco1p in E428/ U1/ST5 is ϳ50 g/mg mitochondrial protein. This value is raised by another factor of 1.7 in SMP, the starting material used for the purification. The results of a typical fractionation are summarized in Table I and Fig. 2. The extraction of the SMPs with deoxycholate solubilizes 70% of the protein almost half of which is lost during the ammonium sulfate fraction- Mitochondria were prepared by the method of Glick and Pon (19) from the wild type strain W303-1A (W303) and from the transformants E428/U1/ST23 (ST23) and E428/U1/ST44 (ST44) (see Fig. 1 for details of the pG41/ST23 and pG41/ST44 plasmids). One-half of the mitochondria (Mt) at a protein concentration of 8 mg/ml were diluted with 4 volumes of 0.6 M sorbitol, 20 mM Hepes, pH 7.5, and the other half was converted to mitoplasts (Mp) by dilution with 4 volumes of 20 mM Hepes, pH 7.5. Both mitochondria and mitoplasts were incubated on ice for 1 h in the absence (Ϫ) or presence (ϩ) of 0.1 mg/ml proteinase K. After the addition of phenylmethylsulfonyl fluoride to a final concentration of 1 mM, the samples were centrifuged at 14,000 rpm for 15 min, and the supernatants from the mitoplasts (S) were saved. The mitochondrial and mitoplast pellets were suspended in 100 l of 0.6 M sorbitol, and 20 mM Hepes, pH 7.5, precipitated by the addition of 0.1 volume of 50% trichloroacetic acid and centrifuged at 14,000 rpm for 5 min. The proteins recovered in the supernatant from the mitoplasts were also precipitated with trichloroacetic acid. The precipitates were rinsed with water, dissolved in sample buffer, and separated on a 12% polyacrylamide gel (15). Two times more of the supernatant fraction was loaded. Following transfer to nitrocellulose filter, the blots were probed with antibody against cytochrome b 2 (b2) and Sco1p.

FIG. 7. Carbonate extraction of native and mutant Sco1p.
Mitochondria were isolated from the wild type strain W303-1A (WT) from the transformants E428/U1/ST5 (ST5) and E428/U1/ST44 (ST44) (see Fig. 1 for details of the pG41/ST5 and pG41/ST44 plasmids). The mitochondria were adjusted to 0.1 M sodium carbonate at a final protein concentration of 5 mg/ml. After incubation on ice for 15 min, a small sample (M) was saved, and the rest was centrifuged at 50,000 rpm for 30 min. Equivalent volumes of the starting sample (M), the membrane pellets (P), and soluble supernatant fraction (S) were separated on a 12% polyacrylamide gel (15), transferred to nitrocellulose membrane, and probed with antibody against Sco1p. The arrow in the margin identifies Sco1p. The blot with the samples from the wild type strain was exposed 50 times longer. ation. Further losses occur at each succeeding step resulting in the recovery of only 2-3% of the starting material. The procedure yields 1.5 mg of Sco1p/gram of SMP protein. The purity of Sco1p is ϳ95% based on scans of silver-stained gels.
Properties of Purified Sco1p-The copper content of purified Sco1p was determined by atomic absorption and corrected for adventitious copper in the buffer used to dissolve the protein. The values obtained for three different preparations are reported in Table II. The purity of two of the preparations used for the copper analysis is shown in Fig. 2, A and B. The copper content ranged from 0.7-1.0 mol/mol of Sco1p and was not significantly different in preparations obtained from E428/U1/ ST5 or E428/U1/ST28. The supplementation of the medium with different concentrations of copper did not raise the copper content above 1 mol/mol protein. The purified protein was also incubated under anaerobic conditions in the presence of cuprous chloride alone or in combination with purified Cox17p (3). Neither condition led to any increase in the amount of protein-bound copper. Finally, the incubation of purified Sco1p in the presence of EDTA caused only a moderate decrease in the copper content, indicating that the copper is bound with high affinity. These results confirm that native Sco1p is a copper-binding protein. The several preparations used for the copper assays suggest a stoichiometry of 1 copper/molecule of protein, a value that is in agreement with the stoichiometry of copper recently reported for a soluble fragment of Sco1p expressed in E. coli (10).
Sequence of Mature Sco1p-The sequence of the amino-terminal six residues (ESNGKK) of Sco1p purified from E428/U1/ ST5 matches the sequence deduced from the gene sequence (2) starting with the serine at residue 42. The preceding residue 41 based on the gene sequence should be a glutamine instead of the glutamic acid indicated by the protein sequence. This finding suggests that following cleavage of the presequence, the amino-terminal Gln42 of the mature protein is deaminated.
Concentration of Sco1p in Mitochondria-To estimate the mitochondrial concentration of Sco1p, a standard curve was obtained relating known amounts of the purified protein to the signal detected by Western blot analysis. The mitochondria from a wild type strain and from the E428/U1/ST5 transformant were similarly analyzed on the same Western blot (Fig.  3). The concentration of Sco1p in wild type mitochondria was calculated to be 0.8 g (27 pmol) of Sco1p per milligram of protein. This value is 2-3 times lower than the concentration of Cox17p (80 pmol/mg protein) determined previously (3). The concentration of Sco1p in E428/U1/ST5 mitochondria was 52 g (1.7 nmol) per milligram of protein, indicating a 60-fold overexpression from the multicopy plasmid.
Molecular Weight of Native Sco1p-The mass of Sco1p was estimated from its sedimentation relative to hemoglobin in sucrose gradients (18). The size of Sco1p was determined by sedimentation analysis in sucrose gradients of wild type and E428/U/ST5 mitochondrial extracts and of the purified protein.
Based on its sedimentation relative to hemoglobin (Fig. 4), native Sco1p has a mass of 88 kDa. The fact that the overexpressed and purified protein sedimented similarly to Sco1p extracted from wild type mitochondria indicates that it is not stably associated with other proteins. The mass of the mature Sco1p monomer is 28.7 kDa, suggesting that the native protein is a homotrimer.
Lack of Complementation of the sco1 Mutant by Soluble Sco1p Directed to the Matrix or Intermembrane Compartment-Sco1p is an intrinsic inner membrane protein with a single membrane-spanning domain (11) and a topology such that the carboxyl-terminal region containing the active site faces the intermembrane space (6). Sco1p behaves as a water-soluble protein when expressed from a gene lacking the sequence coding for the transmembrane domain (11). The water-soluble form of Sco1p was found not to be functional as evidence by its inability to complement sco1 mutants (11). These observations suggested that the maturation of subunit 2 might require that Sco1p be present in the inner membrane. 2 However, in that study, the location of the soluble Sco1p was not determined. Hence, if Sco1p lacking the transmembrane domain is transported to the matrix, the mislocalization could also account for failure to complement the mutant. To address this question, we first examined the compartment in which soluble Sco1p is located. Resistance against protease K (Fig. 6) indicates that the soluble protein is transported to the matrix compartment. The matrix localization implies that the hydrophobic transmembrane domain acts as a stop-transfer sequence.
The dependence of Sco1p function on its localization and/or membrane association was further examined by directing the soluble protein to the intermembrane space. The sequence of SCO1 starting from codon 41 was fused to the sequence encoding the cytochrome c 1 presequence (12). The gene was further modified by removing the sequence coding for the transmembrane domain. The cytochrome c 1 presequence consists of an amino-terminal mitochondrial targeting signal followed by a hydrophobic sorting sequence (20). The targeting signal directs the amino terminus to the matrix where it is cleaved by the matrix-processing protease. The hydrophobic part of the presequence anchors the precursor to the inner membrane (21) after which cleavage by the Imp protease (22) causes the release of the mature amino terminus in the intermembrane space (21). The predication was that substitution of the cytochrome c 1 bipartite signal for its normal import signal would cause Sco1p to be localized in the intermembrane space (Fig. 5C). The intermembrane localization of Sco1p expressed from this construct (pG41/ST44) was confirmed by its proteinase K sensitivity in mitoplasts but not mitochondria (Fig. 6). Most of the intermembrane Sco1p was solubilized by alkaline extraction of mitochondria with carbonate (Fig. 7). However, the protein probably has some residual hydrophobic character, because it is only partially released when mitochondria are converted to mitoplasts (Fig. 6). The retargeted soluble Sco1p failed to rescue the mutant, indicating that its postulated function in copper transfer probably requires that it be anchored to the membrane.
It is of interest that sco1 mutants also fail to be complemented by the SCO1 gene in pG41/ST24. The gene in this plasmid consists of the cytochrome c 1 presequence fused to the entire SCO1 sequence coding for the mature protein including the transmembrane domain. The product of this gene is processed to the mature-size Sco1p that is located in the intermembrane space, but unlike the native Sco1p, it is not located as an intrinsic membrane protein (data not shown). This indicates that the transmembrane domain of Sco1p is a stop-transfer rather than a membrane-targeting/insertion sequence (21). The inability of this gene to complement the sco1 mutant emphasizes the importance of both compartmentation and membrane topology for Sco1p activity.