Abstract
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 approximately 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.
Highlights
(1, 3) and is an example of a larger group of cytoplasmic proteins that target copper to different cellular compartments [4]
We estimate that Sco1p constitutes 0.08% total mitochondrial proteins in wild type yeast and 5% in the transformant used for the purification
The evidence obtained with constructs expressing Sco1p lacking the membrane-spanning domain or having its normal import signal substituted with the leader of cytochrome c1 shows that the localization and orientation of the protein are essential for its function
Summary
Yeast Strains and Media—Sco1p was purified from two different strains of S. cerevisiae. The gene in the resultant plasmid (pG41/ ST24) codes for the following sequence at the junction of cytochrome c1 and Sco1p: glu-ala 2 met-thr-ala-ala-Asp-gln-ser-asn-gly where the arrow demarcates the processing site in cytochrome c1, the lowercase residues are part of the cytochrome c1 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]. 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).
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