Abstract Saccharomyces cerevisiae Sco1p is believed to be involved in the transfer of copper from the carrier Cox17p to the mitochondrial cytochrome c oxidase sub- units 1 and 2. We here report on the results of a mutational analysis of Sco1p. The two cysteine residues of a poten- tial metal-binding motif (CxxxC) are essential for protein function as shown by their substitution by alanines. Chi- meras consisting of Sco1p and its homolog S. cerevisiae Sco2p restrict the specificity of Sco1p function to the N- terminal half of the protein. A candidate region for confer- ring specificity on Sco1p is a stretch of hydrophobic amino acids, which act as a membrane anchor. In line with this suggestion is the result that alterations of individual amino acids within this region impair Sco1p function. Key words Yeast Sco1p · Copper · Cytochrome c oxidase · Mitochondria Introduction In the yeast Saccharomyces cerevisiae copper is trans- ported across the plasma membrane by Ctr1p and trans- ferred to small cytosolic copper-binding proteins such as Atx1p, Lys7p or Cox17p (Valentine and Gralla 1997). At- tachment of copper to the final acceptor proteins like the cytosolic CuZn superoxide dismutase (Sod1p), a multi- copper oxidase in a post-Golgi compartment (Fet3p), or mitochondrial cytochrome c oxidase (COX), requires spe- cific mediator proteins (Yuan et al. 1997). A candidate me- diator for the attachment of copper to COX subunits 1 and 2 is the nuclearly encoded Sco1p, which is anchored by a single trans-membrane (TM) segment of about 17 amino acids to the inner mitochondrial membrane (Buchwald et al. 1991). Sco1p was originally identified as a protein re- quired for the assembly of COX (Schulze and Rödel 1989). The two largest COX subunits, Cox1p and Cox2p, which carry heme and copper as prosthetic groups and constitute the catalytic centre of the enzyme, are rapidly degraded in the absence of a functional SCO1 gene (Krummeck and Rödel 1990). A first clue to the function of Sco1p was ob- tained by Glerum et al. (1996 b), who detected that over- expression of Sco1p in combination with slightly elevated concentrations of copper in the growth medium can par- tially suppress the deficiency of Cox17p. Cox17p is a cop- per-binding protein in both the cytosol (Glerum et al. 1996 a) and the mitochondrial inter-membrane space (Beers et al. 1997), suggesting that Sco1p might be involved in the at- tachment of copper to COX (Glerum et al. 1996 b). This view is supported by the presence of a potential metal-bind- ing motif CxxxC (Glerum et al. 1996 b). Such a motif, with a characteristic spacing of cysteine residues, has been shown to constitute a copper-binding site in Cox2p (Co- ruzzi and Tzagoloff 1979) and an iron-binding site in fer- redoxins (Bruschi and Guerlesquin 1988). The CxxxC mo- tif of Sco1p is located in the carboxy-terminal part of the protein which protrudes into the mitochondrial inter-mem- brane space (Krummeck 1992; Beers et al. 1997). In the course of the yeast genome sequencing project a gene with a high degree of similarity to SCO1 (overall aa identity of 53.8%) was detected (Smits et al. 1994) (see Fig. 2). The function of its gene product, Sco2p, is enig- matic: The protein is localized in the mitochondrial mem- brane (Glerum et al. 1996 b) as expected from the presence of an amino-terminal sequence reminescent of mitochon- drial targeting sequences and a single predicted trans-mem- brane segment. The position of this hydrophobic stretch and the distribution of charged flanking amino-acid resi- dues are almost identical in Sco1p and in Sco2p, suggest- ing a similar topology. Like Sco1p the larger C-terminal part, which probably protrudes into the inter-membrane space, carries the potential metal-binding motif CxxxC. Curr Genet (1999) 35: 103–108 © Springer-Verlag 1999 Received: 30 October / 10 December 1998 Anja Rentzsch · Gaby Krummeck-Weiß Angelika Hofer · Anne Bartuschka · Kai Ostermann Gerhard Rödel Mitochondrial copper metabolism in yeast: mutational analysis of Sco1p involved in the biogenesis of cytochrome c oxidase ORIGINAL PAPER A. Rentzsch · G. Krummeck-Weiß · A. Hofer · A. Bartuschka K. Ostermann · G. Rödel () Institut für Genetik, Technische Universität Dresden, Mommsenstraße 13, D-01062 Dresden, Germany e-mail: Gerhard.Roedel@mailbox.tu-dresden.de Tel.: +49-351-4633010 Fax: +49-351-4637725 Communicated by L. A. Grivell