Mol Gen Genet (1989) 216:3743 © Springer-Verlag 1989 Accumulation of the cytochrome c oxidase subunits I and II in yeast requires a mitochondrial membrane-associated protein, encoded by the nuclear SC01 gene Marion Sehulze and Gerhard Riidel* Institut ffir Genetik und Mikrobiologie der Universit/it Mfinchen, Maria-Ward-Strasse 1 a, D-8000 Mfinchen 19, Federal Republic of Germany Summary. The yeast nuclear SCO1 gene is required for accumulation of the mitochondrially synthesized cyto- chrome c oxidase subunits I and II (COXI and COXII). We cloned and characterized the SC01 gene. It codes for a 0.9 kb transcript. DNA sequence analysis predicts a 33 kDa protein. As shown by in vitro transcription and translation experiments in combination with import studies on isolated mitochondria, this protein is matured into a 30 kDa polypeptide which is tightly associated with a mito- chondrial membrane. The possible function of the SC01 gene product in the assembly of cytochrome c oxidase is discussed. Key words: DNA sequence - PET gene - Saccharomyces eerevisiae Mitochondrial import - cytochrome c oxidase Introduction Yeast cytochrome c oxidase is a multi-protein complex, composed of nine different subunits (Power et al. 1984). The three large subunits (COXI, COXII, COXIII), which are generally believed to represent the catalytic domain of cytochrome c oxidase (Capaldi et al. 1983), are coded by mitochondrial DNA, while the six smaller subunits (COXIV, COXV, COXVI, COXVII, COXVIIa, COXVIII) are coded by nuclear genes (Schatz and Mason 1974). With the exception of COXVII, all the respective structural genes have been isolated and characterized (Bonitz et al. 1980; Coruzzi and Tzagoloff 1979; Fox 1979; Thalenfeld and Tzagoloff 1980; Maarse et al. 1984; McEwen et al. 1986; Koerner etal. 1985; Sbraphin etal. 1985; Wright etal. 1984, 1986; Patterson and Poyton 1986). Biogenesis of cytochrome c oxidase is subjected to com- plex regulation. Heme and oxygen have been shown to be essential for accumulation and assembly of the subunits (Saltzgaber-Mtiller and Schatz 1978; Woodrow and Schatz 1979). In addition, synthesis of cytochrome c oxidase is inhibited by the presence of glucose (" glucose-repression"; Mahler et al. 1975). Several mutations have been described which interfere with the formation of cytochrome c oxidase. * Present address. Institut ffir Pathologic und Rechtsmedizin der Universit/it Ulm, Labor ffir Molekulare Biologic und Allgemeine Pathologic, Neuherbergstrasse 11, D-8000 Mfinchen45, Federal Republic of Germany Offprint requests to." M. Schulze Beside mutations in structural genes, mutations have been identified in genes which are somehow involved in the syn- thesis and/or assembly of the subunits, preferentially of the three mitochondrially synthesized polypeptides. Of 34 nuclear complementation groups, which are mini- mally required for formation of cytochrome c oxidase, 17 are specifically involved in expression of either COXI, COXII, COXIII or COXI and cytochrome b (COB; McEwen et al. 1986). With the exception of mutants in three complementation groups, which lack COX1 or COX2 tran- scripts, all these mutants show mature-sized mitochondrial COX transcripts and are therefore affected in a post-tran- scriptional step in the expression of the mitochondrially- coded cytochrome c oxidase subunits (Kloeckener-Gru- issem et al. 1987). This suggests that the mutant phenotype is caused by a failure to translate the respective mRNAs or by a rapid degradation of the newly synthesized protein. In case of pet494 and pet54 mutants, which both lack COXIII, synthesis of COXIII can be restored by mitochon- drial gene rearrangements, which fuse the COXIII structur- al gene to 5'-untranslated sequences of other mitochondrial genes (Costanzo and Fox 1986; Costanzo et al. 1986). This result clearly demonstrates that the gene products of PET494 and PET54 are necessary to activate COX3 mRNA translation rather than to prevent rapid degradation of newly synthesized COXIII. Similar specific translation fac- tors for COX2 mRNA (PETlll; Poutre and Fox 1987) and COB mRNA (CBSI and CBS2; R6del et al. 1985; R6del 1986) have been reported. We recently described a yeast nuclear gene, SCO1, which is required for COXII accumulation (Schulze and R6del 1988). Deletion of a 1.7 kb DNA fragment of chro- mosome II, which contained the complete SCO1 gene, re- sulted in a concomittant loss of COXII and COXI. This experiment gave no answer as to whether the observed phe- notype resulted from loss of SCO1 alone or whether (par- tial) deletion of another gene, immediately adjacent to SC01, is responsible for loss of COXI. In this paper we report the nucleotide sequence of the SCO1 gene and show that it is required for both COXI and COXII accumulation. In vitro transcription" and trans- lation yields a polypeptide of molecular weight 33 000 dal- ton, which is in excellent agreement with the molecular weight of the protein predicted from the nucleotide se- quence. We further show by in vitro import studies into isolated mitochondria and subsequent carbonate extraction that this protein represents the precursor form of a mito-