Identification of the structural gene for yeast cytochrome c oxidase subunit II on mitochondrial DNA.

SLONIMSKI From the Centre de Gkn6tique Moldculaire du Centre National de la Recherche Scientifique, F-91190 Gif-sur-Yvette, France The mitochondrially translated polypeptides were ana- lyzed in yeast mutants which specifically lacked cytochrome c oxidase as a result of a mutation in mitochondrial DNA. In an earlier study, the mutations have been mapped in one of three distinct loci designated OXI 1, OX1 2, and OX1 3. The OX1 1 locus is now shown to code for the mitochon- drially translated Subunit II by the following criteria: (a) in two OX1 1 mutants, cytochrome c oxidase Subunit II is replaced by a shorter mitochondrially made polypeptide which is not detected in the wild type parent; these “new” polypeptides can be identified as fragments of Subunit II by their weak, but significant cross-reaction with an antiserum against Subunit II from wild type cells. (b) When these two OX1 1 mutants are reverted by an additional mutation of mitochondrial DNA, Subunit II regains its strong interac- tion with anti-Subunit II serum, but its electrophoretic mobility on dodecyl sulfate-polyacrylamide gels is increased over that of wild type Subunit II. Electrophoresis in the presence of the cationic detergent, cetyltrimethylammo- nium bromide, determination of the free electrophoretic mobility, and peptide mapping by limited proteolysis indi- cate that the increased mobility of Subunit II in the revert- ants does not merely reflect abnormal binding of dodecyl sulfate. We conclude that the OX1 1 locus contains the structural gene for cytochrome oxidase Subunit II. Mitochondria contain a genetic system that participates in their own formation (l-4). The main function of this system appears to be the synthesis of at least nine hydrophobic polypeptides which are tightly associated with the mitochon- drial inner membrane. Three of these polypeptides are associ- * This work was supported by Grant 3. 2350. 74 from the Swiss National Science Foundation, a fellowship from the Sandoz Foun- dation, and Grant 75-7-0748 from DQl&tion G&&ale B la Re- cherche Scientifique. Int&actions Mol&ulaires en Biologie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U. S. C. Section 1734 solely to indicate this fact. Present address, National Institutes of Health, Building 37, Bethesda, Md. 20014. § To whom correspondence should be addressed. ated with cytochrome c oxidase, two to four (depending on the organism) with the ATPase complex, and at least one cytochrome b (1, 2, 4). The function of the remaining polypep- tides is still unknown. While it is firmly established that these polypeptides are translated on mitochondrial ribosomes, no structural gene for any of these polypeptides has yet been identified (2-4). For many years it was even debated whether these structural genes were localized on nuclear rather than mitochondrial DNA (2). Recent experiments with cell-free systems pro- grammed with mitochondrial DNA (5) or RNA (6) strongly suggest, however, that at least some of the mitochondrial translation products are coded by mitochondrial DNA. This is also implied by studies which exploited the differences in electrophoretic mobility of several mitochon- drial translation products from different yeast strains (7). However, none of these efforts have led to the identification of a gene coding for a functionally defined mitochondrially made polypeptide. The present study is based on the recent demonstration (8, 9) that mutations in three loci on yeast mitochondrial DNA can lead to the selective loss of cytochrome oxidase. These loci (termed OX1 1, 2, and 3) have been mapped (9), but it remained unclear whether they were structural or regula- tory genes for cytochrome oxidase. We now report evidence which indicates that the OX1 1 locus is the structural gene for cytochrome oxidase Subunit II.