Two mutants with specific defects in cyto- It has been established from recent studies with yeast (1, 2) and Neurospora crassa (3, 4) that the cytochrome oxidase complex of mitochondria contains both mitochondrially and cytoplasmically synthesized subunit proteins. In both organisms the enzyme has been shown to consist of seven distinct polypeptides, the three largest being mitochondrial translation products (1, 2, 4). Although it is generally assumed that the mitochondrial products are coded for by mitochondrial DNA, this has not been experimentally verified. A simple and direct demonstration of this relationship would be the isolation of cytoplasmic mutants with specific lesions in enzymes known to have mitochondrially synthesized proteins. In this communication we report two mutants of Saccharomyces cerevisiae which show non-Mendelian segregation and whose primary defect is in the cytochrome oxidase complex. Biochemical analyses indicate that both mutants lack the visible absorption bands of cytochromes a and a3 and that one of the mutants is missing the largest subunit of the oxidase. MATERIALS AND METHODSThe haploid prototrophic strain of a mating type, S. cerevisiae D273-1OB, characterized by Sherman et al. (5) Isolation of Mutants. The details of the screening procedure used to isolate the mutants will be described elsewheret. Briefly, the selection method consisted of analyzing mitochondrial functions in a class of mutants that were unable to grow on glycerol but were still capable of mitochondrial translation.Complementation Test. The tester strain used was Saccharomyces carlsbergensis CB11 (a MAL6 adel) which was subjected to two cycles of ethidium bromide treatment under conditions that should have deleted all of the mitochondrial DNA (6).

A selection procedure is described which permits a large number of Saccharomyces cerevisiae mutants to be screened for specific lesions in mitochondrial respiratory enzymes and the adenosine triphosphatase. The method has been used to isolate nuclear mutant strains with specific lesions in coenzyme QH2-cytochrome c reductase, cytochrome oxidase, and adenosine triphosphatase. In addition, two cytoplasmic mutants have been found whose primary defect is in cytochrome oxidase, and others have been found that show variable degrees of abnormalities in their mitochondrial translation products.

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