Properties of cytoplasmic mutants of Saccharomyces cerevisiae with specific lesions in cytochrome oxidase.

Abstract

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).

COX15 Codes for a Mitochondrial Protein Essential for the Assembly of Yeast Cytochrome Oxidase

“…G4 mutants are respiratory deficient as a result of recessive mutations in a nuclear gene, causing a specific deficiency in cytochrome oxidase. The enzymatic properties of N7-211, a representative of this complementation group have already been reported in an earlier mutant screen (20). Based on assays of respiratory chain enzymes and spectral analysis of mitochondrial cytochromes, this mutant was shown to have a specific cytochrome oxidase deficiency (20).…”

“…Difference spectra of reduced versus oxidized cytochromes of mitochondrial extracts were taken at room temperature (20). Cytochrome oxidase activity was measured as described previously (20).…”

“…Mitochondria were prepared from the respiratory competent strain W303-1A, from two cox15 mutants (aW303⌬COX15 and WL⌬COX15), and from the transformant WL⌬COX15/Int, which harbors a chromosomally integrated copy of COX15 fused to a bacterial sequence encoding a biotinylation signal. Mitochondria at a protein concentration of 5-7 mg/ml were extracted with potassium deoxycholate under conditions that quantitatively solubilize all the cytochromes (20). Difference spectra of extracts reduced with sodium hydrosulfite and oxidized with potassium ferricyanide were recorded at room temperature.…”

“…The LEU2 and HIS3 disrupted alleles were substituted for the normal chromosomal copies of the gene in the respiratory competent haploid strains LL20 (20) and W303-1A, respectively. Respiratory deficient transformants were verified to be complemented by a o tester strain but not by G4 mutants, confirming the presence of the disruption alleles.…”

“…The insert of this plasmid was dissected at an internal BamHI site into two fragments. Of the two, only the shorter 2.3-kb fragment starting at the internal BamHI site, and extending to the distal end of the insert, (20). Mitochondria were prepared and samples representing 50 g of protein were separated by PAGE on a 7.5-15% polyacrylamide gel (37).…”