A Novel Respiration-deficient Mutant of Saccharomyces cerevisiae

Abstract

Abstract A novel class of respiration-deficient (RD) yeast mutants has been isolated by a procedure designed to enrich for mitochondrial mutations. Four such independently isolated mutants exhibit a phenotype that differs from the classical petite mutant and does not show complementation with a DNA-less (p0) member of the latter class. One of these mutants, strain 73/1 was subjected to further analysis and found to exhibit mitochondrial inheritance on the basis of: (a) lack of this complementation; (b) non-Mendelian segregation of the RD trait after meiosis of diploids formed by mating with standard auxotrophic testers; (c) segregation on mitosis of these diploids, with the RD trait exhibiting vegetative dominance; (d) elimination of the trait and of c upon conversion to a p0 mutant by prolonged growth in the presence of ethidium bromide. Examination of the phenotype disclosed the absence in cells and mitochondria of cytochrome oxidase, cytochrome aa3, and of NADH- and succinate-cytochrome c reductases, and the presence of significant amounts of cytochromes b and c1. These two cytochromes appear reducible by succinate to a small and by NADH to a considerable extent. The mitochondrial ATPase in the mutant is sensitive to the specific inhibitor Dio-9 but resistant to oligomycin at concentrations that inhibit the wild type enzyme ≥50%. Unlike cytoplasmic petites, mitochondria of mutant 73/1 contain a functional protein-synthesizing system. Its cells appear capable not only of mitochondrial polypeptide chain initiation as measured by the formation of formylmethionyl puromycin, but also of incorporating labeled amino acids into mitochondrial proteins in the presence of cycloheximide, a process inhibited by chloramphenicol and acriflavin. The presence of a functional mitochondrial system of protein synthesis is confirmed by the elimination of the cytochrome b peak at 560 nm upon prolonged growth (six generations) in the presence of chloramphenicol. This conversion to a petite phenocopy is reversed completely upon removal of the inhibitor.