Mitochondrial mutants of Succhuromyces cerevisiue defective in cytochrome b were analyzed genetically and biochemically in order to elucidate the role of the mitochondrial genetic system in the biosynthesis of this cytochrome.The mutants mapped between OLIl and OLZ2 on mitochondrial DNA in a region called COB. A fine structure map of the COB region was constructed by rho-deletion mapping and recombination analysis.The combined genetic and biochemical data indicate that the COB region is mosaic and contains at least five distinct clusters of mutants, A-E, with A being closest to OLZ2 and E being closest to OLIl. Clusters A, C and E are probably coding regions for apocytochrome b, whereas clusters B and D seem to be involved in as yet unknown functions. These conclusions rest on the following evidence.1. Most mutants in clusters A, C and E have specifically lost cytochrome b. Many of them accumulate smaller mitochondrial translation products ; some of these were identified as fragments of apocytochrome b by proteolytic fingerprinting. The molecular weight of these fragments depends on the map position of the mutant, increasing in the direction OLI2+OLII. The mutant closest to OLIl accumulates an apocytochrome b which is slightly larger than that of wild type.2. A mutant in cluster C exhibits a spectral absorption band of cytochrome b that is shifted 1.5 nm to the red.3. Mutants in clusters B and D are pleiotropic. A majority of them are conditional and lack the absorption bands of both cytochrome b and cytochrome uu3; these mutants also fail to accumulate apocytochrome b and subunit I of cytochrome c oxidase and instead form a large number of abnormal translation products whose nature is unknown.4. Zygotic complementation tests reveal at least two complementation groups: The first group includes all mutants in cluster B and the second group includes mutants in clusters (A + C + D + E).
The yeast mitochondrial translation product of M , 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]~ulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bcl complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J; Biol. Chem. 250 (1977) 8236-82421 and by immunoprecipitation with a specific antiserum against apocytochrome b.New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apocytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.
1. In non-fermentable substrates growth of mutant tsm-8 cells of Saccharomyces cerevisiae is restricted to about one generation after shift from 23 to 35 degrees C. Non-permissive conditions (35 degrees C, glycerol) cause a gradual decrease in respiration to about 20% of the activity at permissive temperature 23 degrees C). 2. Anaerobically grown and glucose-repressed mutant cells exhibit a decreased adaptation rate of mitochondrial functions to aerobic growth and non-fermentative growth, even at 23 degrees C, as revealed by determination of respiratory rates and mitochondrial protein synthesis. 3. At 35 degrees C, rho+ cells of mutant tsm-8 are converted to p- cells within 6-8 generations of growth, in all fermentable substrates tested. Drugs or antibiotics as nalidixic acid, acriflavin, chloramphenicol and erythromycin, bongkrecic acid, antimycin and FCCP, as well as anaerobiosis, have little or no influence on this kinetics. A heat shock does not yield rho- petites to a significant extent. 4. Reversion of tsm-8 cells to wild type function, which occurs spontaneously with a frequency of 10(-8), is found to be due to a mitochondrial mutational event.
Mitochondrial DNA, protein and ATP syntheses persist at non-permissive temperature (35 "C) in the mitochondrial, conditionally rho-petites forming yeast mutant, tsm8. Protein and ATP syntheses, however, are diminished during prolonged incubation at 35 "C in non-fermentable substrate.Mitochondrial RNA synthesis decreases rapidly to a residual constant level of about 10 % of the initial value after the shift to 35 "C. The decrease is reversed by returning to permissive conditions. Evidence is presented that this temperature-induced decrease in mitochondrial transcription rate is effected by a mutationally altered regulatory process rather than by temperature sensitivity of mitochondrial RNA polymerase. It is concluded that rho-petite formation in mutant tsm8 is not effected by complete inhibition of macromolecular and ATP syntheses but is correlated with a reduction in mitochondrial transcription.Considerable progress towards the identification of mitochondrial genes and gene products has been made by the genetic and biochemical analysis of recently described mit-mutants of Saccharomyces cerevisiae. It has been shown that mit-mutations mapping in different regions of mitochondrial DNA (mtDNA) cause deficiencies in respiratory complexes I11 or IV or in the ATPase complex. In some mutants an alteration in, or a lack of, a (mitochondrially synthesized) protein component of one or the other of these complexes could be related to the mit-mutation Different from these mit-mutants which are deficient in one or the other functional complex, is a conditional mit-mutant previously described by this laboratoy [ 5 ] . Pleiotropic deficiencies in mitochondrial respiratory functions and a lack in several mitochondrially synthesized components of the inner membrane were observed when cells of this mutant, tsm8, were grown at 35 "C [6]. At this temperature growth on non-fermentable substrate of mutant cells was arrested, whereas on fermentable substrates growth is not limited but results in a rapid conversion of cells into rho-Detailed genetic analysis has revealed that mutation tsm8 is a mutation in a mitochondrial gene locus different from previously known mit-loci and also [l-41. Abbreviation. FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone.different from the loci determining drug resistance/ sensitivity or coding for mitochondrial rRNA [8,9]. The analysis of mitochondrial proteins and functions affected by mutation tsm8 thus promises to lead to the identification of a new mitochondrial gene product. Furthermore, the knowledge of gene product and functions altered by mutation tsm8 may help to understand the exceptionally high rho factor instability observed in this mutant.This paper intends to present a study of mitochondrial macromolecular syntheses in mutant tsm8. The results indicate a conditional reduction in expression of part of or all mitochondrial genes in this mutant, which may cause the pleiotropic effects observed in mutant cells at 35 "C, including the exceptionally high rate of the formation of rho-peti...
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