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Journal ArticleDOI

Biogenesis of Mitochondria, XVIII. A New Class of Cytoplasmically Determined Antibiotic Resistant Mutants in Saccharomyces cerevisiae

TL;DR: Results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins.
Abstract: New mutant yeasts resistant to the antibiotics chloramphenicol and mikamycin were isolated. They are mitochondrial mutants, characterized by several criteria as cytoplasmically determined. Biochemical studies show that amino acid incorporation into protein in vitro by mitochondria isolated from cells resistant or sensitive to mikamycin or chloramphenicol is inhibited by these antibiotics. Although aerobically-grown resistant strains of Saccharomyces cerevisiae are not affected by mikamycin or chloramphenicol, it is found that the mitochondrial protein-synthesizing system of anaerobically grown cells is inhibited in vivo. Cross resistance among the antibiotics chloramphenicol, mikamycin, erythromycin, lincomycin, carbomycin, and spiramycin is reported. All erythromycin resistant mutants, unlike the others, are resistant to erythromycin in vivo and in vitro. The results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins. We further suggest that conformational changes, either in the membranes or ribosomes, are likely to account for the observed antibiotic cross resistances.
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Journal ArticleDOI
TL;DR: The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions and results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.
Abstract: The rules that govern complementation of mutant and wild-type mitochondrial genomes in human cells were investigated under different experimental conditions. Among mitochondrial transformants derived from an individual affected by the MERRF (myoclonus epilepsy associated with ragged red fibers) encephalomyopathy and carrying in heteroplasmic form the mitochondrial tRNA(Lys) mutation associated with that syndrome, normal protein synthesis and respiration was observed when the wild-type mitochondrial DNA exceeded 10% of the total complement. In these transformants, the protective effect of wild-type mitochondrial DNA was shown to involve interactions of the mutant and wild-type gene products. Very different results were obtained in experiments in which two mitochondrial DNAs carrying nonallelic disease-causing mutations were sequentially introduced within distinct organelles into the same human mitochondrial DNA-less (rho 0) cell. In transformants exhibiting different ratios of the two genomes, no evidence of cooperation between their products was observed, even 3 months after the introduction of the second mutation. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which mitochondria carrying a chloramphenicol resistance-inducing mitochondrial DNA mutation were introduced into chloramphenicol-sensitive cells. A plausible interpretation of the different results obtained in the latter two sets of experiments, compared with the complementation behavior observed in the heteroplasmic MERRF transformants, is that in the latter, the mutant and wild-type genomes coexisted in the same organelles from the time of the mutation. This would imply that the way in which mitochondrial DNA is sorted among different organelles plays a fundamental role in determining the oxidative-phosphorylation phenotype in mammalian cells. These results have significant implications for mitochondrial genetics and for studies on the transmission and therapy of mitochondrial DNA-linked diseases.

170 citations


Cites result from "Biogenesis of Mitochondria, XVIII. ..."

  • ...However, a dominance of the CAPS allele would be in contrast to the available evidence from bacterial systems indicating that the CAPS ribosomes are not "frozen" by CAP on the polysomes and are therefore not expected to block the passage of the resistant ones (18); also, the well-documented isolation of CAPr mutants of Saccharomyces cerevisiae (4, 7) and mammalian cells (44, 48) under selective conditions, which would require the initial expression of single mutant mtDNA molecules mixed with wild-type molecules, argues strongly against a dominance of the CAPS allele....

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Journal ArticleDOI
TL;DR: This review focuses mainly on work with the yeast Saccharomyces cerevisiae, which is the organism of choice for drawing up an inventory of all nuclear genes involved in mitochondrial biogenesis and for the identification of their counterparts in other organisms.
Abstract: All proteins encoded by mitochondrial DNA (mtDNA) are dependent on proteins encoded by nuclear genes for their synthesis and function. Recent developments in the identification of these genes and the elucidation of the roles their products play at various stages of mitochondrial gene expression are covered in this review, which focuses mainly on work with the yeast Saccharomyces cerevisiae. The high degree of evolutionary conservation of many cellular processes between this yeast and higher eukaryotes, the ease with which mitochondrial biogenesis can be manipulated both genetically and physiologically, and the fact that it will be the first organism for which a complete genomic sequence will be available within the next 2 to 3 years makes it the organism of choice for drawing up an inventory of all nuclear genes involved in mitochondrial biogenesis and for the identification of their counterparts in other organisms.

154 citations

Journal ArticleDOI
TL;DR: The presence of a nuclear gene(s) in a strain D6, influencing certain facets of the recombination process in both homosexual and heterosexual crosses has been demonstrated and this behaviour is superimposed upon, and clearly separable from the effects due to the mitochondrial gene ω.
Abstract: Summary1.Two loci OIand OIIlocalized on the mitochondrial DNA and conferring oligomycin resistance in S. cerevisiae have been demonstrated. The two loci show a high frequency of recombination with each other.2.In the majority of OR strains examined the mutation was found to map at the OIlocus.3.Mapping studies have indicated that the OIand OIIloci are either unlinked or very weakly linked to each other. Both of these loci also appear to be essentially unlinked from the ω-RI-RII-RIIIsegment of the mitochondrial genome specifying mitoribosomal functions.4.Analysis of crosses involving mutants at either the OIor OIIloci and a series of ϱ− petites variously deleted in known mitochondrial genes has demonstrated that the two oligomycin resistance loci OIand OIIare separable. Assuming that the non deleted segment of the mitochondrial DNA is continuous, the results suggest the gene order ω-RI-RII-RIII-OI-OII.5.The rules applicable to the system and previously delineated from studies of the RI, RIIand RIIIloci in oligomycin sensitive strains have been shown to apply equally to strains carrying OR alleles at the OIand OIIloci. Oligomycin resistance alleles at both the O loci, OIand OII, are present in both ω+ and ω− strains.6.The presence of a nuclear gene(s) in a strain D6, influencing certain facets of the recombination process in both homosexual and heterosexual crosses has been demonstrated and this behaviour is superimposed upon, and clearly separable from the effects due to the mitochondrial gene ω.

151 citations

Journal ArticleDOI
TL;DR: It is proposed that Mba1 functions as a ribosome receptor that cooperates with Oxa1 in the positioning of the ribosomes exit site to the insertion machinery of the inner membrane.
Abstract: The genome of mitochondria encodes a small number of very hydrophobic polypeptides that are inserted into the inner membrane in a cotranslational reaction. The molecular process by which mitochondrial ribosomes are recruited to the membrane is poorly understood. Here, we show that the inner membrane protein Mba1 binds to the large subunit of mitochondrial ribosomes. It thereby cooperates with the C-terminal ribosome-binding domain of Oxa1, which is a central component of the insertion machinery of the inner membrane. In the absence of both Mba1 and the C-terminus of Oxa1, mitochondrial translation products fail to be properly inserted into the inner membrane and serve as substrates of the matrix chaperone Hsp70. We propose that Mba1 functions as a ribosome receptor that cooperates with Oxa1 in the positioning of the ribosome exit site to the insertion machinery of the inner membrane.

144 citations


Cites background from "Biogenesis of Mitochondria, XVIII. ..."

  • ...The tight association of fungal mitochondrial ribosomes with the inner membrane is known for a long time as the addition of detergent is necessary for their isolation (Bunn et al, 1970; Borst and Grivell, 1971)....

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  • ...…ribosomes remain membrane-associated in the absence of both Mba1 and Oxa1 In contrast to cytosolic ribosomes, mitochondrial ribosomes are tightly associated with membranes and, at least in fungi, can only be released by treatment with detergents (Bunn et al, 1970; Borst and Grivell, 1971)....

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Journal ArticleDOI
TL;DR: The rules applicable to the mitochondrial genetic system and previously delinated from the studies of the ω-RI-RII-RIII segment specifying mitoribosomal functions have been shown to apply equally to PR strains.
Abstract: Summary1.A mutation (PR) conferring resistance to paromomycin is shown to be located on the mit-DNA. At the cellular level it increases the resitance 30 to 40 fold and increases the sensitivity to chloramphenicol 2 fold. It is suggested that the PR acts at the mitoribosomal level.2.The rules applicable to the mitochondrial genetic system and previously delinated from the studies of the ω-RI-RII-RIIIsegment specifying mitoribosomal functions have been shown to apply equally to PR strains. ω+ PR strains have been constructed from ω− PR mutants.3.Homo- and heterosexual crosses involving 3 and 4 mutations located at the loci RI, RIII, OI, PIand conferring resistance respectively to chloramphenicol, erythromycin, oligomycin, and paromomycin have been performed in all possible cis trans configurations. PR and OR mutations present similar features of recombination which are different from those shown by the CR and ER mutations. PR and OR are not included in the ω-RI-RII-RIIIsegment and are not linked or only very loosely linked to it. PR and OR are not linked together.4.Main features of mitochondrial multifactorial crosses are analysed in terms of the frequency and the distribution of different classes of recombinants. In homosexual crosses a positive coincidence is observed for all combinations of three or four markers. In heterosexual crosses the polarity of recombination is shown to depend on the segment considered. An excess of the allele of a third marker brought by the ω− parent is observed among any polar recombinants for two others.

132 citations