Mitochondrial resistance to miconazole in Saccharomyces cerevisiae

Summary One mutant of mitochondrial origin resistant to miconazole has been isolated and characterized in S. cerevisiae. The mutation is linked to the locus oli1, the structural gene for subunit 9 of ATPase on mitochondrial DNA. Miconazole inhibited the mitochondrial ATPase of the wild type while the enzyme of the resistant mutant was insensitive to this effect. Levels of ATP decreased to one-third of the control in the wild type in the presence of miconazole, while they were unaffected in the mutant.Abbreviations MNNG N-methyl-N-nitrosoguanidine - Mic^s/Mic^r phenotypic sensitivity/resistance to miconazole - M ₁ ^R mitochondrial locus conferring miconazole resistance - rho⁺/rho^- grand/cytoplasmic petite - rho^o cytoplasmic petite deleted of all mitochondrial DNA - w⁺ mitochondrial locus conferring polarity of recombination     

Localization of mucidin-resistant locus muc3 on mitochondrial DNA with respect to ubiquinol-cytochrome c reductase deficient box loci. Locus muc3 is allelic to box2

Summary Genetic relations between mitochondrial mucidin-resistant locus muc3 and ubiquinol-cytochrome c reductase-deficient box loci have been studied by recombination and petite deletion analysis. It was found that the locus muc3 maps in the segment of mitochondrial DNA corresponding to the locus box2. The results suggest the participation of box2/muc3 locus in the sequences of the structural gene for cytochrome b.     

Comparative studies of the effects of acridines and other petite inducing drugs on the mitochondrial genome of Saccharomyces cerevisiae.

Summary The effects of the acridines euflavine and proflavine on mitochondrial DNA (mtDNA) replication and mutation inSaccharomyces cerevisiae have been compared. In contrast to previous results we found that under our conditions proflavine can indeed induce high levels (>80%) of petite mutants, although six times less efficiently than euflavine. The parameters measured for mutagenesis of the mitochondrial genome and inhibition of mtDNA replication in whole cells suggest that the modes of action of euflavine and proflavine are very similar. After extended (18h) treatment of growing cells with each drug the percentage loss of mtDNA or genetic loci was almost coincidental with the extent of petite induction.It was found that proflavine is equally as effective as euflavine in inhibiting mtDNA replication in isolated mitochondria in contrast to the differential between the drugs observed in vivo. However, proflavine and euflavine inhibit cellular growth at almost the same concentrations. It is therefore proposed that there is some intracellular permeability barrier which impedes proflavine access to the mitochondrial DNA replicating system.The petites induced by euflavine (and proflavine) are characterized by there being a preferential induction ofrho ⁰ petites lacking mtDNA as opposed torho ^- petites retaining mtDNA. This is in contrast to the relative proportions of such petites induced by ethidium bromide or berenil. A scheme for the production of petites by euflavine is presented, in which euflavine inhibits the replication of mtDNA, but does not cause direct fragmentation of mtDNA (unlike ethidium bromide and berenil). The proposed scheme explains the production of the high frequency ofrho ^o cells, as well as therho ^- cells induced by euflavine. The scheme also accounts for previous observations that euflavine only mutants growing cultures, and that the buds, but not mother cells, become petite.     

Biogenesis of Mitochondria: Analysis of Deletion of Mitochondrial Antibiotic Resistance Markers in Petite Mutants of Saccharomyces cerevisiae

Yeast strains carrying markers in several mitochondrial antibiotic resistance loci have been employed in a study of the retention and deletion of mitochondrial genes in cytoplasmic petite mutants. An assessment is made of the results in terms of the probable arrangement and linkage of mitochondrial genetic markers. The results are indicative of the retention of continuous stretches of the mitochondrial genome in most petite mutants, and it is therefore possible to propose a gene order based on co-retention of different markers. The order par, mik1, oli1 is suggested from the petite studies in the case of three markers not previously assigned an unambiguous order by analysis of mitochondrial gene recombination. The frequency of separation of markers by deletion in petites was of an order similar to that obtained by recombination in polar crosses, except in the case of the ery1 and cap1 loci, which were rarely separated in petite mutants. The deletion or retention of the locus determining polarity of recombination (ω) was also demonstrated and shown to coincide with deletion or retention of the ery1, cap1 region of the mitochondrial genome. Petites retaining this region, when crossed with rho⁺ strains, display features of polarity of recombination and transmission similar to the parent rho⁺ strain. By contrast a petite determined to have lost the ω⁺ locus did not show normal polarity of marker transmission. Differences were observed in the relative frequency of retention of markers in a number of strains and also when comparing petites derived spontaneously with those obtained after ultraviolet light mutagenesis. By contrast, a similar pattern of marker retention was seen when comparing spontaneous with ethidium bromide-induced petites.     

Mapping of the mitochondrial 16S ribosomal RNA gene and its expression in the cytoplasmic petite mutants ofSaccharomyces cerevisiae

The 16S ribosomal RNA gene of yeast mitochondria was titrated in various cytoplasmic petite mutants by DNA-RNA hybridization. The gene was located close to the prolyl transfer RNA gene. The properties of the rho^? strains suggest that the gene order would be: - P_I - 16S - prolyl tRNA - valyl tRNA - (tRNAs) - R_I - R_III -; the 23S ribosomal gene is far from the 16S one. Several petite mutants were found which have retained, in addition to many transfer RNA genes, both of the 23S and 16S ribosomal RNA genes. The two genes seem to be transcribed in these mutants.     

Regulatory interactions between mitochondrial genes: Exon and intron phenotypes observed in vivo can be expressed in vitro

In Saccharomyces cerevisiae the mitochondrial gene responsible for the specification of apocytochrome b (cob-box) is believed to consist of both coding and intervening sequences. Mutations in the latter give rise to pleiotropic phenotypes in vivo, lacking not only cytochrome b but also subunit I of cytochrome oxidase, and producing sets of novel polypeptides. The experiments described here have examined 15 different mit^? mutants in this region and demonstrate that these results are faithfully reproduced by isolated mitochondria in vitro. This inference also applies to other types of mutational lesions in coding segments of the cob-box gene and of the gene oxi3, responsible for the specification of subunit I.     

Maintenance and Integrity of the Mitochondrial Genome: a Plethora of Nuclear Genes in the Budding Yeast

Instability of the mitochondrial genome (mtDNA) is a general problem from yeasts to humans. However, its genetic control is not well documented except in the yeast Saccharomyces cerevisiae. From the discovery, 50 years ago, of the petite mutants by Ephrussi and his coworkers, it has been shown that more than 100 nuclear genes directly or indirectly influence the fate of the rho⁺ mtDNA. It is not surprising that mutations in genes involved in mtDNA metabolism (replication, repair, and recombination) can cause a complete loss of mtDNA (rho⁰ petites) and/or lead to truncated forms (rho⁻) of this genome. However, most loss-of-function mutations which increase yeast mtDNA instability act indirectly: they lie in genes controlling functions as diverse as mitochondrial translation, ATP synthase, iron homeostasis, fatty acid metabolism, mitochondrial morphology, and so on. In a few cases it has been shown that gene overexpression increases the levels of petite mutants. Mutations in other genes are lethal in the absence of a functional mtDNA and thus convert this petite-positive yeast into a petite-negative form: petite cells cannot be recovered in these genetic contexts. Most of the data are explained if one assumes that the maintenance of the rho⁺ genome depends on a centromere-like structure dispensable for the maintenance of rho⁻ mtDNA and/or the function of mitochondrially encoded ATP synthase subunits, especially ATP6. In fact, the real challenge for the next 50 years will be to assemble the pieces of this puzzle by using yeast and to use complementary models, especially in strict aerobes.     

Genetics of oxidative phosphorylation: petite deletion mapping of the Oli 2 region of the mitochondrial genome of Saccharomyces cerevisiae

Summary Petite deletion mapping has been carried out for the Oli 2 region of the mitochondrial genome of Saccharomyces cerevisiae to produce a fine structure genetic map. Previously unlocated mit ^- mutants together with the drug resistant loci Oli 2 and Oss 1 have been ordered between the cytochrome oxidase and apocytochrome b genes.As a result of this study a series of isogenic p ^- clones have been isolated spanning the Oli 2 region.     

Cytochrome b-deficiency in a mitochondrial muc1muc2 recombinant of Saccharomyces cerevisiae

Summary Cytochrome b-deficient muc1muc2 recombinants of Saccharomyces cerevisiae carrying two specific mitochondrial mucidin-resistant mutations were not able to grow on nonfermentable substrates with the exception of lactate. Crosses of the selected muc1muc2 recombinant strain H331 with different box mutants yielded progenies of mucidin-resistant colonies in a pattern which demonstrates the double mutation origin of the recombinant cells.Only 20% of the wild type cytochrome b content was spectrally detected in the muc1muc2 recombinant, which, however, contained normal amounts of cytochromes c and a when grown under derepressing conditions. The respiratory activity of mitochondria isolated from the muc1muc2 recombinant was limited at the level of cytochrome b and was specifically resistant to high concentrations of mucidin. Electrophoretic analysis of the radioactive products of mitochondrial protein synthesis revealed no differences in apparent molecular weight and amount of radioactivity accumulated in apocytochrome b of the wild and recombinant strains.These results indicate that the accumulation in the same strain of the two mucidin-resistance mutations muc1muc2 results in an altered assembly of the noncovalently attached heme to the doubly mutated form of apocytochrome b.Publication n⁰ 1663 of the Biology Division of the Commission of European Communities. Financial help of the Fonds National de la Recherche Fondamentale Collective is also acknowledged     

Assembly of the mitochondrial membrane system. DNA sequence and organization of the cytochrome b gene in Saccharomyces cerevisiae D273-10B

The mitochondrial genomes of cytoplasmic "petite" (rho-) mutants of Saccharomyces cerevisiae have been used to sequence the cytochrome b gene. A continuous sequence of 6.2 kilobase pairs has been obtained from 71.4 to 80.2 units of the wild type map. This region contains all the cytochrome b mutations previously assigned to the cob1 and cob2 genetic loci. Analysis of the DNA sequence has revealed that in the strain D273-10B, the cytochrome b gene is composed of three exons. The longest exon (b1) codes for the first 252 to 253 amino acids from the NH2-terminal end of the protein. The next two exons (b2 and b3) code for 16 to 18 and 115 to 116 amino acids, respectively. The complete cytochrome b polypeptide chain consists of 385 amino acids. Based on the amino acid composition, the yeast protein has a molecular weight of 44,000. The three exon regions of the cytochrome b gene are separated by two introns. The intron between b1 and b2 is 1414 nucleotides long and contains a reading frame that is continuous with the reading frame of exon b1. This intron sequence is potentially capable of coding for another protein of 384 amino acid residues. The second intron is 733 nucleotides long. This sequence is rich in A + T and includes a G + C cluster that may be involved in processing of the cytochrome b messenger. The organization of the cytochrome b region in S. cerevisiae D273-10B is somewhat less complex than has been reported for other yeast strains i which exon b1 appears to be further fragmented into three smaller exons.     

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.     

Biogenesis of mitochondria 27

Summary The isolation and characterization of five new mutants affecting mitochondrial protein synthesis in S. cerevisiae is reported. Each mutation confers in vivo resistance to the macrolide antibiotic spiramycin which acts by inhibiting mitochondrial protein synthesis in sensitive yeast. The mutants are distinguishable on the basis of their in vivo cross resistance to other antibiotics, their biochemical properties and genetic behaviour. Genetic analysis indicates the mode of inheritance to be nuclear for one mutation and cytoplasmic for the other four. Recombination analysis performed on crosses between different cytoplasmic determinants, together with data from monofactorial crosses of each determinant with sensitive strains, demonstrates at least two and possibly three cytoplasmic genetic loci conferring spiramycin resistance.The protein synthesizing activities of mitochondria isolated from the mutant strains range in response to spiramycin and other antibiotics from strong resistance through partial resistance to complete sensitivity. Based on this data the mutants showing strong antibiotic resistance in vitro might be simply classified as mitochondrial ribosome mutants and mutants sensitive in vitro as mitochondrial membrane mutants; however mutants showing partial resistances are not so readily accommodated in either class. The diverse biochemical properties cannot be correlated with the different genetic loci described; indeed three mutations, each resulting in different biochemical behaviour appear to occur at the same locus. The results are interpreted as providing further evidence for an earlier proposal of mitochondrial membrane-ribosome interactions.     

Temperature-sensitive respiratory-deficient mitochondrial mutations: Isolation and genetic mapping

Summary In order to find new genetic loci and functions on the yeast mitochondrial DNA, especially mutations affecting the mitochondrial protein synthesis apparatus, temperature sensitive mutants have been isolated after MnCl₂ mutagenesis and mitochondrial and nuclear mutants classified according to their pattern of recombination with three rho^- tester strains.Eighteen cold- and heat-sensitive respiratory deficient mitochondrial mutants have been isolated and localized on the mitochondrial genome by deletion mapping using 113 rho^- strains. Eight of them appear to represent new loci, among which some are probably mutations of the tRNA and rRNA genes.     

Mammalian mitochondrial mutants selected for resistance to the cytochrome b inhibitors HQNO or myxothiazol

Mouse LA9 cell lines were selected for increased resistance to either HQNO or myxothiazol, inhibitors of electron transport which bind to the mitochondrial cytochrome b protein. Two phenotypically distinguishable HQNO-resistant mutants were recovered while the myxothiazol-resistant isolates had a common phenotype. All three mutant phenotypes were transmitted cytoplasmically in cybrid crosses. Biochemical studies further established that for all three mutant types, resistance at the cellular level was paralleled by an increase in inhibitor resistance of mitochondrial succinate-cytochrome c oxidoreductase, the respiratory complex containing cytochrome b. As with the previously described mitochondrial antimycin-resistant mutant, the initial biochemical and genetic studies indicated that these mutations occur within the mitochondrial cytochrome b gene. This conclusion was strongly supported by the results of mtDNA restriction fragment analyses in which it was found that one HQNO-resistant mutant had undergone a small insertion or duplication in the apocytochrome b gene. Finally, all four mitochondrial cytochrome b mutants have been analyzed in both cell plating studies and succinate-cytochrome c oxidoreductase assays to determine the pattern of cross-resistance to inhibitors of cytochrome b other than the one used for selection.     

Regulatory interactions between mitochondrial genes. I. Genetic and biochemical characterization of some mutant types affecting apocytochrome b and cytochrome oxidase.

The region of the mtDNA containing the structural gene for apocytochrome b is called the cob or box region. There is evidence that the same region is also involved in the regulation of cytochrome oxidase. We have isolated eight mit- mutants in this region and have ordered them using petite deletion mapping. Four of these mutants appear to map outside the boxII region on the oli2-proximal end. Analysis of restriction endonuclease fragments of the mtDNA from peptides used in the deletion mapping suggests a minimum size of 3.1 x 10(3) base pairs for the whole cob region. Although none of our mutants contained any cytochrome b or cytochrome b-linked activities, polypeptides apparently related to apocytochrome b were present in some but not all mutants. Additional regulatory effects (both positive and negative) on cytochrome oxidase by virtue of control of its subunit I were also observed. In addition to these phenotypic traits, some of the mutants accumulated novel, mitochondrially translated polypeptides not seen in wild type.     

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.     

New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction

Summary A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii. The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants. Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type. Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C. reinhardtii. We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin. The mutants all have point mutations in the mitochondrial gene for apocytochrome b. Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested. In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche. The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic. The genetic approach presented here confirms C. reinhardtii as a model system that is unique among plant cells.     

Decreased amounts of core proteins I and II and the iron-sulfur protein in mitochondria from yeast lacking cytochrome b but containing cytochrome c1

The effect of cytochrome b on the assembly of the subunits of complex III into the inner mitochondrial membrane has been studied in four mutants of yeast that lack a spectrally detectable cytochrome b and do not synthesize apocytochrome b. Quantitative analysis of intact mitochondria by immunoprecipitation or immunoblotting techniques with specific antisera revealed that the core proteins and the iron-sulfur protein were decreased 50% or more in the mitochondria from the mutants as compared to the wild type. Sonication of wild-type mitochondria did not result in any decrease in any of these proteins from the membrane; however, sonication of mitochondria from the four mutants resulted in a further decrease in the amount of these proteins suggesting that they are not as tightly bound to the mitochondrial membrane in the absence of cytochrome b. By contrast, the amounts of cytochrome c1 in the mitochondria, as determined both spectroscopically and immunologically, were not significantly affected by the absence of cytochrome b. In addition, no loss of cytochrome c1 was observed after sonication of the mitochondria suggesting that this protein is tightly bound to the membrane. These results suggest that the processing and/or assembly of these subunits of complex III into the mitochondrial membrane is affected by the absence of cytochrome b.     

The Fate of Mitochondrial Loci in Rho minus Mutants Induced by Ultraviolet Irradiation of SACCHAROMYCES CEREVISIAE: Effects of Different Post-Irradiation Treatments

Three main features regarding the loss of mitochondrial genetic markers among rho^- mutants induced by ultraviolet irradiation are reported: (a) the frequency of loss of six loci examined increases with UV dose; (b) preferential loss of one region of the mitochondrial genome observed in spontaneous rho^- mutants is enhanced by UV; and (c) the loss of each marker results from large deletions. Marker loss in rho^- mutants was also investigated under conditions that modulate rho^- induction. Liquid holding of irradiated exponential or stationary phase cells, as well as a split-dose regime applied to stationary phase cells, results in rho^- mutants in which the loss of markers is correlated with rho^- induction: the more sensitive the cells are to rho^- induction, the more frequent are the marker losses among rho^- clones derived from these cells. This correlation is not found in exponential-phase cells submitted to a split-dose treatment, suggesting that a different mechanism is involved in the latter case. It is known that UV-induced pyrimidine dimers are not excised in a controlled manner in mitochondrial DNA. However, our studies indicate that an accurate repair mechanism (of the recombinational type?) can lead to the restoration of mitochondrial genetic information in growing cells.