Biogenesis of mitochondria

Summary The action of ethidium bromide and berenil on the mitochondrial genome of Saccharomyces cerevisiae has been compared in three types of study: (i) early kinetics (up to 4 h) of petite induction by the drugs in the presence or absence of sodium dodecyl sulphate; (ii) genetic consequences of long-term (8 cell generations) exposure to the drugs; (iii) inhibition of mitochondrial DNA replication, both in whole cells and in isolated mitochondria.The results have been interpreted as follows. Firstly, the early events in petite induction differ markedly for the two drugs, as indicated by differences in the short-term kinetics. After some stage a common pathway is apparently followed because the composition of the population of petite cells induced after long-term exposure are very similar for both ethidium bromide and berenil. Secondly, both drugs probably act at the same site to inhibit mitochondrial DNA replication, in view of the fact that a petite strain known to be resistant to ethidium bromide inhibition of mitochondrial DNA replication was found to have simultaneously acquired resistance to berenil. From consideration of the drug concentrations needed to inhibit mitochondrial DNA replication in vivo and in vitro it is suggested that in vivo permeability barriers impede the access of ethidium bromide to the site of inhibition of mitochondrial DNA replication, whilst access of berenil to this site is facilitated. The site at which the drugs act to inhibit mitochondrial DNA replication may be different from the site(s) involved in early petite induction. Binding of the drugs at the latter site(s) is considered to initiate a series of events leading to the fragmentation of yeast mitochondrial DNA and petite induction.     

Biogenesis of mitochondria 36

Summary The isolation and characterisation of a mutant affecting the assembly of mitochondrial ATPase is reported. The mutation confers resistance to oligomycin and venturicidin and sensitivity of growth on nonfermentable substrates to low temperature (19°). Genetic analysis indicates that the phenotype is due to a single mutation located on the mitochondrial DNA which is probably allelic with the independently isolated oligomycin resistance mutation [oli1-r].Growth of the mutant at the non-restrictive temperature (28°) yields mitochondria in which the ATPase appears more sensitive to oligomycin than that of the sensitive parental strain. However, when the enzyme is isolated free from the influence of the membrane strong resistance to oligomycin is evident. These data suggest that the component responsible for the oligomycin resistance of the ATPase is part of or subject to interaction with the mitochondrial inner membrane.Measurements of the ATPase content of mitochondria indicate that ATPase production is impaired during growth at 19° C. In addition, studies of the maximum inhibition of mitochondrial ATPase activity by high concentrations of oligomycin suggest a selective lesion in ATPase assembly at low temperature. The nett result is that during growth at 19° only about 10% of the normal level of ATPase is produced of which less than half is membrane integrated and thus capable of oxidative energy production.We propose that the mutation affects a mitochondrially synthesised membrane sector peptide of the ATPase which defines the interaction of F₁ ATPase with specific environments on the mitochondrial inner membrane.     

Biogenesis of mitochondria 48

Summary Commercial preparations of mikamycin have been shown to act as both inhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds.A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Complex III.The mitochondrial [mik 1-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mik 1 has therefore been renamed ana1 to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the ana1 determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the ana1 locus is part of a gene specifying a membrane component of Complex III.     

Biogenesis of mitochondria 51

Summary An examination of the effect of the aminoglycoside antibiotics paromomycin and neomycin on mitochondrial ribosome function in yeast has been made. Both antibiotics are potent inhibitors of protein synthesis in isolated mitochondria. With isolated mitochondrial ribosomes programmed with polyuridylic acid (poly U), the drugs are shown to inhibit polyphenylalanine synthesis at moderately high concentrations (above 100 g/ml). At lower concentrations (about 10 g/ml), paromomycin and neomycin cause a 2–3 fold stimulation in the extent of misreading of the UUU codons in poly U, over and above the significant level of misreading catalyzed by the ribosomes in the absence of drugs.Comparative studies have been made between a paromomycin sensitive strain D585-11C and a mutant strain 4810P carrying the parl-r mutation in mtDNA, which leads tohigh resistance to both paromomycin and neomycin in vivo. A high level of resistance to these antibiotics is observed in strain 4810P at the level of mitochondrial protein synthesis in vitro. Whilst the degree of resistance of isolated mitochondrial ribosomes from strain 4810P judged by the inhibition of polyphenylalanine synthesis by paromomycin and neomycin is not extensive, studies on misreading of the poly U message promoted by these drugs demonstrate convincingly the altered properties of mitochondrial ribosomes from the mutant strain 4810P. These ribosomes show resistance to the stimulation of misreading of the codon UUU brought about by paromomycin and neomycin in wild-type mitochondrial ribosomes. Although strain 4810P was originally isolated as being resistant to paromomycin, in all the in vitro amino acid incorporation systems tested here, the 4810P mitochondrial ribosomes show a higher degree of resistance to neomycin than to paromomycin.It is concluded that the parl-r mutation in strain 4810P affects a component of the mitochondrial ribosome, possibly by altering the 15S rRNA or a protein of the small ribosomal subunit. The further elucidation of the functions in the ribosomes that are modified by the parl-r mutation was hampered by the inability of current preparations of yeast mitochondrial ribosomes to translate efficiently natural messenger RNAs from the several sources tested.     

Biogenesis of mitochondria 44

Summary A comparative study of eight independently isolated mitochondrial oligomycin resistant mutants obtained from three laboratories show a variety of phenotypes based on cross resistance to venturicidin and sensitivity to low temperature. Analysis of recombination between pairs of markers indicate the existence of at least three genetic classes; class A, cross resistant to venturicidin and including the mutations O III, [oli1-r], [OLG1-R], [tso-r]; class B, mutations O _I, [oli17-r], [OLG2-R]; and class C, the mutation O _II. The recombination data is consistent with mutations of each class residing in three separate genes, although mutations of class A and B show very close linkage.Recombination in non-polar crosses has demonstrated that markers of all three classes are linked to the mik1 locus in the configuration (AB)-mik1-C. The mapping of this segment with respect to other markers of the mitochondrial genome and the order of classes A and B was established by analyses of co-retention frequencies of markers in primary petite isolates as well as by analysis of marker overlap of genetically and physically defined petite genomes. The unambiguous order ery1-A-B-mik1-C-par was obtained. DNA-DNA hybridization studies using mtDNA isolated from selected petites confirms this map and estimates the physical separation of markers. A reasonable correlation exists in this region of the genome between distances estimated physically by hybridization and genetically by frequency of recombination in non-polar crosses.It is postulated that the oligomycin-mikamycin linkage group represents a cluster of genes involved in determining a number of mitochondrial membrane proteins associated with the mitochondrial ATPase and respiratory complex III.This work was supported by the Australian Research Grants Committee, Project D65/15930     

Biogenesis of mitochondria 47

Summary This paper consolidates and refines the physical map of genetic loci previously established in our laboratory, by molecular analysis of seven genetically characterized new petites (deletion mutants of mtDNA). A modified DNA-DNA hybridization procedure employing filters simultaneously bound with mtDNA from two different petites has been used to measure the overlaps in mtDNA sequences between the different petite mutants.Thus, by analysis of three new petites carrying the antibiotic-resistance loci, ery1, cap1 and par1 on their mitochondrial genomes, it has now been possible to improve our estimation of the maximum distance between the cap1 and ery1 loci. The cap1, ery1 loci, and the 21S ribosomal RNA gene have now been mapped within 5 units in the same region (map position 0 to 5 units). Similarly, by analysis of four new petites carrying the O _II and/or par1 loci on their mtDNAs, the map position of the O _II locus is also more accurately determined within 2 units in a region (map position 34 to 36 units) between the par1 and ana1 loci. The positions of other loci including par1, the 15S ribosomal RNA gene, and some mit ^- loci are also discussed.We have thus extended our library of genetically and molecularly defined petite mutants, resulting in a set of petites having overlapping regions distributed throughout the entire wild-type mitochondrial genome, consistent with the idea that yeast mtDNA is physically circular.     

Biogenesis of mitochondria 34

Summary Mutants of the yeast Saccharomyces cerevisiae have been isolated in this laboratory which show increased resistance to a number of structurally and functionally unrelated antibiotics such as mikamycin, chloramphenicol, oligomycin and tetracycline (Bunn et al., 3971). When a multiply resistant haploid strain was crossed to an antibiotic sensitive strain, the resultant diploid progeny were completely resistant to chloramphenicol and oligomycin. However, the progeny showed different responses to mikamycin depending upon the concentration of antibiotic, all showed resistance to 25 g/ml but only about half were resistant to high levels of mikamycin (>100 g/ml). Detailed genetic analyses has shown that resistance to high levels of mikamycin is the result of a phenotypic interaction between two mutations, one nuclear and the other mitochondrial. The nuclear mutation by itself confers resistance to a number of antibiotics including chloramphenicol, oligomycin and mikamycin at a level of 25 g/ml. The mitochondrial mutation increases cellular resistance to mikamycin from 3 g/ml to about 8 g/ml. When the two mutations occur together in a cell, resistance to mikamycin is increased to at least 800 g/ml, the limit of solubility. Thus, the phenotypie interaction between these two mutations is not additive but synergistic.When cells containing the cytoplasmic [mik1-r] mutation are treated with ethidium bromide to produce ^° cells (no mtDNA), the [mik1-r] determinant is lost, indicating that this mutation is located in the mitochondrial DNA. Recombination analyses with other mitochondrial markers indicates a marker order of [oli1-r mik1-r ery1-r] with [mik1-r] showing tighter linkage to the [oli1-r] marker.     

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.     

Biogenesis of mitochondria. 52

Summary The characteristics of recombination of several petite (rho ^-) mutants of S. cerevisiae that retain the -influenced region of the mitochondrial genome, identified by the markers cap1-r, ery1-r and tsr1, are described. The petites were derived from an ^– grande (rho ⁺) strain and those petites which retain all three markers show recombination properties similar to those of the ^- parental strain. However, other rho ^- mutants that retain the cap1 and ery1 loci but have lost the tsr1 locus, which is located between cap1 and ery1, show markedly different properties of mitochondrial transmission and recombination, consistent with the presence of ⁺ alleles. The association of an internal deletion between the cap1 and ery1 loci with a change in phenotype provides additional evidence for the location of between these two loci.Although the petites deleted for the tsr1 locus exhibited the recombination properties of ⁺ strains, it was not possible to transmit this characteristic to rho ⁺ recombinant cells. Experiments on the kinetics of elimination by ethidium bromide of the cap1 and eryl markers from the petites and measurements of the buoyant densities of their mtDNA species did not indicate major changes (such as selective sequence repetition) in the sequences of the mtDNAs. The possible nature of the changes in the mtDNAs of these petites is discussed in light of recent studies on the physical nature of the alleles.