Ruth M. Hall

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance.

A series of isonuclear oligomycin‐resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial olil gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross‐resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.

Factors affecting petite induction and the recovery of respiratory competence in yeast cells exposed to ethidium bromide.

SummaryWhen growing cultures of S. cerevisiae are treated with high concentrations of ethidium bromide (>50 μg/ml), three phases of petite induction may be observed: I. the majority of cells are rapidly converted to petite, II. subsequently a large proportion of cells recover the ability to form respiratory competent clones, and III. slow, irreversible conversion of all cells to petite. The extent of recovery of respiratory competence observed is dependent on the strain of S. cerevisiae employed and the temperature and the carbon source used in the growth medium. The effects of 100 μg/ml ethidium bromide are also produced by 10 μg/ml ethidium bromide in the presence of the detergent, sodium dodecyl sulphate, and recovery is also observed when cells are treated with 10 μg/ml ethidium bromide under starvation conditions. Genetic analysis of strain differences indicates that a number of nuclear genes influence petite induction by ethidium bromide.In one strain, S288C, petite induction by 100 μg/ml ethidium bromide is extremely slow under certain conditions. Mitochondria isolated from S288C lack the ethidium bromide stimulated nuclease activity found in D243-4A, a strain which shows triphasic kinetics of petite formation. This enzyme may, therefore, be responsible for the initial phase of rapid petite formation.

BIOGENESIS OF MITOCHONDRIA

SummaryThe 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 49 identification and mapping of a new mitochondrial locus (tsr 1) which maps within the polar region of the yeast mitochondrial genome

SummaryAn enrichment procedure which facilitates the isolation of conditional respiratory-deficient mutants of Saccharomyces cerevisiae is reported. Detailed genetic analysis of one mutant which exhibits a respiratory deficient phenotype at low temperature (18°C) is also presented. The phenotype is due to a single lesion at a new locus, tsr1, located on the mitochondrial DNA. By analysis of locus retention patterns in a set of physically characterized petite strains, the tsr1 mutation has been mapped within the segment 0–5 map units on the physical map of the yeast mitochondrial genome. This segment of the mitochondrial DNA also contains the cap1 and ery1 loci and the cistron for the mitochondrial 21S rRNA. Studies of the frequencies of co-retention of markers in petite populations, and of the frequencies of recombination of markers in non-polar crosses (ω+ × ω+), demonstrate linkage of the tsr1 locus to both the cap1 and ery1 loci. The degree of linkage indicates that tsr1 is closer to the ery1 locus. Comparison of pairwise recombination frequencies for these three markers indicate the order cap1-tsr1-ery1. The tsr1 locus lies within the segment of the mitochondrial genome which is influenced by the polarity locus ω, and analysis of transmission and recombination frequencies and polarities in a polar (ω+ × ω-) cross show that the behaviour of the tsr1 locus is similar to that of ery1. However striking features of this cross are that the recombination frequency between tsr1 and ery1 is comparable to that observed in non-polar crosses, and that the polarity for recombination between tsr1 and cap1 or ery1 is extremely low.

Evidence for a functional association of DNA synthesis with the membrane in mitochondria of Saccharomyces cerevisiae

We have studied the effect of membrane fatty acid composition on replicative DNA synthetic activity in mitochondria isolated from Saccharomyces cerevisiae. Cells containing different levels of membrane unsaturated fatty acids were obtained by growth of a fatty acid desaturase mutant of Saccharomyces cerevisiae in glucose-limited chemostat cultures supplemented with various concentrations of Tween 80. Arrhenius plots of DNA synthetic activity in isolated mitochondria show a discrete discontinuity at specific temperature which are dependent on the membrane unsaturated fatty acid content of the mitochondria. This indicates a functional association of DNA replication with the mitochondrial membrane in Saccharomyces cerevisiae.

Induction of respiratory incompetent mutants by unsaturated fatty acid depletion in Saccharomyes cerevisiae

Abstract Constant levels of cellular unsaturated fatty acids were obtained by growing a fatty acid desaturase mutant of Saccharomyces cerevisiae in glucose limited chemostat cultures supplemented with various concentrations of Tween 80. An increase in the frequency of cytoplasmic respiratory incompetent mutants was observed in cultures growing at low cellular levels of unsaturated fatty acids. This effect has been shown to result from an increase in the rate of mutation as the cellular unsaturated fatty acid level is decreased. The majority of induced petite mutants are ϱ° (contain no mitochondrial DNA).

The action of structural analogues of ethidium bromide on the mitochondrial genome of yeast

We have studied the effects on the yeast mitochondrial genome of four analogues of ethidium bromide, in which the phenyl moiety has been replaced by linear alkyl chains of lengths varying from seven to fifteen carbon atoms. These analogues are more efficient than ethidium bromide in inducing petite mutants inSaccharomyces cerevisiae. The drugs also cause a loss of mtDNA from the cellsin vivo; however these analogues are in fact less effective inhibitors of mitochondrial DNA replicationper se, as shown by directin vitro studies. It is concluded that these analogues are more efficient than ethidium bromide in causing the fragmentation of mitochondrial DNA inS. cerevisiae.

Biogenesis of mitochondria. 52

SummaryThe characteristics of recombination of several petite (rho-) mutants of S. cerevisiae that retain the ω-influenced region of the mitochondrial genome, identified by the markers cap1-r, ery1-r and tsr1, are described. The petites were derived from an ω− grande (rho+) strain and those petites which retain all three markers show recombination properties similar to those of the ω- 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 ω+ alleles. The association of an internal deletion between the cap1 and ery1 loci with a change in ω phenotype provides additional evidence for the location of ω between these two loci.Although the petites deleted for the tsr1 locus exhibited the recombination properties of ω+ 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 ω alleles.

Mitochondrial Biogenesis: The Identification of Mitochondrial Genes and Gene Products

Elucidation of the biogenesis of subcellular organelles may be considered as one of the significant challenges in modern cell biology. Brief reflection on the problem of the biogenesis of mitochondria illustrates this thesis. The mitochondrion is a complex organelle with inner and outer membrane systems of still largely unknown composition; these membranes enclose a matrix space which is comprised of some hundreds of enzymes and small molecules of great functional and molecular diversity.