A nuclear mutation defective in mitochondrial recombination in yeast.

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.     

Respiratory-competent yeast mitochondrial DNAs generated by deleting intergenic regions

Two respiratory-competent yeast strains having mitochondrial (mt) DNA characterized by single non-overlapping deletions, encompassing intergenic sequences, have been crossed. Diploid daughter clones have been screened by electrophoresis of mtDNA fragments, and a respiratory-competent clone (ER8.75), having a recombinant small mtDNA with both parental deletions, has been detected. ER8.75 mtDNA lacks around 20% of wild-type intergenic sequences, encompassing three ori/rep sequences. This mutant could be helpful in analyzing the organelle genome and, particularly, the function of intergenic sequences.     

Nuclear origin of specific yeast mitochondrial aminoacyl-tRNA synthetases.

Hydroxylapatite chromatographies of mitochondrial and total enzymes from a rho+ yeast, or from the related rho degrees mitochondrial DNA-less mutant, show the occurrence in the mitochondrial enzyme of one Phe-, one Met-, one Leu-tRNA synthetase peak which elutes distinctly from the cytoplasmic counterpart and charges well mitochondrial tRNA, whereas the cytoplasmic enzyme does not. The measurement of the mitochondrial synthetases activities in various enzymatic extracts shows that they are not repressed in rho+ cells grown on 10% glucose and that they are concentrated in the mitochondria (Phe- and Met- tRNA synthetases) but are also present outside the mitochondria. It is concluded that yeast mitochondrial protein biosynthesis involves the nuclear coded mitochondrial specific Phe-, Met- and Leu-tRNA synthetases and that the entrance of the synthetases into the mitochondria needs no factor depending on the mitochondrial DNA.     

A nuclear mutation conferring aurovertin resistance to yeast mitochondrial adenosine triphosphatase.

A single gene nuclear yeast mutant was isolated whose mitochondrial F1-ATPase was resistant to the specific F1 inhibitor aurovertin. The mutant enzyme was not cross-resistant to other F1 inhibitors. The binding of aurovertin to F1 and to the two largest F1 subunits (alpha and beta) was measured by enhancement of aurovertin fluorescence. Aurovertin bound to wild type F1-ATPase and to its monomeric beta subunit with about the same binding constant. It failed to bind to wild type alpha subunit or to either F1 or F1 subunits from the mutant. The aurovertin-resistant mutant thus contains an altered nuclear gene which specifies the structure of the beta subunit of F1.     

Two related genes encoding extremely hydrophobic proteins suppress a lethal mutation in the yeast mitochondrial processing enhancing protein.

The processing enhancing protein of mitochondria (PEP) is an essential component that has been shown to participate in proteolytic removal of NH2-terminal signal peptides from precursor proteins imported into the mitochondrial matrix. Using a yeast strain bearing a PEP mutation that renders it temperature-sensitive, an approach of genetic suppression was taken in order to identify additional components that could be involved with protein import: high copy plasmids comprising a yeast genomic library were tested for ability to suppress the 37 degrees C growth defect. Two plasmids were isolated, pSMF1 and pSMF2, which suppressed the growth defect nearly as well as the cloned PEP gene itself. Sequence analysis of the rescuing genes predicted extremely hydrophobic proteins with sizes of 63 and 60 kDa, respectively. Remarkably, the predicted SMF1 and SMF2 products are 49% identical to each other overall. To test the requirement for SMF1 and SMF2, the chromosomal genes were disrupted. Individual disruption was without effect, but cells in which both genes were disrupted grew poorly. When mitochondria were prepared from the double disruption strain grown in a nonfermentable carbon source, they were morphologically normal but defective for translocation of radiolabeled precursor proteins. SMF1 protein was provisionally localized to the mitochondrial membranes using epitope tagging. We suggest that SMF1 and SMF2 are mitochondrial membrane proteins that influence PEP-dependent protein import, possibly at the step of protein translocation.     

Transmission of the yeast mitochondrial genome to progeny: The impact of intergenic sequences

Summary In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs. In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked, loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin. These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains. Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome. In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker. These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole. More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences. These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome. However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts. Therefore, in a more general sense, variations in the amount and structure of intergenic sequences in various yeasts may reflect processes that have been of selective advantage in the metabolism of individual mitochondrial DNA in a particular environment and that have not drastically interrupted the respiratory phenotype.     

A specific point mutation in the mitochondrial genome of Caucasians with MELAS

Summary The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS) exhibits a specific heteroplasmic AG transition in the tRNA^Leu at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.     

A cold-sensitive cytoplasmic mutation of Saccharomyces cerevisiae affecting assembly of the mitochondrial 50S ribosomal subunit.

Summary A cytoplasmic mutant of Saccharomyces cerevisiae (E23-1) has been isolated that is resistant to erythromycin and cold sensitive for growth on nonfermentable carbon sources at 18°. Genetic analysis has shown that both of these properties probably result from a single mutation at the rib2 locus which maps close to or within the gene for the 21S rRNA of the mitochondrial 50S ribosomal subunit. Electrophoresis of total RNA extracted from purified mitochondria demonstrated that the 21S and 14S rRNA species from both mutant and wild-type cells were present in roughly equimolar quantities regardless of growth temperature. The mutant is therefore not defective in the synthesis of the 21S rRNA. Sucrose gradient analysis of the mitochondrial ribosomes in Mg²⁺-containing buffers revealed that approximate values for the ratio of 50S to 37S subunits were 1:1 for wild-type cells grown at either 18° or 32°, 0.5:1 for the mutant grown at 32° and 0.2:1 for the mutant grown at 18°. The subunit ratios were approximately 1:1 when Ca²⁺-containing buffers were used, however, In alls cases, 50S particles from the mutant grown at 18° lacked or contained markedly reduced amounts of two distinctive protein components that were present in the mutant at 32° and in the wild-type at both temperatures. In addition, no intact 21S RNA could be recovered from the mitochondrial ribosomes of the mutant grown at the restrictive temperature, even in the presence of Ca²⁺. These findings indicate that mitochondrial 50S ribosomal subunits produced by the mutant at 18° are structurally defective and raise the possibility that the defect results from an alteration in the gene for 21S rRNA.A preliminary report of this work was presented at the meeting on The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, August 18–22, 1977     

A mutation altering the kinetic responses of the yeast mitochondrial F1-ATPase

Nucleotide-binding proteins, including the mitochondrial F1-ATPase, the ras proteins, and the G-proteins, contain a homologous glycine-rich sequence that is thought to constitute part of the active site. This study reports the effects of a single amino acid replacement of Thr197 to Ser197, which is located at the hinge region of this putative loop, in the yeast Saccharomyces cerevisiae F1-ATPase. This replacement resulted in a 3-fold increase in the specific activity of the enzyme, eliminated the stimulatory effects of oxyanions, and modulated the effects of the inhibitor NaN3 while having little effect on the uni-site ATPase. These results indicate a role of the glycine-rich loop in many of the kinetic responses of the F1-ATPase.     

Pleiotropic mutations within two yeast mitochondrial cytochrome genes block mRNA processing.

The mRNAs from two yeast mitochondrial genes cob-box (cytochrome b) and oxi-3 (cytochrome oxidase 40,000 dalton subunit) are processed from large (7-10 kb) precursors. Certain mutations in each gene block the maturation of the RNAs from both genes at a variety of specific steps. The pleiotropic cytochrome b mutants seem to lack a functional trans-acting RNA required for the processing of both messengers. In contrast, the oxi-3 mutants may act by producing an activity that inhibits specific steps.     

Ziram, a sulfhydryl reagent and specific inhibitor of yeast mitochondrial dehydrogenases.

The fungicide zinc dimethyldithiocarbamate (ziram) is a sulfhydryl reagent which inhibits specifically the growth of the yeast Saccharomyces cerevisiae on nonfermentable substrates. In isolated mitochondria, the uncoupled as well as the state 3 oxidations of succinate, α-ketoglutarate, ethanol, and malate plus pyruvate are sensitive to ziram concentrations of 10 to 30 μm. The oxidations of isocitrate, of external NADH, of α-glycerophosphate, and of ascorbate plus tetramethylphenylenediamine exhibit a lower sensitivity to ziram. Succinate, α-ketoglutarate, and pyruvate dehydrogenases activities are 50% inhibited by concentration of ziram lower than 10 μm. At the same concentrations, neither the mitochondrial transports of succinate, ADP, or phosphate nor oxidative phosphorylation and adenosine triphosphatase activities are modified. The kinetic study of the inhibition by ziram of succinate dehydrogenase activity shows that ziram is noncompetitive with succinate and produces sigmoidal inhibitions of state 3 and of uncoupled oxidation of succinate by intact mitochondria. Inhibition of succinate:phenazine methosulfate oxidoreductase activity yields exponential kinetics. However sigmoidal-type inhibition is observed when succinate dehydrogenase activity is stimulated by ATP.     

Crucial mitochondrial impairment upon CDC48 mutation in apoptotic yeast

Mutation in CDC48 (cdc48(S565G)), a gene essential in the endo-plasmic reticulum (ER)-associated protein degradation (ERAD) pathway, led to the discovery of apoptosis as a mechanism of cell death in the unicellular organism Saccharomyces cerevisiae. Elucidating Cdc48p-mediated apoptosis in yeast is of particular interest, because Cdc48p is the highly conserved yeast orthologue of human valosin-containing protein (VCP), a pathological effector for polyglutamine disorders and myopathies. Here we show distinct proteomic alterations in mitochondria in the cdc48(S565G) yeast strain. These observed molecular alterations can be related to functional impairment of these organelles as suggested by respiratory deficiency of cdc48(S565G) cells. Mitochondrial dysfunction in the cdc48(S565G) strain is accompanied by structural damage of mitochondria indicated by the accumulation of cytochrome c in the cytosol and mitochondrial enlargement. We demonstrate accumulation of reactive oxygen species produced predominantly by the cytochrome bc1 complex of the mitochondrial respiratory chain as suggested by the use of inhibitors of this complex. Concomitantly, emergence of caspase-like enzymatic activity occurs suggesting a role for caspases in the cell death process. These data strongly point for the first time to a mitochondrial involvement in Cdc48p/VCP-dependent apoptosis.