Mitochondrial Genome Integrity Research Articles

Mitochondrial Genome Integrity Mutations Uncouple the Yeast Saccharomyces cerevisiae ATP Synthase

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the alpha-, beta-, and gamma-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the gamma-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk.

Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA

Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Δ cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Δ background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

Stability and Association with the Cytomatrix of Mitochondrial DNA in Spontaneously Immortalized Mouse Embryo Fibroblasts Containing or Lacking the Intermediate Filament Protein Vimentin

To extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim(+) and vim(-) fibroblasts derived from wild-type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim(+/+) and Vim(-/-) fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim(+) cells was lower by a factor of 0.6 than that in four clones of vim(-) cells. However, during serial subcultivation up to high passage numbers, the vim(+) and vim() fibroblasts increased their mtDNA copy number 1.5- and 2.5-fold, respectively. While early-passage cells of the vim(+) and vim(-) fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim(+) and vim() cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high-passage vim(-) fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (gamma) detected in vim(+) fibroblasts by Triton X-100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim(+) and vim(-) fibroblasts were equally active in generating free radicals, the vim(-) cells exhibited higher levels of immunologically detectable 8-oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim(-) fibroblasts only one point mutation was detected in the mtDNA D-loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large-scale mtDNA deletions were found in high-passage vim(-) fibroblasts, but not in low-passage vim(-) cells and vim(+) cells of both low and high passage. Large mtDNA deletions were also induced in young vim(-) fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim(+) cells. These results indicate that in immortalized vim(-) fibroblasts the mitochondrial genome is prone to large-scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin.

Oxidative DNA Damage Causes Mitochondrial Genomic Instability in Saccharomyces cerevisiae

Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

Protein release in a Saccharomyces cerevisiae mutant does not depend on mitochondrial genome integrity and function

A mutant allele the of Saccharomyces cerevisiae NUD1 gene causes a thermosensitive phenotype of the yeast cells and increases release of proteins into the culture medium at the restrictive temperature (37 degrees C). The release of proteins does not depend on the process of oxidative phosphorylation, because protein release is not stopped in the presence of inhibitors of oxidative phosphorylation. NUD1 transcription is not under retrograde regulation. This type of transcriptional regulation depends on the nuclear Rtg transcription factors and on the integrity of the mitochondrial genome.

Intramolecular recombination and deletions in mitochondrial DNA of senescent, a nuclear-gene mutant of Neurospora crassa exhibiting “death” phenotype

In Neurospora crassa, a nuclear-gene mutant, senescent, derived from a phenotypically normal wild isolate of Neurospora intermedia exhibits a ‘death’ phenotype. Regardless of the composition of the culture medium, the mycelium ceases to grow in 2–6 subcultures at 26 °C and 1 or 2 subcultures at 34 °C. Senescence of vegetative mycelium is associated with deficiencies in cytochromes aa 3 and b and reduced oxygen uptake. The restriction fragment analysis of mitochondrial DNA from senescing mycelia showed deletions and gross sequence rearrangements. Analysis of mitochondrial DNA of ( sen + sen +) heterokaryons constructed with “excess” sen cytoplasm suggested correlation between mtDNA deletions and senescence. Three novel sen-specific EcoRI fragments of sizes 3.6, 3.9, and 4.4 kb were cloned, sequenced, and analyzed. Nucleotide sequences of the sen-specific EcoRI fragments suggested that deletions were a consequence of intramolecular recombination between EcoRI-5 and -10 and/or between EcoRI-8 and -10. The recombination junctions were close to stretches of GC-rich- PstI palindromic sequences that potentially form stable hairpin structures and might facilitate recombination between homologous repeats as short as 6–10 bp. These observations suggest that the wild-type ( sen +) allele encodes a factor that protects the mitochondrial genome from undergoing intramolecular recombination and deletions. In this respect sen + (linkage group V) has a function similar to nd + (linkage group I) and the two gene products probably have mutually exclusive roles in suppressing cruciform-associated and homologous recombination, respectively, thus safeguarding mitochondrial genome integrity. The sen + allele most likely codes for a factor involved in recombination, repair or replication of the mitochondrial genome, or a transcription factor that regulates the expression of genes affiliated with mitochondrial DNA metabolism.

Downregulation of the human Lon protease impairs mitochondrial structure and function and causes cell death

Lon now emerges as a major regulator of multiple mitochondrial functions in human beings. Lon catalyzes the degradation of oxidatively modified matrix proteins, chaperones the assembly of inner membrane complexes, and participates in the regulation of mitochondrial gene expression and genome integrity. An early result of Lon downregulation in WI-38 VA-13 human lung fibroblasts is massive caspase 3 activation and extensive (although not universal) apoptotic death. At a later stage, the surviving cells fail to divide, display highly abnormal mitochondrial function and morphology, and rely almost exclusively on anaerobic metabolism. In a selected subpopulation of cells, the mitochondrial mass decreases probably as a result of mitochondrial inability to divide. At this final point the Lon-deficient cells are not engaged anymore in apoptosis, and are lost by necrosis or “mitoptosis.” Our results indicate that mitochondrial Lon is required for normal survival and proliferation; a clear impetus for Lon's evolutionary conservation.

Epidermal Growth Factor Up-regulates the Transcription of Mouse Lon Homology ATP-Dependent Protease Through Extracellular Signal-Regulated Protein Kinase- and Phosphatidylinositol-3-Kinase-dependent Pathways

Epidermal growth factor (EGF) induces tumorigenic transformation of mouse epidermal cells (JB6 P +). We cloned a full-length EGF-responsive cDNA in JB6P + cells; EGF up-regulated mRNA expression of this gene 5- to 6-fold. The deduced amino acid sequence of this cDNA exhibited 84 and 96% homology with human and rat Lon homology ATP-dependent protease, respectively, and all conserved domains of Lon, such as ATPase/protease domains, are present in the mouse gene, indicating that this gene is mouse Lon. EGF increased the transcriptional rate without affecting the mRNA stability of m-Lon. The level of m-Lon in irreversibly transformed mouse epidermal cells (JB7) was 3.4-fold higher than that in parental JB6 P + cells. Similarly, human mammary epithelial cells overexpressing the proto-oncogene ErbB2 expressed significantly higher levels of Lon than normal mammary epithelial cells. EGF failed to regulate Lon expression in ERK-deficient JB6 P − cells or cells that expressed the dominant-negative p85 P13-K regulatory unit. Furthermore, selective chemical blockers for MEK1 and P13-K (PD98059 and LY294002) inhibited EGF-mediated induction. Mitochondria-localized Lon protease plays a critical role in the regulation of mitochondrial gene expression and genome integrity. Disruption of mitochondrial homeostasis is a general characteristic of tumorigenic transformation. Thus, the role of Lon in tumor promotion warrants further study.

The nature of enzymes involved in uracil-DNA repair: isoform characteristics of proteins responsible for nuclear and mitochondrial genomic integrity.

The absence of uracil from DNA genomes is a consequence of enzyme functions that eliminate intracellular dUTP pools and that purposefully recognize and remove uracil moieties from DNA. These enzymatic functions are dUTP nucleotidohydrolase (dUTPase) and uracil-DNA glycosylase (UDG), respectively. There are distinct nuclear and mitochondrial isoforms of each of these enzymes in human cells. The mitochondrial isoform of dUTPase (DUT-M) begins as a 31 kilodalton precursor protein containing an arginine-rich, amino-terminal presequence required for targeting to the mitochondria. This precursor is processed into a 23 kilodalton protein that resides, in mature form, in the mitochondria. The nuclear isoform of dUTPase (DUT-N) is an 18 kilodalton protein. Both species of dUTPase are nearly identical except for their amino-termini. Analysis of protein expression reveals that DUT-M is constitutive and independent of cell cycle phase or proliferation status of the cell. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication. The common sequence for both nuclear and mitochondrial isoforms includes a cyclin-dependent kinase consensus site. However, only the nuclear form appears to be phosphorylated at this site in vivo. Studies on dUTPase genomic organization reveal that both isoforms are encoded by the same gene. Isoform specific transcripts arise through the use of alternate 5' exons. Uracil-DNA glycosylase (UDG1) is but one of a growing family of enzymes that repairs potentially mutagenic events caused by uracil in DNA. Human cells contain two isoforms of UDG1 which are also nearly identical except for their amino termini. One isoform (UDG1-M), which is constitutively expressed, is targeted to the mitochondria. This form originates as a 35,000 dalton precursor and is N-terminally processed to a mature 29,000 dalton protein as it transits into the mitochondria. The other isoform is targeted to the nucleus and its expression is a function of cellular proliferation status. As with dUTPase, UDG1 isoform specific transcripts arise through the use of alternate 5prie; exons. Both of these enzymatic functions are a unique illustration, in humans, of the use of alternate exons to generate differentially expressed proteins targeted to different organelles. There are questions as to whether the nuclear isoform of UDG (UDG1-N) is also processed (at the N-terminus) to a lower molecular weight form. Polyclonal antisera generated to the unique N-terminal region of this isoform, reveals that UDG1-N exists as a 36,000 dalton protein in human cell nuclei. Since the epitope for this antibody resides in the first 24 amino acids of UDG1-N, it is apparent that the majority of this isoform is not processed and retains its amino terminus. Evidence also indicates that UDG1-N exists as a serine/threonine phosphoprotein and that phosphorylation occurs in the unique N-terminal region. This was initially deduced from the observation that nuclear UDG1-N migrates as multiple bands on SDS-PAGE and as a single band subsequent to phosphatase treatment. Cdc2 kinase is at least one of the enzymes that can phosphorylate UDG1-N. This review will summarize the current information on isoform characteristics of both dUTPase and uracil-DNA glycosylase. It will also focus on evidence for phosphorylation and speculate as to the purpose of these post-translational events.

Protein overexport in a Saccharomyces cerevisiae mutant depends on mitochondrial genome integrity and function

The wild-type yeast Saccharomyces cerevisiae (S. cerevisiae) is able to export less than 1 percent of the protein to be secreted. The reasons for retention of most of the secretory proteins on the cell surface of S. cerevisiae are unknown. Recently, temperature-sensitive (ts) mutants of S. cerevisiae showing an oversecretion phenotype were isolated. In order to study the influence of the mitochondrial genome status on protein export in yeast cells, we have isolated several types of respiratory impaired mitochondrial mutants of either the parental S. cerevisiae strain or their derivative ts protein-overexporting mutants. In this paper we demonstrate by quantitative analyses of exported proteins and by SDS-PAGE analysis that protein overexport in ts mutants requires mitochondrial genome integrity and function.

Germline passage of mitochondria: quantitative considerations and possible embryological sequelae.

Using a semi-quantitative review of published electron micrographs, we have explored the passage of mitochondria from one generation to the next through the cytoplasm of the human female germ cell. We propose a testable hypothesis that the mitochondria of the germline are persistently 'haploid' (effectively carrying just one mitochondrial chromosome per organelle). For mitochondria, the passage through germ cell differentiation, oogenesis, follicle formation and loss could constitute a restriction/amplification/constraint event of a type previously demonstrated for asexual purification and refinement of a nonrecombining genome. At the restriction event (or 'bottleneck') in the human primordial germ cell, which differentiates in embryos after gastrulation, there appear to be <10 mitochondria per cell. From approximately 100 or so such cells, a population of > or =7 x 10(6) oogonia and primary oocytes is produced in the fetal ovaries during mid-gestation, with mitochondria numbering up to 10000 per cell, implying a massive amplification of the mitochondrial genome. A further 10-fold or greater increase in mitochondrial numbers per oocyte occurs during adult follicular growth and development, as resting primordial follicles develop to preovulatory maturity. So few are the numbers of oocytes that fertilize and successfully cleave to form an embryo of the new generation, that biologists have long suspected that a competitive constraint lies behind the generational completion of this genetic cycle. I propose that maintaining the integrity of mitochondrial inheritance is such a strong evolutionary imperative that features of ovarian follicular formation, function, and loss could be expected to have been primarily adapted to this special purpose. To extend the hypothesis further, the imperative of maintaining mitochondrial genomic integrity in a population could explain why women normally become sterile a number of years before there is depletion of ovarian follicles and endocrine ovarian failure (i.e. why there is 'an oopause' preceding the menopause). Plausible explanations might also follow for several well-known and puzzling reproductive difficulties, including recurrent miscarriage, unexplained infertility, and persistent failure of IVF embryos to cleave or to implant. Current experimental laboratory manoeuvres that might circumvent mitochondrial shortcomings (such as cytoplasmic transfusion and karyoplast exchange) are examined and possible clinical hazards identified.

Protein degradation in mitochondria

The biogenesis of mitochondria and the maintenance of mitochondrial functions depends on an autonomous proteolytic system in the organelle which is highly conserved throughout evolution. Components of this system include processing peptidases and ATP-dependent proteases, as well as molecular chaperone proteins and protein complexes with apparently regulatory functions. While processing peptidases mediate maturation of nuclear-encoded mitochondrial preproteins, quality control within various subcompartments of mitochondria is ensured by ATP-dependent proteases which selectively remove non-assembled or misfolded polypeptides. Moreover, these proteases appear to control the activity- or steady-state levels of specific regulatory proteins and thereby ensure mitochondrial genome integrity, gene expression and protein assembly.

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae.

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.

Hypoxia‐induced mitochondrial and nuclear DNA damage in the rat brain

In humans, cerebral hypoxia is a common component of severe brain insults, including trauma, stroke, and perinatal asphyxia. Oxidative stress and free radicals incidental to cerebral hypoxia are implicated in damaging macromolecules, leading to collapse of cellular homeostasis and cell death. Neuronal DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Here, we measured hypoxia-induced damage and repair in nuclear and mitochondrial DNA in rat hippocampus and cortex. Two highly sensitive quantitative polymerase chain reaction (QPCR) assays were used to measure DNA damage. One assay measures the integrity of the entire mitochondrial genome and the other the integrity of nuclear DNA. The latter is a novel assay, developed in our laboratory, which utilizes the high copy number of short interspersed DNA elements (SINEs) residing in introns and untranslated regions of mammalian genes. A unique feature of the SINE-mediated QPCR is its ability to amplify simultaneously long random segments of DNA. Consequently, the SINE assay offers sufficient sensitivity for detecting DNA damage at levels that are compatible with the cellular capacity for DNA repair, and are likely to be consistent with cellular survival and therefore adequate for studying the DNA damage response in the brain. In rats, we found that exposure to an atmosphere of 4% oxygen for 30 min resulted in induction of DNA damage in nuclear and to a greater extent, in mitochondrial DNA. Following a 3-hr recovery period in ambient air, dissimilar repair kinetics for nuclear and mitochondrial DNA were measured. J. Neurosci. Res. 58:262–269, 1999. © 1999 Wiley-Liss, Inc.

Hypoxia-induced mitochondrial and nuclear DNA damage in the rat brain

In humans, cerebral hypoxia is a common component of severe brain insults, including trauma, stroke, and perinatal asphyxia. Oxidative stress and free radicals incidental to cerebral hypoxia are implicated in damaging macromolecules, leading to collapse of cellular homeostasis and cell death. Neuronal DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Here, we measured hypoxia-induced damage and repair in nuclear and mitochondrial DNA in rat hippocampus and cortex. Two highly sensitive quantitative polymerase chain reaction (QPCR) assays were used to measure DNA damage. One assay measures the integrity of the entire mitochondrial genome and the other the integrity of nuclear DNA. The latter is a novel assay, developed in our laboratory, which utilizes the high copy number of short interspersed DNA elements (SINEs) residing in introns and untranslated regions of mammalian genes. A unique feature of the SINE-mediated QPCR is its ability to amplify simultaneously long random segments of DNA. Consequently, the SINE assay offers sufficient sensitivity for detecting DNA damage at levels that are compatible with the cellular capacity for DNA repair, and are likely to be consistent with cellular survival and therefore adequate for studying the DNA damage response in the brain. In rats, we found that exposure to an atmosphere of 4% oxygen for 30 min resulted in induction of DNA damage in nuclear and to a greater extent, in mitochondrial DNA. Following a 3-hr recovery period in ambient air, dissimilar repair kinetics for nuclear and mitochondrial DNA were measured.

Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer's disease.

To determine whether pathogenic mutations in mtDNA are involved in phenotypic expression of Alzheimer's disease (AD), the transfer of mtDNA from elderly patients with AD into mtDNA-less (rho0) HeLa cells was carried out by fusion of platelets or synaptosomal fractions of autopsied brain tissues with rho0 HeLa cells. The results showed that mtDNA in postmortem brain tissue survives for a long time without degradation and could be rescued in rho0 HeLa cells. Next, the cybrid clones repopulated with exogenously imported mtDNA from patients with AD were used for examination of respiratory enzyme activity and transfer of mtDNA with the pathogenic mutations that induce mitochondrial dysfunction. The presence of the mutated mtDNA was restricted to brain tissues and their cybrid clones that formed with synaptosomes as mtDNA donors, whereas no cybrid clones that isolated with platelets as mtDNA donors had detectable mutated mtDNA. However, biochemical analyses showed that all cybrid clones with mtDNA imported from platelets or brain tissues of patients with AD restored mitochondrial respiration activity to almost the same levels as those of cybrid clones with mtDNA from age-matched normal controls, suggesting functional integrity of mtDNA in both platelets and brain tissues of elderly patients with AD. These observations warrant the reassessment of the conventional concept that the accumulation of pathogenic mutations in mtDNA throughout the aging process is responsible for the decrease of mitochondrial respiration capacity with age and with the development of age-associated neurodegenerative diseases.

The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria.

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI and COB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.

Allele-specific expression of the Mgi- phenotype on disruption of the F1-ATPase delta-subunit gene in Kluyveromyces lactis.

Kluyveromyces lactis is a petite-negative yeast that does not form viable mitochondrial genome-deletion mutants (petites) when treated with DNA-targeting drugs. Loss of mtDNA is lethal for this yeast but mutations at three loci termed MGI, for mitochondrial genome integrity, can suppress this lethality. The three loci encode the alpha-, beta- and gamma-subunits of mitochondrial F1-ATPase. In this study we report the isolation and characterization of the KlATPdelta gene encoding the delta-subunit of F1-ATPase. The deduced protein contains 158 amino acids showing 72% identity to the protein from Saccharomyces cerevisiae and a putative mitochondrial targeting sequence of 23 amino acids. Disruption of the gene causes cells to become respiratory deficient while the introduction of ATPdelta from S. cerevisiae restores growth on glycerol. Cells with a disrupted ATPdelta gene, like strains with disruptions of alpha-, beta- and gamma-F1-subunits, do not produce petite mutants when treated with ethidium bromide. However, unlike strains with disruptions in the three largest F1-subunits, disruption of ATPdelta in the presence of some mgi alleles does not abolish the Mgi- phenotype. By contrast, elimination of ATPdelta in other mgi strains removes resistance to ethidium bromide and rho0 mutants are not formed. Hence the ATPdelta subunit of F1-ATPase, while not mandatory for a Mgi- phenotype, aids some mgi alleles in suppressing rho0 lethality.

Regulated protein degradation in mitochondria.

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of the Escherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, the m- and the i-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, the m-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.

The mitochondrial genome integrity gene, MGI1, of Kluyveromyces lactis encodes the beta-subunit of F1-ATPase.

In a previous report, we found that mutations at the mitochondrial genome integrity locus, MGI1, can convert Kluyveromyces lactis into a petite-positive yeast. In this report, we describe the isolation of the MGI1 gene and show that it encodes the beta-subunit of the mitochondrial F1-ATPase. The site of mutation in four independently isolated mgi1 alleles is at Arg435, which has changed to Gly in three cases and Ile in the fourth isolate. Disruption of MGI1 does not lead to the production of mitochondrial genome deletion mutants, indicating that an assembled F1 complex is needed for the "gain-of-function" phenotype found in mgi1 point mutants. The location of Arg435 in the beta-subunit, as deduced from the three-dimensional structure of the bovine F1-ATPase, together with mutational sites in the previously identified mgi2 and mgi5 alleles, suggests that interaction of the beta- and alpha- (MGI2) subunits with the gamma-subunit (MGI5) is likely to be affected by the mutations.