Studies of human, mouse and yeast homologues indicate a mitochondrial function for frataxin.

Friedreich's ataxia is due to loss of function mutations in the gene encoding frataxin (FRDA). Frataxin is a protein of unknown function. In situ hybridization analyses revealed that mouse frataxin expression correlates well with the main site of neurodegeneration, but the expression pattern is broader than expected from the pathology of the disease. Frataxin mRNA is predominantly expressed in tissues with a high metabolic rate, including liver, kidney, brown fat and heart. We found that mouse and yeast frataxin homologues contain a potential mitochondrial targeting sequence in their N-terminal domains and that disruption of the yeast gene results in mitochondrial dysfunction. Finally, tagging experiments demonstrate that human frataxin co-localizes with a mitochondrial protein. Friedreich's ataxia is therefore a mitochondrial disease caused by a mutation in the nuclear genome.

Aconitase and mitochondrial iron-sulphur protein deficiency in Friedreich ataxia.

Friedreich ataxia (FRDA) is a common autosomal recessive degenerative disease (1/50,000 live births) characterized by a progressive-gait and limb ataxia with lack of tendon reflexes in the legs, dysarthria and pyramidal weakness of the inferior limbs. Hypertrophic cardiomyopathy is observed in most FRDA patients. The gene associated with the disease has been mapped to chromosome 9q13 (ref. 3) and encodes a 210-amino-acid protein, frataxin. FRDA is caused primarily by a GAA repeat expansion within the first intron of the frataxin gene, which accounts for 98% of mutant alleles. The function of the protein is unknown, but an increased iron content has been reported in hearts of FRDA patients and in mitochondria of yeast strains carrying a deleted frataxin gene counterpart (YFH1), suggesting that frataxin plays a major role in regulating mitochondrial iron transport. Here, we report a deficient activity of the iron-sulphur (Fe-S) cluster-containing subunits of mitochondrial respiratory complexes I, II and III in the endomyocardial biopsy of two unrelated FRDA patients. Aconitase, an iron-sulphur protein involved in iron homeostasis, was found to be deficient as well. Moreover, disruption of the YFH1 gene resulted in multiple Fe-S-dependent enzyme deficiencies in yeast. The deficiency of Fe-S-dependent enzyme activities in both FRDA patients and yeast should be related to mitochondrial iron accumulation, especially as Fe-S proteins are remarkably sensitive to free radicals. Mutated frataxin triggers aconitase and mitochondrial Fe-S respiratory enzyme deficiency in FRDA, which should therefore be regarded as a mitochondrial disorder.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

In-frame recombination between the yeast H(+)-ATPase isogenes PMA1 and PMA2: insights into the mechanism of recombination initiated by a double-strand break.

Chimeric PMA1::PMA2 sequences, placed under the control of the PMA1 promoter, were constructed by in vivo recombination between a gapped linearized plasmid containing the PMA2 gene and four different fragments of the PMA1 gene. Correct in-frame assembly of the PMA sequences was screened by the expression of the lacZ reporter gene fused to the PMA2 coding region. Restriction and sequencing analysis of 35 chimeras showed that in all cases, the hybrid sequences was obtained as fusions between continuous sequences specific to PMA1 and PMA2, separated by a region of identity. In all but three cases, the junction sequences were not located at regions of greatest identity. Strikingly, depending on the PMA1 fragment used, junction distribution fell into two categories. In the first, the junctions were scattered over several hundreds of nucleotides upstream of the extremity of the PMA1 fragment, while in the second, they were concentrated at this extremity. Analysis of the alignment of the PMA1 and PMA2 sequences suggests that the distribution is not related to the size of the region of identity at the PMA1-PMA2 boundary but depends on the degree of identity of the PMA genes upstream of the region of identity, the accumulation of successive mismatches leading to a clustered distribution of the junctions. Moreover, the introduction of seven closely spaced mismatches near the end of a PMA1 segment with an otherwise-high level of identity with PMA2 led to a significantly increased concentration of the junctions near this end. These data show that a low level of identity in the vicinity of the common boundary stretch is a strong barrier to recombination. In contrast, consecutive mismatches or regions of overall moderate identity which are located several hundreds of nucleotides upstream from the PMA1 end do not necessarily block recombination.

Two antigens recognized by autologous cytolytic T lymphocytes on a melanoma result from a single point mutation in an essential housekeeping gene.

We have pursued our analysis of antigens recognized by autologous cytolytic T lymphocytes (CTLs) on the melanoma cells of patient LB33. This patient enjoys an unusually favorable evolution, which is associated with a strong and sustained antitumor CTL response. We reported previously the analysis of two melanoma cell lines, MEL.A and MEL.B, which were derived from metastases removed from the patient at 5 years' distance. Autologous CTL clones derived from blood lymphocytes recognized several antigens presented by different HLA class I molecules on MEL.A. The MEL.B cells resisted lysis by these CTLs because they have lost expression of most HLA molecules, suggesting that they were selected in vivo by the anti-MEL.A CTL response. One of the MEL.A antigens was shown to result from a point mutation in the tumor. Here we report the cloning of a gene that encodes two other MEL.A antigens. This new gene, MUM-2, is expressed ubiquitously. In the melanoma cells of patient LB33, it contains a point mutation that changes one amino acid in the translated protein. Two different antigenic peptides, one presented to CTL by HLA-B44 molecules and another by HLA-C6 molecules, overlap and contain the mutated residue. Gene MUM-2 is homologous to an essential yeast gene, bet5, that was recently shown to be implicated in the vesicular transport of proteins from the endoplasmic reticulum to the Golgi. In a mutant yeast with a disrupted bet5 gene, both the wild-type and the mutated MUM-2 genes could complement for bet5 function. These results indicate that the antigenic mutation does not destroy the function of the protein, a function that is conserved in eukaryotic cells. The identification of these antigens suggests that point mutations could be the major cause of the strong immunogenicity of MEL.A cells.

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe.

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.

Analysis of the trinucleotide CAG repeat from the human mitochondrial DNA polymerase gene in healthy and diseased individuals.

The human nuclear gene (POLG) for the catalytic subunit of mitochondrial DNA polymerase (DNA polymerase gamma) contains a trinucleotide CAG microsatellite repeat within the coding sequence. We have investigated the frequency of different repeat-length alleles in populations of diseased and healthy individuals. The predominant allele of 10 CAG repeats was found at a very similar frequency (approximately 88%) in both Finnish and ethnically mixed population samples, with homozygosity close to the equilibrium prediction. Other alleles of between 5 and 13 repeat units were detected, but no larger, expanded alleles were found. A series of 51 British myotonic dystrophy patients showed no significant variation from controls, indicating an absence of generalised CAG repeat instability. Patients with a variety of molecular lesions in mtDNA, including sporadic, clonal deletions, maternally inherited point mutations, autosomally transmitted mtDNA depletion and autosomal dominant multiple deletions showed no differences in POLG trinucleotide repeat-length distribution from controls. These findings rule out POLG repeat expansion as a common pathogenic mechanism in disorders characterised by mitochondrial genome instability.

Human genetic diseases: a cross-talk between man and yeast.

A sequence similarity search has been carried out against the complete Saccharomyces cerevisiae genome to identify the yeast homologues of human disease-associated genes. Using the BLAST algorithm (Basic Local Alignment Search Tool), it was found that 52 out of the 170 disease genes identified without reference to chromosomal map position and 22 of the 80 (27.5%) positionally cloned genes match yeast genes with a P-value of

New features of mitochondrial DNA replication system in yeast and man.

In this review, we sum up the research carried out over two decades on mitochondrial DNA (mtDNA) replication, primarily by comparing this system in Saccharomyces cerevisiae and Homo sapiens. Brief incursions into systems of other organisms have also been achieved when they provide new information.S. cerevisiae and H. sapiens mitochondrial DNA (mtDNA) have been thought for a long time to share closely related architecture and replication mechanisms. However, recent studies suggest that mitochondrial genome of S. cerevisiae may be formed, at least partially, from linear multimeric molecules, while human mtDNA is circular. Although several proteins involved in the replication of these two genomes are very similar, divergences are also now increasingly evident. As an example, the recently cloned human mitochondrial DNA polymerase beta-subunit has no counterpart in yeast. Yet, yeast Abf2p and human mtTFA are probably not as closely functionally related as thought previously. Some mtDNA metabolism factors, like DNA ligases, were until recently largely uncharacterized, and have been found to be derived from alternative nuclear products. Many factors involved in the metabolism of mitochondrial DNA are linked through genetic or biochemical interconnections. These links are presented on a map. Finally, we discuss recent studies suggesting that the yeast mtDNA replication system diverges from that observed in man, and may involve recombination, possibly coupled to alternative replication mechanisms like rolling circle replication.

Isolation and characterization of ten mutator alleles of the mitochondrial DNA polymerase-encoding MIP1 gene from Saccharomyces cerevisiae.

Ten mutator alleles of MIP1, the gene encoding mitochondrial (mt) DNA polymerase, have been isolated after in vitro random mutagenesis. Five mutations causing a 100-400-fold increase in the frequency of erythromycin-resistant (ErR) mt mutants in yeast mapped to the 3'-5' exonuclease (Exo) domain, and mainly to the three conserved motifs Exo1, Exo2 and Exo3 of this domain, highlighting the importance of proofreading in accurate mt DNA replication. The essential role of the invariant glutamate at the Exo1 site was confirmed and the participation of four amino acids (aa) in the 3'-5' Exo function revealed. Another mutation that is located between the Exo1 and Exo2 sites produced an extremely strong mutator phenotype associated with impaired DNA replication, but could be assigned neither to a conserved aa nor to a conserved portion of the 3'-5' exonuclease domain. The importance of the polymerization domain in accurate mt DNA replication was pointed out by three mutator mutations. Two of these severely impaired mt DNA replication and were assigned to a subdomain of the polymerase which probably corresponds to the 'fingers' module of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk). The third, which did not alter the efficiency of DNA replication, was located at the active center of the polymerization reaction. Finally, the mutation, R1001I, mapped to the C-terminal part of the MIP1 protein which has no counterpart in prokaryotic DNA polymerases.

Mitochondrial DNA polymerases from yeast to man: a new family of polymerases.

We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma). Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.

Mass-murdering: deletion of twenty-three ORFs from Saccharomyces cerevisiae chromosome XI reveals five genes essential for growth and three genes conferring detectable mutant phenotype.

In the frame of the European Network for Functional Analysis (EUROFAN), two regions from chromosome XI covering 54kb have been subjected to 'mass-murder'. Ten deletions covering 23 novel open reading frames (ORFs) were constructed in haploid and diploid strains. Six deletions were lethal in haploid strains. One deletion caused slow germination of spores and slow cellular growth, and another one was associated with both cellular growth thermosensitivity and poor growth on glycerol. These two defects were assigned to two different genes. All mutant phenotypes were complemented by a single gene, enabling us to identify five genes essential for vegetative growth, three genes with detectable phenotype and 15 dispensable genes under standard physiological conditions.

Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain.

Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects. We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild-type and YFH1 deletant strains. Under these conditions, the activity of the respiratory complexes is restored. However, the activity of the mitochondrial aconitase, a 4Fe-4S cluster-containing protein, remains low. The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur. Yfh1p might control the synthesis of iron-sulfur clusters in mitochondria.

Deletion of the yeast homologue of the human gene associated with Friedreich's ataxia elicits iron accumulation in mitochondria.

Deletion of YDL120, the yeast homologue of the human gene responsible for Friedreich's ataxia, elicits decreased cellular respiration associated with decreased cytochrome c oxidase activity and, in certain nuclear backgrounds, mitochondrial DNA is lost. In the null mutants, the cellular growth is highly sensitive to oxidants, such as H2O2, iron and copper. However, only ferrous sulfate elicits loss of mitochondrial DNA. Mitochondria of the null mutants contain 10 times more iron than wild-type. The neurodegeneration observed in Friedreich's ataxia can be well explained on the basis of a mitochondrial iron overload responsible for an increased production of highly toxic free radicals.

The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae.

The currently available yeast mitochondrial DNA (mtDNA) sequence is incomplete, contains many errors and is derived from several polymorphic strains. Here, we report that the mtDNA sequence of the strain used for nuclear genome sequencing assembles into a circular map of 85,779 bp which includes 10 kb of new sequence. We give a list of seven small hypothetical open reading frames (ORFs). Hot spots of point mutations are found in exons near the insertion sites of optional mobile group I intron-related sequences. Our data suggest that shuffling of mobile elements plays an important role in the remodelling of the yeast mitochondrial genome.

Search for protein partners of mitochondrial single-stranded DNA-binding protein Rim1p using a yeast two-hybrid system.

RIM1 is a nuclear gene of the yeast Saccharomyces cerevisiae coding for a protein with single-stranded DNA-binding activity that is essential for mitochondrial genome maintenance. No protein partners of Rim1p have been described so far in yeast. To better understand the role of this protein in mitochondrial DNA replication and recombination, a search for protein interactors by the yeast two-hybrid system was performed. This approach led to the identification of several candidates, including a putative transcription factor, Azf1p, and Mph1p, a protein with an RNA helicase domain which is known to influence the mutation rate of nuclear and mitochondrial genomes.

Repair of mitochondrial DNA in Saccharomyces cerevisiae. Induction of cytoplasmic petite mutants in a nuclear mutant exhibiting thermosensitive mitochondrial deoxyribonuclease activity.

Four nuclear thermosensitive mutants have been obtained in which induction of up 100% cytoplasmic petite mutants (rho-) is observed upon cell incubation at 36 degrees C. For a given incubation time at 36 degrees C, the percentage of rho- is increased by preliminary gamma-ray irradiation. Under these conditions, the induction of rho- is a linear function of the irradiation dose. The retention of genetic information by rho- and of mitochondrial DNA synthesis in vivo and in vitro exclude that the mutants are deficient in the replication of mitochondrial DNA. The degradation of mitochondrial DNA labeled with [3H]dTTP in isolated mitochondria, has been monitored at 26 degrees C and at 36 degrees C after addition of 0.5% Triton X-100 in the presence or in the absence of ethidium bromide. In assays carried out at 26 degrees C, the degradation of mitochondrial DNA is similar in the parental strain and in the mutant gamma s rho 2. However, at 36 degrees C, the degradation of mitochondrial DNA is slower in the mutant. We have shown that a mitochondrial membrane deoxyribonuclease acting on double-stranded DNA at acid pH is thermosensitive in the mutant. Analysis of the meiotic segregants of a tetrad issued from the cross of the mutant with an isogenic parental strain shows co-segregation of rho- induction and of nuclease thermosensitivity in a 2:2 Mendelian pattern. These results suggest that a mitochondrial deoxyribonuclease is involved in the repair of damages caused to mitochondrial DNA by elevated temperature and by x-rays.

Endonucleases in yeast mitochondria: apurinic and manganese-stimulated deoxyribonuclease activities in the inner mitochondrial membrane of Saccharomyces cerevisiae.

An apurinic endonuclease activity has been characterized in yeast mitochondrial. It is dependent on Mg2+, stimulated by about 50% in the presence of 50 mM NaCl and inhibited at higher NaCl concentrations. It is located in the inner mitochondrial membrane and requires high concentrations of detergent (1.5-3% Triton X-100) to be extracted. The same treatment extracts several other endonuclease activities: the two Mg2+-dependent endonuclease activities cleaving double-stranded DNA at pH 7.5 and 5.4 respectively, the ethidium-bromide-stimulated endonuclease activity, the endonuclease activity cleaving single-stranded DNA at pH 7.l5 [Jacquemin-Sablon et al. (1979) Biochemistry, 18, 119-127], and a manganese-stimulated deoxyribonuclease activity cleaving double-stranded DNA at pH 7.5 which has been discovered during the present work. Another endonuclease activity cleaving double-stranded DNA at pH 7.5 in the presence of Mg2+, slightly stimulated by low NaCl concentrations and inhibited by ethidium bromide is extracted from the membrane pellet remaining after the treatment with 1.5% Triton X-100 by a second treatment with 1.5% Triton X-100 plus 1 M KCl. The presence in the mitochondrial membrane of this apurinic endonuclease activity indicates that, like nuclear and prokaryotic DNA, yeast mitochondrial DNA is also subject to specialized repair systems.

The 31-kDa polypeptide is an essential subunit of the vacuolar ATPase in Saccharomyces cerevisiae.

The VMA4 gene encodes a 26.6-kDa hydrophilic polypeptide which exhibits 34% sequence identity with the E subunit of the vacuolar ATPase from bovine kidney microsomes. The chromosomal VMA4 gene was inactivated by a 171-base pair deletion followed by insertion of the URA3 gene within the coding sequence. Null vma4 haploid mutants are viable. However, their growth is considerably slowed down specially in non-acidic conditions; they are cold sensitive and thermo-sensitive, exhibit poor growth on glycerol medium, and do not accumulate in their vacuole the red pigment of ade2 strains. No bafilomycin-sensitive ATPase is detected in a vacuolar fraction. These properties shared by null mutants in the A, B, and C subunits of the vacuolar ATPase show that the VMA4 polypeptide is also an essential component of the vacuolar ATPase which has been conserved from yeast to mammals. The tightly linked VMA4 and MIP1 (encoding the mitochondrial DNA polymerase) genes are divergently transcribed from face-to-face promoters. About 250 base pairs upstream of the VMA4 gene, Homoll and RPG consensus for the binding of TUF (RAP/GRF1) protein are present, suggesting that the VMA4 gene belongs to this large family of genes involved in cellular growth and division whose transcription is regulated by the TUF protein.