Françoise Foury

Assembly of the mitochondrial membrane system

SummaryNineteen mutants of S. cerevisiae exhibiting a double deficiency in cytochrome oxidase and coenzyme QH2-cytochrome c reductase (also cytochrome b deficient) have been studied. The mutants have been crossed to a set of ρ− tester strains with different segments of mitochondrial DNA. The mutants have also been crossed to mit− testers with defined genetic lesions. In addition, crosses were performed with a respiratory competent strain to ascertain whether mitotic and meiotic segregants could be isolated with only one of the two enzymatic deficiencies.The ρ− testers allowed the doubly deficient mutants to be separated into two classes. Mutants in class 1 were not restored by any of the ρ− testers and appeared to have separate mutations, one in cytochrome oxidase and the other in cytochrome b. Mutants in class 2 were restored by a set of ρ− clones whose retained segments of mitochondrial DNA contained the cytochrome b but not the cytochrome oxidase loci. These appeared to behave as single hit mutations. Further studies, however, indicated that both class 1 and class 2 mutants carried separate mutations in two different loci. Mitotic and meiotic segregants with a single enzymatic deficiency could be isolated. In a number of strains, the mutations were mapped in known cytochrome oxidase and cytochrome b loci. The apparent discrepancy of the ρ− tests for the class 2 mutants was shown to be probably due to a high unstability in one of the mutations.It has been concluded that all the doubly deficient strains carry two mutations in previously described cytochrome oxidase and cytochrome b loci. This conclusion argues against the existence of a single gene on mitochondrial DNA that controls the biosynthesis of the two respiratory enzymes.

In Vivo Functional Analysis of the Human Mitochondrial DNA Polymerase POLG Expressed in Cultured Human Cells

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3′-5′ (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.

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 <e(-40). The percentage of the disease genes identified by positional cloning which bear homology to yeast is similar to that of a random collection of human cDNAs. The biochemical and physiological functions of the large majority of these human genes remain poorly understood and, even though a strict conservation of function cannot safely be assessed from structural homology analysis without the support of experimental and three-dimensional data, functional analogies can often be established between the human and yeast genes.

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild‐type plasmid library by transforming a pif1 null strain to growth on glycerol at the non‐permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single‐stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T‐antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix‐localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single‐stranded (ss) DNA‐dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5′ to 3′ direction with respect to the ss DNA on which it binds first.

Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity.

The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N‐terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3′‐5′ exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred‐fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild‐type enzyme possesses a mismatch‐specific 3′‐5′ exonuclease activity. This activity was decreased by approximately 500‐fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild‐type mtDNA polymerase efficiently catalyses elongation of singly‐primed M13 DNA to the full‐length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3′‐5′ exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.

Mitochondrial functional interactions between frataxin and Isu1p, the iron-sulfur cluster scaffold protein, in Saccharomyces cerevisiae

Friedreichs ataxia is caused by a deficit in the mitochondrial protein frataxin. The present work demonstrates that in vivo yeast frataxin Yfh1p and Isu1p, the mitochondrial scaffold protein for the Fe–S cluster assembly, have tightly linked biological functions, acting in concert to promote the Fe–S cluster assembly. A synthetic lethal screen on high iron media with the mild G107D yfh1 mutant has specifically identified Isu1p. Analysis of the cellular phenotypes resulting from pairwise combinations of yfh1 and isu1 mutations, and cross‐linking experiments in isolated mitochondria provide evidence for a direct interaction between Yfh1p and Isu1p.

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.

Cloning and sequencing of the PIF gene involved in repair and recombination of yeast mitochondrial DNA.

The nuclear gene PIF of Saccharomyces cerevisiae is required for both repair of mitochondrial DNA (mtDNA) and recognition of a recombinogenic signal characterized by a 26‐bp palindromic AT sequence in the ery region of mtDNA. This gene has been cloned in yeast by genetic complementation of pif mutants. Its chromosomal disruption does not destroy the genetic function of mitochondria. The nucleotide sequence of the 3.5‐kb insert from a complementing plasmid reveals an open reading frame encoding a potential protein of 857 amino acids and Mr = 97,500. An ATP‐binding domain is present in the central part of the gene and in the carboxy‐terminal region a putative DNA‐binding site is present. Its alpha helix‐turn‐alpha helix motif is found in DNA‐binding proteins such as lambda and lactose repressors which recognize symmetric sequences. Significant amino acid homology is observed with yeast RAD3 and E. coli UvrD (helicase II) proteins which are required for excision repair of damaged DNA.

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.