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The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP-Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP-Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F 0 F 1 -ATP synthase at elevated temperatures. The ⌬fmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37°C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the ⌬fmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports ␣-ketoglutarate and ␣-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the ␣-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.In the inner mitochondrial membrane, the F 0 F 1 -ATP synthase performs the late step of the oxidative phosphorylations. This hetero-oligomer uses the electrochemical transmembrane proton gradient generated by the respiratory chain to catalyze ATP synthesis from ADP and inorganic phosphate (for reviews, see Refs. 1 and 2). It is composed of two distinct domains, a membrane-integrated F 0 domain containing a proton channel and a hydrophilic peripheral F 1 domain bearing the catalytic sites for ATP synthesis.This enzyme comprises about 20 different subunits for an overall molecular mass approaching 600 kDa. The genes encoding these proteins are located part in the nucleus and part in the mitochondrion itself. The subunits of nuclear origin are synthesized in the cytoplasm and then imported into the mitochondrion (for review, see Ref.3), whereas the mitochondrial DNA-encoded subunits are synthesized within the mitochondrion. This genetic compartmentalization makes the assembly of the ATP synthase complex a particularly intricate process. Studies in Saccharomyces cerevisiae have shown that specific proteins, usually called assembly factors, not belonging to the final complex, are required in the different steps of the enzyme biogenesis. Three such proteins, Atp11p, Atp12p, and Fmc1p, were shown to facilitate the assembly of the ATP synthase F 1 component (4 -6), which is made of five different nuclear-encoded proteins in the ␣ 3 ␤ 3 ␥␦⑀ stoichiometry (7). The ␣ and ␤ subunits alternate in ...

During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].

Mitochondrial diseases are rare diseases most often linked to energy in the form of ATP-depletion. The high number of nuclear- and mitochondrial-DNA-encoded proteins (>500), required for ATP production and other crucial mitochondrial functions such as NADH re-oxidation, explains the increasing number of reported disorders. In recent years, yeast has revealed to be a powerful model to identify responsible genes, to study primary effects of pathogenic mutations and to determine the molecular mechanisms leading to mitochondrial disorders. However, the clinical management of patients with mitochondrial disorders is still essentially supportive. Here we review some of the most fruitful yeast mitochondrial disorder models and propose to subject these models to highthroughput chemical library screening to prospect new therapeutic drugs against mitochondrial diseases.