Claire Duyckaerts

Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae.

The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the ‘positive inside rule’ approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane α‐helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.

Biochemical, genetic and molecular characterization of new respiratory-deficient mutants in Chlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.

Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria.

The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed.

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt− parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.

Kinetic study of the aspartate/glutamate carrier in intact rat heart mitochondria and comparison with a reconstituted system.

The homologous exchange of external [14C] aspartate/internal aspartate catalyzed by the aspartate/glutamate carrier of rat heart mitochondria was investigated using aspartate-loaded, glutamate-depleted mitochondria. An inhibitor-stop technique was developed for kinetic studies by applying pyridoxal phosphate. Direct initial rate determinations from the linear phase of [14C] aspartate uptake were insufficiently accurate at high external and/or low internal substrate concentrations. Therefore, the full time-course of [14C] aspartate uptake until reaching isotope equilibrium was fitted by a single exponential function and was used to calculate reliable initial steady-state rates. This method was applied in bisubstrate analyses of the antiport reaction for different external and internal aspartate concentrations. The kinetic patterns obtained in double reciprocal plots showed straight lines converging on the abscissa. This result is consistent with a sequential antiport mechanism. It implies the existence of a catalytic ternary complex that is formed by the translocator and substrate molecules bound from both sides of the membrane. The Km values for aspartate were clearly different for the external and the internal sides of the membrane, 216 +/- 23 microM and 2.4 +/- 0.5 mM, respectively. These values indicated a definite transmembrane asymmetry of the carrier. The same asymmetry became evident when investigating the isolated protein from bovine heart mitochondria after reconstitution into liposomes. In this case the Km values for external and internal aspartate were determined to be 123 +/- 11 microM and 2.8 +/- 0.6 mM, respectively. This comparison demonstrates a right-side out orientation of the carrier after insertion into liposomal membranes. The sequential transport mechanism of the aspartate/glutamate carrier, elucidated both in proteoliposomes and in mitochondria, also seems to be a common characteristic of other mitochondrial antiport carriers.

Oxygen consumption of equine articular chondrocytes: Influence of applied oxygen tension and glucose concentration during culture

We investigated the oxygen (O2) uptake of equine articular chondrocytes to assess their reactions to anoxia/re‐oxygenation. They were cultured under 5% or 21% gas phase O2 and at glucose concentrations of 0, 1.0 or 4.5 g/L in the culture medium (n = 3). Afterwards, the O2 consumption rate of the chondrocytes was monitored (oxymetry) before and after an anoxia period of 25 min. The glucose consumption and lactate release were measured at the end of the re‐oxygenation period. The chondrocytes showed a minimal O2 consumption rate, which was hardly changed by anoxia. Independently from the O2 tension, glucose uptake by the cells was about 30% of the available culture medium glucose, thus higher for cells at 4.5 g/L glucose (n= 3). Lactate release was also independent from O2 tension, but lower for cells at 4.5 g/L glucose (n= 3). Our observations indicated that O2 consumption by equine chondrocytes was very low despite a functional mitochondrial respiratory chain, and nearly insensitive to anoxia/re‐oxygenation. But the chondrocytes metabolism was modified by an excess of O2 and glucose.

Kinetic Mechanisms of the Adenylic and the Oxoglutaric Carriers: a Comparison

Translocations by exchange through membranes must obey to one of the two general types of mechanisms: the ping-pong mechanism and the sequential mechanism.

Oxoglutarate translocator of rat-heart mitochondria: regulation by aspartate

The oxoglutarate translocator participates in the transfer of reducing equivalents from the cytosol to the mitochondrial respiratory chain via the malateaspartate shuttle, by a cyclic process exchanging external malate for internal 2oxoglutarate [ 11. The catalytic components of the shuttle (the cytosofic and matricial glutamate-oxaloacetate transaminases and malate dehydrogenases, and the oxoglutarate and glutamate-aspartate translocators) act in sequence: one of the products is a substrate of the following step. Do allosteric effecters regulate the operation of the shuttle? This paper describes regulatory effects of aspartate on the oxoglutarate-translocator step of the shuttle working in the opposite direction (efflux of reducing equivalents from the mitochondrial matrix).

Transient modifications of respiratory capacity in thymic cells during murine radioleukemogenesis

The evolution of mitochondrial oxidative phosphorylation was studied during cancer induction in a model of thymic radiolymphomagenesis in C57BL/Ka mice. During the preneoplastic period, thymuses displayed an increase of the cytochrome c oxidase activity and oxygen consumption together with oxidative DNA damage assessed by the presence of the 8-hydroxydeoxyguanine DNA base modification. These transient changes in mitochondrial functional activity were not observed in thymuses of mice rescued from lymphoma development by a bone marrow graft, suggesting an important role of mitochondria for neoplastic transformation in this model, which might therefore be of interest to test the utilization of antioxidants for the prevention of radiation-induced malignancies.

Kinetic mechanisms of mitochondrial carriers catalysing exchange reactions

The single-binding site or ping-pong mechanism is widely accepted for exchange reactions, catalysed by mitochondrial carriers.However, when the most relevant approach to discriminate between mechanisms,i.e., kinetic study is used, the ping-pong mechanism is eliminated in favour of the sequential or ternary complex mechanism implying two binding sites simultaneously accessible to both internal and external substrates. This is the case for the oxoglutarate carrier, the aspartate/glutamate carrier and there are very strong presumptive evidences for the adenylic carrier.