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International audience; The transfer of metabolites through the mitochondrial membranes is a vital process that is highly controlled and regulated by the inner membrane. A variety of metabolites, nucleotides, and cofactors are transported across the inner mitochondrial membrane (IMM) by a superfamily of membrane transporters which are known as the mitochondrial carrier family (MCF) or the solute carrier family 25 (SLC25 protein family). In humans, the MCF has 53 members encoded by nuclear genes. Members of the SLC25 family of transporters, which is the largest group of solute carriers, are also known as mitochondrial carriers (MCs). Because MCs are nuclear-coded proteins, they must be imported into the IMM. When compared with normal cells, the mitochondria of cancer cells exhibit significantly increased transmembrane potentials and a number of their transporters are altered. SLC25 members were identified as potential biomarkers for various cancers. The objective of this review is to summarize what is currently known about the involvement of mitochondrial SLC25 carriers in associated diseases. This review suggests that the SLC25 family could be used for the development of novel points of attack for targeted cancer therapy.

The mitochondrial phosphate carrier (PiC) of mammals, but not the yeast one, is synthesized with a presequence. The deletion of this presequence of the mammalian PiC was reported to facilitate the import of the carrier into yeast mitochondria, but the question as to whether or not mammalian PiC could be functionally expressed in yeast mitochondria was not addressed. In the present study, we first examined whether the defective growth on a glycerol plate of yeast cells lacking the yeast PiC gene could be reversed by the introduction of expression vectors of rat PiCs. The introduction of expression vectors encoding full-length rat PiC (rPiC) or rPiC lacking the presequence (ΔNrPiC) was ineffective in restoring growth on the glycerol plates. When we examined the expression levels of individual rPiCs in yeast mitochondria, ΔNrPiC was expressed at a level similar to that of yeast PiC, but that of rPiC was very low. These results indicated that ΔNrPiC expressed in yeast mitochondria is inert. Next, we sought to isolate “revertants” viable on the glycerol plate by expressing randomly mutated ΔNrPiC, and obtained two clones. These clones carried either of two mutations, F267S or F282S; and these mutations restored the transport function of ΔNrPiC in yeast mitochondria. These two Phe residues were conserved in human carrier (hPiC), and the transport function of ΔNhPiC expressed in yeast mitochondria was also markedly improved by their substitutions. Thus, substitution of F267S or F282S was concluded to be important for functional expression of mammalian PiCs in yeast mitochondria.

International audience; Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O2 consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.

[ES]Tesis doctoral sobre el estudio de la Apoptosis, una forma de muerte celular programada y regulada genéticamente por el que las células inducen su propia muerte en respuesta a determinados estímulos.

The antiestrogen tamoxifen (TAM) inhibits the growth of different estrogen receptor (ER)-negative cells. Recently, multiple effects of TAM on mitochondrial bioenergetic functions have been pointed to explain its ER-independent cell death mechanisms. We have shown that TAM and its major active metabolite 4-hydroxytamoxifen (OHTAM) induce depolarization of the mitochondrial membrane potential ([Delta][Psi]) and uncouple the mitochondrial respiration, depressing the oxidative phosphorylation efficiency. To clarify the biochemical mechanisms underlying the changes in the regulation of ATP synthesis and yield, in this work we evaluated the alterations of mitochondrial adenine nucleotides induced by both drugs and ascertained whether such changes could reflect a specific inhibition of either the adenine nucleotide translocase (ANT) or the phosphate carrier, as well as the activation of ATP hydrolysis due to [Delta][Psi] depolarization. We found that both antiestrogens caused a concentration-dependent decrease in mitochondrial ATP levels. Mitochondrial ADP and AMP were concomitantly increased with a subsequent decrease in the ATP/ADP or ATP/AMP ratios. The total concentration of adenine nucleotides also changed. Additionally, both drugs decreased the ANT content of mitochondria, inhibited the phosphate carrier and induced ATP hydrolysis. However, the effects of TAM were more drastic than those induced by OHTAM. Therefore, the depletion of ATP might result from an activation of ATP catabolism, as well as from a decrease in the mitochondrial content of ANT and partial inhibition of the phosphate carrier. Our data may explain the ER-independent effects and cytotoxicity of both drugs and, in agreement with other previous studies, suggest that OHTAM is much less toxic to mitochondria than TAM. http://www.sciencedirect.com/science/article/B6TCP-49C5CK6-6/1/f5b08afedd3f5563da8601805f04bc8f

After heterologous expression in E. coli, functionally active phosphate carrier (PIC) from Saccharomyces cerevisiae mitochondria was purified and reconstituted into giant liposomes and used for patch clamp experiments. Single channel currents across excised patches revealed an anion channel function of the PIC protein. Besides the three transport modes known to date, namely phosphate/phosphate exchange, phosphate/OH− exchange and mercurial-induced unidirectional transport, this channel activity represents the fourth transport mode of the PIC. The PIC channel activity was sensitive towards phosphate as its physiological substrate. Phosphate (10 mM) blocked in a specific but reversible manner the PIC channel, suggesting a phosphate-dependent conformational change of the protein into the carrier mode. Furthermore, the current through the channel and its gating activity were affected by divalent cations. In the presence of Ca2+ and Mg2+, the channel displayed a mean conductance of 25±5 pS whereas 40±10 pS was observed in the absence of divalent cations. Also, the dwell times in either the open or closed state of the PIC channel appeared to be prolonged in the presence of Ca2+ and Mg2+. The observed PIC channel characteristics are discussed with respect to previously reported electrophysiological in situ measurements on anion channels of the inner mitochondrial membrane. Similarities of the PIC channel to the inner mitochondrial anion channel (IMAC) have been found.

Comparison of the sequence of the human mitochondrial phosphate carrier (PiC) gene with cDNA clones characterised from a human heart cDNA library suggested the existence of two isoforms of the PiC, which were generated by alternative splicing of exon IIIA or exon IIIB and which differed in 13 amino acids [Dolce et al. (1994) J. Biol. Chem. 269,10451]. In this work the expression of isoforms A and B of the PiC was investigated in different bovine tissues by Northern blot analysis using two probes that are specific for bovine exon IIIA and exon IIIB, respectively. Isoform A is highly expressed in heart and skeletal muscle. Isoform B is ubiquitously expressed in all tissues that were examined, although at different levels. The tissue-specific expression pattern of the two PiC isoforms is similar to that reported for the isoforms of several mitochondrial proteins required for energy production.

The sequences of three mitochondrial carriers involved in energy transfer, the ADP/ATP carrier, phosphate carrier and uncoupling carrier, are analyzed. Similarly to what has been previously reported for the ADP/ATP carrier and the uncoupling protein, now also the phosphate carrier is found to have a tripartite structure comprising three similar repeats of approx. 100 residues each. The three sequences show a fair overall homology with each other. More significant homologies are found by comparing the repeats within and between the carriers in a scheme where the sequences are spliced into repeats, which are arranged for maximum homology by allowing possible insertions or deletions. A striking conservation of critical residues, glycine, proline, of charged and of aromatic residues is found throughout all nine repeats. This is indicative of a similar structural principle in the repeats. Hydropathy profiles of the three proteins and a search for amphipathic α-spans reveal six membrane-spanning segments for each carrier, providing further support for the basic structural identity of the repeats. The proposed folding pattern of the carriers in the membrane is exemplified with the phosphate carrier. A possible tertiary arrangement of the repeats and the membranespanning helices is shown. The emergence of a mitochondrial carrier family by triplication and by divergent evolution from a common gene of about 100 residues is discussed.

ATP synthesis rate, measured as the variation in external Pi concentration, varied as a linear function of either delta mu H+ or delta Gpin, in such a manner that the delta Gpin/delta mu H+ ratio increased while VATP increased. We also observed a linear dependence of the flux control coefficient of the Pi carrier on delta pH. All the results presented can be explained by a relatively large delta pH drop when VATP increases.

The general procedure for the isolation of the phosphate carrier from mitochondria involves solubilization with non-ionic detergents and chromatography on hydroxylapatite [l-3]. With high resolution SDS-gel electrophoresis this preparation can be separated into 4-5 protein bands [2]. Further purification, in particular removal of the ADP/ATP-carrier, was obtained by chromatography on Celite [2], on Mersalyl-Ultrogel [3,4] and by using Triton X-l 14 instead of Triton X-100 [5]. However, after all these procedures the purified phosphate carrier fraction still contained 4-5 protein bands in the &.-region of 30 000-35 000 [5]. Affi-Gel 501 (an organomercurial agarose gel), Dowex AG l-X8 and hydroxylapatite (Bio-Gel HTP) were obtained from Bio-Rad; [32P]phosphate (carrier free) from Amersham; [3H]NEM from New England Nuclear; acrylamide, N,N’methylenebisacrylamide, SDS, Triton X-100 and NEM from Serva; scintillation liquid (Maxifluor) from J. Baker; L-cY-phosphatidylcholine (from egg yolk, Type X-E) from Sigma and cardiolipin from Serdary.

The phosphate carrier has been isolated from beef heart mitochondria in the presence of cardiolipin and reconstituted in asolectin vesicles. It has been found that 100 microM doxorubicin and 100 microM Br-daunomycin inhibit the unidirectional phosphate uptake in the reconstituted liposomes to the same extent as N-ethylmaleimide. The inhibition by Br-daunomycin is not due to covalent interaction with the carrier. The specific interaction between doxorubicin and cardiolipin is responsible for the inhibition of the phosphate carrier. Br-daunomycin interacts with 3 mitochondrial proteins of apparent Mr approximately 45 000, approximately 35 000 and approximately 30 000.