Identification of mitochondrial carriers in Saccharomyces cerevisiae by transport assay of reconstituted recombinant proteins".

The inner membranes of mitochondria contain a family of carrier proteins that are responsible for the transport in and out of the mitochondrial matrix of substrates, products, co-factors and biosynthetic precursors that are essential for the function and activities of the organelle. This family of proteins is characterized by containing three tandem homologous sequence repeats of approximately 100 amino acids, each folded into two transmembrane α-helices linked by an extensive polar loop. Each repeat contains a characteristic conserved sequence. These features have been used to determine the extent of the family in genome sequences. The genome of Saccharomyces cerevisiae contains 34 members of the family. The identity of five of them was known before the determination of the genome sequence, but the functions of the remaining family members were not. This review describes how the functions of 15 of these previously unknown transport proteins have been determined by a strategy that consists of expressing the genes in Escherichia coli or Saccharomyces cerevisiae, reconstituting the gene products into liposomes and establishing their functions by transport assay. Genetic and biochemical evidence as well as phylogenetic considerations have guided the choice of substrates that were tested in the transport assays. The physiological roles of these carriers have been verified by genetic experiments. Various pieces of evidence point to the functions of six additional members of the family, but these proposals await confirmation by transport assay. The sequences of many of the newly identified yeast carriers have been used to characterize orthologs in other species, and in man five diseases are presently known to be caused by defects in specific mitochondrial carrier genes. The roles of eight yeast mitochondrial carriers remain to be established. © 2006 Elsevier B.V. All rights reserved

Targeting of the translocator protein 18 kDa (TSPO): A valuable approach for nuclear and optical imaging of activated microglia.

The aim of the present review is to give a concise and updated analysis of the imaging tools for the visualization of activated microglia. After an overview on the important pathologies where activated microglia are involved, we first describe the role played by the translocator protein-18 kDa (TSPO) as an important target for the visualization of activated microglia. Second, imaging tools based on TSPO ligands radiolabeled for positron emission tomography (PET) are summarized with particular emphasis to the TSPO ligands alternative to the standard radioligand [11C]PK11195 or (R)-[11C]PK11195. In this regard, an updated list of 11C- and 18F-labeled TSPO radioligands is shown. Moreover, a detailed analysis based on TSPO ligands bearing fluorescent probes for fluorescence microscopy is also provided. This last optical imaging technique represents an area of large and increasing interest due to the advantages offered by the use of simple instrumentation and safer experimental conditions. The scope and limitations of the nuclear and optical imaging techniques are discussed. Finally, a perspective on the plausible advances in this area is also presented

Recombinant expression of the Ca(2+)-sensitive aspartate/glutamate carrier increases mitochondrial ATP production in agonist-stimulated Chinese hamster ovary cells.

The Ca(2+)-sensitive dehydrogenases of the mitochondrial matrix are, so far, the only known effectors to allow Ca2+ signals to couple the activation of plasma membrane receptors to the stimulation of aerobic metabolism. In this study, we demonstrate a novel mechanism, based on Ca(2+)-sensitive metabolite carriers of the inner membrane. We expressed in Chinese hamster ovary cells aralar1 and citrin, aspartate/glutamate exchangers that have Ca(2+)-binding sites in their sequence, and measured mitochondrial Ca2+ and ATP levels as well as cytosolic Ca2+ concentration with targeted recombinant probes. The increase in mitochondrial ATP levels caused by cell stimulation with Ca(2+)-mobilizing agonists was markedly larger in cells expressing aralar and citrin (but not truncated mutants lacking the Ca(2+)-binding site) than in control cells. Conversely, the cytosolic and the mitochondrial Ca2+ signals were the same in control cells and cells expressing the different aralar1 and citrin variants, thus ruling out an indirect effect through the Ca(2+)-sensitive dehydrogenases. Together, these data show that the decoding of Ca2+ signals in mitochondria depends on the coordinate activity of mitochondrial enzymes and carriers, which may thus represent useful pharmacological targets in this process of major pathophysiological interest

The S. cerevisiae YPR011cp is a mitochondrial carrier of adenosine-5’-phosphosulfate and 3’-phospho-adenosine 5’-phosphosulfate

The S. cerevisiae YPR011c gene is located on chromosome 16 and encodes a protein of unknown function with a sequence containing the characteristic features of the mitochondrial carrier family (MCF). Until now YPR011c has been investigated only in microarray analysis of the genome-wide transcription profile of S. cerevisiae concerning YPR011c is available ([1]; website of Yeast Microarray Global Viewer (YMGV)). In the present study YPR011cp was overexpressed in Escherichia coli, purified and reconstituted into liposomes. Our results demonstrate that YPR011cp is a mitochondrial transporter for adenosine-5’-phosphosulfate (APS) and 3’-phospho-adenosine 5’-phosphosulfate (PAPS). Besides transporting APS and PAPS, recombinant and reconstituted YPR011cp also transports sulfate, phosphate, thiosulfate and pyrosulfate. YPR011cp functions almost exclusively by a counter-exchange mechanism; our transport measurements in the reconstituted system indicate that APS and PAPS may cross the mitochondrial membrane in both directions via YPR011cp in exchange with sulfate or phosphate. This is true only for APS, which is produced by the MET3p (ATP sulfurylase) that has a dual cellular localization: cytosolic and mitochondrial [2]. Having established the transport function in vitro, we investigated the physiological significance of YPR011cp in yeast cells. Upon a temperature shift from 30 to 45 °C, S. cerevisiae cells do not survive in the absence of APS and PAPS [3]. At 45°C using cells lacking YPR011c gene and other mutants we have demonstrated that both cytosolic and mitochondrial APS are crucial to support S. cerevisiae cell survival. In addition, our results strongly suggest that APS produced in mitochondria is transported from the mitochondrial matrix to the cytosol via YPR011cp under thermal stress conditions. Finally, the cellular quantification of methionine and total glutathione suggest that APS-mediated protection may be, at least in part, related to the synthesis of glutathione which is necessary for protecting cells at high temperatures [4] and for replenishing cells with sulfur metabolites. This is the first time that mitochondria are found involved in thermotolerance by mediating the transport of APS to the cytosol, which may be the basis of a signaling mechanism crucial for cell survival at higher temperatures. [1] H.C. Causton, et al. Mol. Biol. Cell 12 (2001) 323–337. [2] A. Sickmann, et al. Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13207–13212. [3] H. Jakubowski, E. Goldman, J. Bacteriol. 175 (1993) 5469–5476. [4] K. Sugiyama, et al., Biochem. J. 352 (2000) 71–78

Identification of the human mitochondrial ATP-Mg/Pi transporter

The functions of several members of the mitochondrial transporter family found in genome sequences are unknown. At the same time there are other transport activities observed in intact mitochondria that have yet to be associated with specific proteins. An example is the reversible counterexchange of ATP-Mg for Pi that accounts for the net uptake or efflux of ATP-Mg, as Pi recycles rapidly through the membrane via the phosphate carrier. By screening human ESTs with the sequence of the human ADP/ATP carrier (AAC1) we selected clones encoding proteins of unknown function and containing Ca 2 + -binding EF-hand motifs in their sequences. The corresponding cDNAs (accession numbers AJ619961, AJ619962 and AJ619963) encode three proteins (named APC1-3) with 66 –75% identical amino acids, and with Ca 2 + -binding motifs in their N-terminal domains and the characteristic features of the mitochondrial carrier family in their C-terminal domains. They were overexpressed in E. coli, purified and reconstituted into liposomes. The recombinant proteins APC1 and APC2 transported ATP-Mg, phosphate, ATP, ADP and, less effieciently, AMP in an electroneutral H + - compensated counterexchange. The APC-mediated transport was inhibited by mercurials, bathophenanthroline, tannic acid and bromocresol purple. Little inhibition was observed with carboxyatractyloside and bonkrekate (powerful inhibitors of the AAC1). The green fluorescence (GFP) protein fused to APC1-3 was found to be targeted to mitochondria. The transport properties of APC1 and APC2 and their targeting to mitochondria demonstrate that they are responsible for the ATP-Mg/Pi exchange described in the past in whole mitochondria. The tissue specificity of the three isoforms shows that at least one isoform is present in all the tissues investigated. By screening the human genome databases with the cDNAs of APC1, APC2 and APC3, the corresponding genes (SLC25A24, SLC25A23 and SLC25A26, respectively) were found. They were located on three chromosomes, 1p13.3, 19p13.3 and 9q34.13; contained10exons separated by nine introns; and all the splicing junctions occurred in the same nucleotide regions, indicating a triplication of a common ancestral gene. The main function of the APC isoforms is probably to catalyze the net uptake or efflux of adenine nucleotides into or from the mitochondria, thus explaining the variation in the matrix adenine nucleotide content, which has been found to change in many physiopathological situations