Mimivirus-Encoded Nucleotide Translocator VMC1 Targets the Mitochondrial Inner Membrane

Mimivirus (Acanthamoeba polyphaga mimivirus) was the first giant DNA virus identified in an amoeba species. Its genome contains at least 979 genes. One of these, L276, encodes a nucleotide translocator with similarities to mitochondrial metabolite carriers, provisionally named viral mitochondrial carrier 1 (VMC1). In this study, we investigated the intracellular distribution of VMC1 upon expression in HeLa cells and in the yeast Saccharomyces cerevisiae. We found that VMC1 is specifically targeted to mitochondria and to the inner mitochondrial membrane. Newly synthesized VMC1 binds to the mitochondrial outer-membrane protein Tom70 and translocates through the import channel formed by the β-barrel protein Tom40. Derivatization of the four cysteine residues inside Tom40 by N-ethylmaleimide caused a delay in translocation but not a complete occlusion. Cell viability was not reduced by VMC1. Neither the mitochondrial membrane potential nor the intracellular production of reactive oxygen species was affected. Similar to endogenous metabolite carriers, mimivirus-encoded VMC1 appears to act as a specific translocator in the mitochondrial inner membrane. Due to its permeability for deoxyribonucleotides, VMC1 confers to the mitochondria an opportunity to contribute nucleotides for the replication of the large DNA genome of the virus.     

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.     

The human uncoupling proteins 5 and 6 (UCP5/SLC25A14 and UCP6/SLC25A30) transport sulfur oxyanions,phosphate and dicarboxylates

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H₂S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H₂S.     

Identification and functional reconstitution of yeast mitochondrial carrier for S-adenosylmethionine

The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier protein family, most of which have not yet been functionally identified. Here the identification of the mitochondrial carrier for S-adenosylmethionine (SAM) Sam5p is described. The corresponding gene has been overexpressed in bacteria and the protein has been reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, (i) the Sam5p-GFP protein was found to be targeted to mitochondria; (ii) the cells lacking the gene for this carrier showed auxotrophy for biotin (which is synthesized in the mitochondria by the SAM-requiring Bio2p) on fermentable carbon sources and a petite phenotype on non-fermentable substrates; and (iii) both phenotypes of the knock-out mutant were overcome by expressing the cytosolic SAM synthetase (Sam1p) inside the mitochondria.     

Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.     

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 alpha-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.     

Identification and functions of new transporters in yeast mitochondria

The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.     

Molecular identification of three Arabidopsis thaliana mitochondrial dicarboxylate carrier isoforms: organ distribution, bacterial expression, reconstitution into liposomes and functional characterization

Screening of the Arabidopsis thaliana genome revealed three potential homologues of mammalian and yeast mitochondrial DICs (dicarboxylate carriers) designated as DIC1, DIC2 and DIC3, each belonging to the mitochondrial carrier protein family. DIC1 and DIC2 are broadly expressed at comparable levels in all the tissues investigated. DIC1-DIC3 have been reported previously as uncoupling proteins, but direct transport assays with recombinant and reconstituted DIC proteins clearly demonstrate that their substrate specificity is unique to plants, showing the combined characteristics of the DIC and oxaloacetate carrier in yeast. Indeed, the Arabidopsis DICs transported a wide range of dicarboxylic acids including malate, oxaloacetate and succinate as well as phosphate, sulfate and thiosulfate at high rates, whereas 2-oxoglutarate was revealed to be a very poor substrate. The role of these plant mitochondrial DICs is discussed with respect to other known mitochondrial carrier family members including uncoupling proteins. It is proposed that plant DICs constitute the membrane component of several metabolic processes including the malate-oxaloacetate shuttle, the most important redox connection between the mitochondria and the cytosol.     

Identification of the mitochondrial carnitine carrier in Saccharomyces cerevisiae

The mitochondrial carrier protein for carnitine has been identified in Saccharomyces cerevisiae. It is encoded by the gene CRC1 and is a member of the family of mitochondrial transport proteins. The protein has been over-expressed with a C-terminal His-tag in S. cerevisiae and isolated from mitochondria by nickel affinity chromatography. The purified protein has been reconstituted into proteoliposomes and its transport characteristics established. It transports carnitine, acetylcarnitine, propionylcarnitine and to a much lower extent medium- and long-chain acylcarnitines.     

Characterization and developmentally regulated localization of the mitochondrial carrier protein homologue MCP6 from Trypanosoma brucei

Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.     

Purification and folding of recombinant bovine oxoglutarate/malate carrier by immobilized metal-ion affinity chromatography

A major obstacle to investigating the structure of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. The oxoglutarate carrier, an intrinsic membrane-transport protein of the inner membranes of bovine-heart mitochondria, has been cloned as a fusion protein containing a C-terminal hexa-histidine tag. This fusion protein has been expressed at an abundant level in a mutant strain of Escherichia coli BL21 (DE3) called C41 (DE3). The protein accumulated as inclusion bodies and none was detected in the bacterial inner membrane. The denatured protein was refolded to reconstitute functional properties similar to the native protein. Solubilized inclusion body protein was immobilized using nickel-chelating affinity chromatography, and purified and refolded in a single step. The protein eluted as a monomer which was stable in mild detergent, at a yield equivalent to 15 mg active protein/liter bacterial culture. The reconstituted fusion protein displayed the same transport characteristics as the wild-type, demonstrating that the tag does not perturb the structure of the protein. The oxoglutarate carrier is one member of an extensive family of mitochondrial transport proteins. These proteins transport many different metabolites across the inner mitochondrial membrane and share a common mechanism of action. Therefore, it is likely that this folding protocol can be applied successfully to other mitochondrial transport proteins, thus providing sufficient protein for extensive crystallization trials with a wide range of family members.     

Cardiolipin,a critical determinant of mitochondrial carrier protein assembly and function

The ability of phospholipids to act as determinants of membrane protein structure and function is probably best exemplified by cardiolipin (CL), the signature phospholipid of mitochondria. Early efforts to reconstitute individual respiratory complexes and members of the mitochondrial carrier family, most notably the ADP/ATP carrier (AAC), often demonstrated the importance of CL. Over the past decade, the significance of CL in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Another protein required for oxidative phosphorylation, AAC, has received comparatively little attention likely stemming from the fact that AACs were thought to function in isolation as either homodimers or monomers. Recently however, AACs have been demonstrated to interact with the respiratory supercomplex, other members of the mitochondrial carrier family, and the TIM23 translocon. Interestingly, many if not all of these interactions depend on CL. As the paradigm for the mitochondrial carrier family, these discoveries with AAC suggest that other members of this large group of important proteins may be more gregarious than anticipated. Moreover, it is proposed that AAC and perhaps additional members of the mitochondrial carrier family might represent downstream targets of pathological states involving alterations in CL.     

An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.     

Mitochondrial carrier homolog 2 (MTCH2): the recruitment and evolution of a mitochondrial carrier protein to a critical player in apoptosis

Recent studies report mitochondrial carrier homolog 2 (MTCH2) as a novel and uncharacterized protein that acts as a receptor-like protein for the truncated BH3-interacting domain death agonist (tBID) protein in the outer membrane of mitochondria. These studies, using mouse embryonic stem cells and fibroblasts as well as mice with a conditional knockout of MTCH2 in the liver, showed that deletion of MTCH2 hindered recruitment of tBID to the mitochondria with subsequent reductions in the activation of pro-apoptotic proteins, mitochondrial outer membrane permeabilization and apoptosis. Sequence analysis shows that MTCH2 is present in all examined multicellular Metazoa as well as unicellular Choanoflagellata, and is a highly derived member of the mitochondrial carrier family. Mitochondrial carriers are monomeric transport proteins that are usually found in the inner mitochondrial membrane, where they exchange small substrates between the mitochondrial matrix and intermembrane space. There are extensive differences between the protein sequences of MTCH2 and other mitochondrial carriers that may explain the ability of MTCH2 to associate with tBID and thus its role in apoptosis. We review the experimental evidence for the role of MTCH2 in apoptosis and suggest that the original transport function of the ancestral MTCH2 mitochondrial carrier has been co-opted by the apoptotic machinery to provide a receptor and signaling mechanism.     

Identification of the mitochondrial GTP/GDP transporter in Saccharomyces cerevisiae

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 16 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates, and cofactors across the inner membrane. Here the identification and functional characterization of the mitochondrial GTP/GDP carrier (Ggc1p) is described. The ggc1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported GTP and GDP and, to a lesser extent, the corresponding deoxynucleotides and the structurally related ITP and IDP by a counter-exchange mechanism. Transport was saturable with an apparent K(m) of 1 microm for GTP and 5 microm for GDP. It was strongly inhibited by pyridoxal 5'-phosphate, bathophenanthroline, tannic acid, and bromcresol purple but little affected by the inhibitors of the ADP/ATP carrier carboxyatractyloside and bongkrekate. Furthermore, in contrast to the ADP/ATP carrier, the Ggc1p-mediated GTP/GDP heteroexchange is H(+)-compensated and thus electroneutral. Cells lacking the ggc1 gene had reduced levels of GTP and increased levels of GDP in their mitochondria. Furthermore, the knock-out of ggc1 results in lack of growth on nonfermentable carbon sources and complete loss of mitochondrial DNA. The physiological role of Ggc1p in S. cerevisiae is probably to transport GTP into mitochondria, where it is required for important processes such as nucleic acid and protein synthesis, in exchange for intramitochondrially generated GDP.     

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.