Identification and metabolic role of the mitochondrial aspartate-glutamate transporter in Saccharomyces cerevisiae.

The malate-aspartate NADH shuttle in mammalian cells requires the activity of the mitochondrial aspartate-glutamate carrier (AGC). Recently, we identified in man two AGC isoforms, aralar1 and citrin, which are regulated by calcium on the external face of the inner mitochondrial membrane. We have now identified Agc1p as the yeast counterpart of the human AGC. The corresponding gene was overexpressed in bacteria and yeast mitochondria, and the protein was reconstituted in liposomes where it was identified as an aspartate-glutamate transporter from its transport properties. Furthermore, yeast cells lacking Agc1p were unable to grow on acetate and oleic acid, and had reduced levels of valine, ornithine and citrulline; in contrast they grew on ethanol. Expression of the human AGC isoforms can replace the function of Agc1p. However, unlike its human orthologues, yeast Agc1p catalyses both aspartate-glutamate exchange and substrate uniport activities. We conclude that Agc1p performs two metabolic roles in Saccharomyces cerevisiae. On the one hand, it functions as a uniporter to supply the mitochondria with glutamate for nitrogen metabolism and ornithine synthesis. On the other, the Agc1p, as an aspartate-glutamate exchanger, plays a role within the malate-aspartate NADH shuttle which is critical for the growth of yeast on acetate and fatty acids as carbon sources. These results provide strong evidence of the existence of a malate-aspartate NADH shuttle in yeast.

The structure of bovine IF(1), the regulatory subunit of mitochondrial F-ATPase.

In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.

Citrin and aralar1 are Ca(2+)-stimulated aspartate/glutamate transporters in mitochondria.

The mitochondrial aspartate/glutamate carrier catalyzes an important step in both the urea cycle and the aspartate/malate NADH shuttle. Citrin and aralar1 are homologous proteins belonging to the mitochondrial carrier family with EF-hand Ca(2+)-binding motifs in their N-terminal domains. Both proteins and their C-terminal domains were overexpressed in Escherichia coli, reconstituted into liposomes and shown to catalyze the electrogenic exchange of aspartate for glutamate and a H(+). Overexpression of the carriers in transfected human cells increased the activity of the malate/aspartate NADH shuttle. These results demonstrate that citrin and aralar1 are isoforms of the hitherto unidentified aspartate/glutamate carrier and explain why mutations in citrin cause type II citrullinemia in humans. The activity of citrin and aralar1 as aspartate/glutamate exchangers was stimulated by Ca(2+) on the external side of the inner mitochondrial membrane, where the Ca(2+)-binding domains of these proteins are localized. These results show that the aspartate/glutamate carrier is regulated by Ca(2+) through a mechanism independent of Ca(2+) entry into mitochondria, and suggest a novel mechanism of Ca(2+) regulation of the aspartate/malate shuttle.

GRIM-19, a cell death regulatory gene product, is a subunit of bovine mitochondrial NADH:ubiquinone oxidoreductase (complex I).

The sequences of 42 subunits of NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria have been described previously. Seven are encoded by mitochondrial DNA, whereas the remaining 35 are nuclear gene products imported into the organelle from the cytoplasm. An additional protein, which does not correspond to any previously known subunit of the complex I assembly, has now been detected. Denaturing gels of subcomplex Ilambda, the hydrophilic arm of complex I, clearly show a hitherto unidentified band, which was digested with trypsin and subjected to mass-spectrometric analysis to provide several peptide sequences, used in cDNA cloning and sequencing. Measurement of the intact protein mass indicated that the N terminus is acetylated. The new complex I subunit (B16.6) is the bovine homolog of GRIM-19, the product of a cell death regulatory gene induced by interferon-beta and retinoic acid, thus providing a new link between the mitochondrion and its electron-transport chain and apoptotic cell death.

Solution structure of a C-terminal coiled-coil domain from bovine IF(1): the inhibitor protein of F(1) ATPase.

Bovine IF(1) is a basic, 84 amino acid residue protein that inhibits the hydrolytic action of the F(1)F(0) ATP synthase in mitochondria under anaerobic conditions. Its oligomerization state is dependent on pH. At a pH value below 6.5 it forms an active dimer. At higher pH values, two dimers associate to form an inactive tetramer. Here, we present the solution structure of a C-terminal fragment of IF(1) (44-84) containing all five of the histidine residues present in the sequence. Most unusually, the molecule forms an anti-parallel coiled-coil in which three of the five histidine residues occupy key positions at the dimer interface.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals.

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Identification of the human mitochondrial oxodicarboxylate carrier. Bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location.

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine.

Identification in Saccharomyces cerevisiae of two isoforms of a novel mitochondrial transporter for 2-oxoadipate and 2-oxoglutarate.

The nuclear genome of Saccharomyces cerevisiae encodes 35 members of a family of membrane proteins. Known members transport substrates and products across the inner membranes of mitochondria. We have localized two hitherto unidentified family members, Odc1p and Odc2p, to the inner membranes of mitochondria. They are isoforms with 61% sequence identity, and we have shown in reconstituted liposomes that they transport the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a strict counter exchange mechanism. Intraliposomal adipate and glutarate and to a lesser extent malate and citrate supported [14C]oxoglutarate uptake. The expression of Odc1p, the more abundant isoform, made in the presence of nonfermentable carbon sources, is repressed by glucose. The main physiological roles of Odc1p and Odc2p are probably to supply 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol where they are used in the biosynthesis of lysine and glutamate, respectively, and in lysine catabolism.

Characterisation of new intracellular membranes in Escherichia coli accompanying large scale over-production of the b subunit of F(1)F(o) ATP synthase.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli F(1)F(o) ATP synthase in the mutant host strains E. coli C41(DE3) and C43(DE3) is accompanied by the proliferation of intracellular membranes without formation of inclusion bodies. Maximal levels of proliferation of intracellular membranes were observed in C43(DE3) cells over-producing subunit b. The new proliferated membranes contained all the over-expressed protein and could be recovered by a single centrifugation step. Recombinant subunit b represented up to 80% of the protein content of the membranes. The lipid:protein ratios and phospholipid compositions of the intracellular membranes differ from those of bacterial cytoplasmic membranes, and they are particularly rich in cardiolipin.

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.

Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH.

Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).

Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance.

The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins that transport metabolites and substrates across the inner membranes of mitochondria. They include three isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the start of our work, the functions of the remaining 30 members of the family were unknown. We are attempting to identify these 30 proteins by overexpression of the proteins in specially selected host strains of Escherichia coli that allow the carriers to accumulate at high levels in the form of inclusion bodies. The purified proteins are then reconstituted into proteoliposomes where their transport properties are studied. Thus far, we have identified the dicarboxylate, succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, together with characterization of yeast knockout strains, has brought insight into the physiological significance of these transporters. The yeast dicarboxylate carrier sequence has been used to identify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequence has been used to establish the sequence of the human ortholog.

Organization and sequence of the gene for the human mitochondrial dicarboxylate carrier: evolution of the carrier family.

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

Identification of the yeast mitochondrial transporter for oxaloacetate and sulfate.

Saccharomyces cerevisiae encodes 35 members of the mitochondrial carrier family, including the OAC protein. The transport specificities of some family members are known, but most are not. The function of the OAC has been revealed by overproduction in Escherichia coli, reconstitution into liposomes, and demonstration that the proteoliposomes transport malonate, oxaloacetate, sulfate, and thiosulfate. Reconstituted OAC catalyzes both unidirectional transport and exchange of substrates. In S. cerevisiae, OAC is in inner mitochondrial membranes, and deletion of its gene greatly reduces transport of oxaloacetate sulfate, thiosulfate, and malonate. Mitochondria from wild-type cells swelled in isoosmotic solutions of ammonium salts of oxaloacetate, sulfate, thiosulfate, and malonate, indicating that these anions are cotransported with protons. Overexpression of OAC in the deletion strain increased greatly the [(35)S]sulfate/sulfate and [(35)S]sulfate/oxaloacetate exchanges in proteoliposomes reconstituted with digitonin extracts of mitochondria. The main physiological role of OAC appears to be to use the proton-motive force to take up into mitochondria oxaloacetate produced from pyruvate by cytoplasmic pyruvate carboxylase.

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.

The sequence, bacterial expression, and functional reconstitution of the rat mitochondrial dicarboxylate transporter cloned via distant homologs in yeast and Caenorhabditis elegans.

The dicarboxylate carrier (DIC) belongs to a family of transport proteins found in the inner mitochondrial membranes. The biochemical properties of the mammalian protein have been characterized, but the protein is not abundant. It is difficult to purify and had not been sequenced. We have used the sequence of the distantly related yeast DIC to identify a related protein encoded in the genome of Caenorhabditis elegans. Then, related murine expressed sequence tags were identified with the worm sequence, and the murine sequence was used to isolate the cDNA for the rat homolog. The sequences of the worm and rat proteins have features characteristic of the family of mitochondrial transport proteins. Both proteins were expressed in bacteria and reconstituted into phospholipid vesicles where their transport characteristics closely resembled those of whole rat mitochondria and of the rat DIC reconstituted into vesicles. As expected from the role of the DIC in gluconeogenesis and ureogenesis, its transcripts were detected in rat liver and kidney, but unexpectedly, they were also detected in rat heart and brain tissues where the protein may fulfill other roles, possibly in supplying substrates to the Krebs cycle.

Identification of the yeast ACR1 gene product as a succinate-fumarate transporter essential for growth on ethanol or acetate.

The protein encoded by the ACR1 gene in Saccharomyces cerevisiae belongs to a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 28 members of the family are unknown. The yeast ACR1 gene was introduced into Escherichia coli on an expression plasmid. The protein was over-produced as inclusion bodies, which were purified and solubilised in the presence of sarkosyl. The solubilised protein was reconstituted into liposomes and shown to transport fumarate and succinate. Its physiological role in S. cerevisiae is probably to transport cytoplasmic succinate, derived from isocitrate by the action of isocitrate lyase in the cytosol, into the mitochondrial matrix in exchange for fumarate. This exchange activity and the subsequent conversion of fumarate to oxaloacetate in the cytosol would be essential for the growth of S. cerevisiae on ethanol or acetate as the sole carbon source.

Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis.

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.

Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein.

The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.

The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence.

The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.