The mitochondrial transporter family (SLC25): physiological and pathological implications
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- Palmieri, F. Pflugers Arch - Eur J Physiol (2004) 447: 689. doi:10.1007/s00424-003-1099-7
The mitochondrial carriers (MCs) shuttle a variety of metabolites across the inner mitochondrial membrane (i.m.m.). In man they are encoded by the SLC25 genes. Some MCs have isoforms encoded by different SLC25 genes, whereas the phosphate carrier has two variants arising from an alternative splicing of SLC25A3. Six MCs have been sequenced after purification, and many more have been identified from their transport and kinetic properties following heterologous over-expression and reconstitution into liposomes. All MCs of known function belong to the same protein family, since their polypeptide chains consist of three tandemly related sequences of about 100 amino acids, and the repeats of the different carriers are homologous. They probably function as homodimers, each monomer being folded in the membrane into six transmembrane segments. The functional information obtained in studies with mitochondria and/or the reconstituted system has helped to gain an insight into the physiological role of the MCs in cell metabolism, as have tissue distribution, the use of knock-out mice (and/or yeast) and over-expression in human cell lines (or yeast) of individual carriers and isoforms. At the same time, the cloning and functional identification of many SLC25 genes has made it possible (i) to identify the genes (and their defects) responsible for some diseases, e.g. Stanley syndrome and Amish microcephaly, and (ii) where the genes were already known, to characterize the function of the gene products and hence understand the molecular basis and the symptoms of the diseases, e.g. hyperornithinaemia, hyperammonaemia and homocitrullinuria (HHH) syndrome and type II citrullinemia. It is likely that further extension and functional characterization of the SLC25 gene family will elucidate other diseases caused by MC deficiency.
KeywordsMitochondriaTransportTransportersCarriersSolute carriersMitochondrial diseasesSLC25 genesMitochondrial carrier genesMitochondrial carrier familyPhysiological role of mitochondrial carriers
peroxisomal adenine nucleotide carrier
Graves’ disease carrier
inner mitochondrial membrane
mitochondrial carrier family
Online Mendelian Inheritance in Man (database)
name of the human mitochondrial solute carrier gene family, assigned by the Human Genome Organisation (HUGO) nomenclature committee
Introduction and brief history of the SLC25 family: discovery and extension
The mitochondrial carriers (MCs), encoded in man by the SLC25 genes, are membrane-embedded proteins that, with one exception, are localized in the inner membranes of mitochondria, catalyse the translocation of solutes across the membrane and belong to a family of carrier proteins, the SLC25 or mitochondrial carrier family (MCF). Their common function is to provide a link between mitochondria and cytosol by facilitating the flux of a large variety of solutes through the permeability barrier of the inner mitochondrial membrane (i.m.m.). This link is indispensable, as many physiological processes require the participation of both intra- and extra-mitochondrial enzyme reactions. Besides this basic function, some MCs play an important role in regulating and maintaining a balance between cytosol and mitochondrial matrix, for example of the phosphorylation and redox potentials. Furthermore, some exert flux control on metabolic pathways. The substrates transported by the MCs vary widely in their structure and size from the smallest, H+, to the largest and most highly charged species transported through membranes, e.g. ATP. The majority of the substrates are anions, but some are cations or zwitterions. Some MCs are present in more-or-less all tissues, whereas others are tissue specific and have a limited distribution, reflecting their importance in special functions. A number have isoforms with different tissue distributions. In addition, all MCs are nuclear-coded proteins and have to be imported into the i.m.m. Functional studies in intact mitochondria have indicated the presence of around 20 carrier systems for the transport of metabolites involved in oxidative phosphorylation, citric acid cycle, fatty acid oxidation, gluconeogenesis, lipogenesis, transfer of reducing equivalents, urea synthesis, amino acid degradation, intramitochondrial DNA, RNA and protein syntheses and other functions occurring between cytosol and mitochondria.
In 1982 the amino acid sequence of the ADP/ATP carrier was determined . This actually was the first primary structure of a solute carrier to be reported. The analysis of the ADP/ATP carrier sequence showed that the whole structure of around 300 amino acids could be divided into three related domains, each about 100 residues in length . This striking pattern of homologous repeats was also observed in the second MC to be sequenced, the uncoupling protein (UCP) . These observations raised speculations that these proteins, and possibly other MCs, might belong to one protein family originating from a common ancestral gene by two-tandem duplication. The validity of this hypothesis was amply demonstrated when the carriers for phosphate, oxoglutarate, citrate and carnitine/acylcarnitines were sequenced (see  and references therein). These six MCs were sequenced after purification by direct amino acid analysis or by DNA sequencing. Following purification of the ADP/ATP carrier in 1974, it took more than 20 years to reach this point, because nearly all the MCs are present in the i.m.m. in very minute amounts.
In this review, after illustrating typical MCF structural and functional characteristics, I will focus on the physiological and pathological implications of each human SLC25 member identified so far. For other important aspects of MC research and for reference to the vast amount of literature, the reader is referred to reviews on MCs in general [5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21] or on particular MCs [22, 23, 24, 25, 26, 27].
Structural characteristics of the SLC25 family
All MCs of known function exhibit a tripartite sequence structure, i.e. they contain three tandemly repeated homologous domains about 100 amino acids in length. Each domain contains two hydrophobic stretches, that are thought to span the membrane as α-helices, separated by hydrophilic regions, and the repeats of one carrier are related to those present in the others. MCs contain the signature sequence motif P-h-D/E-X-h-K/R-X-R/K-(20–30 residues)-D/E-G-(4 residues)-a-K/R-G, where h represents a hydrophobic and a represents an aromatic residue. However, in several MCs the sequence is partially modified in one, two or even all three repeats.
Most of the isolated MC proteins have been shown to be homodimers and this may apply to all members of the MCF. The evidence is based on cross-linking studies, inhibitor binding stoichiometry per monomer and MW determinations by hydrodynamic measurements and blue native gel electrophoresis. The role of the homodimers in the transport mechanism, involving for example a central channel between the monomers or two coordinated channels, one in each monomer, is still being elucidated. If MCs indeed function as dimers, their structure would consist of 12 transmembrane segments, as for the majority of carrier proteins . As for carrier proteins in general, no three-dimensional structures are available.
Functional characteristics of the SLC25 family members and structure-function studies
MCs can be divided into electrogenic (or electrophoretic) and electroneutral transporters. So far, three well-characterized carriers have been shown to be electrophoretic. The ADP/ATP and aspartate/glutamate carriers catalyse exchanges that result in charge imbalance because they transport ADP3− for ATP4− and aspartate− for glutamate− plus H+, respectively, across the mitochondrial membrane. The third, the uncoupling protein, catalyses the unidirectional transport of H+. Charge balance can be achieved by cotransport (symport) of solutes, countertransport of solutes and uniport of electroneutral metabolites. The carriers for phosphate (Pi), glutamate and pyruvate (the latter not yet identified at the molecular level) facilitate the transport of anions together with an equivalent amount of H+ (anion/H+ symport) or in exchange for OH− (anion/OH− antiport). Other carriers catalyse exchange of anions or cations. The oxoglutarate carrier, for example, transports oxoglutarate2− for malate2− and the ornithine carrier ornithine for lysine, arginine or H+. In some cases, electroneutrality is imposed by simultaneous carrier-mediated H+ movement. The citrate carrier, for example, transports H-citrate2− against malate2− and the human ornithine carrier can transport ornithine+ against citrulline plus H+.
The MCs utilize, as their driving force, the concentration gradient of the solutes and/or the H+ electrochemical potential generated across the i.m.m. by the respiratory chain. Since the electrical component of the electrochemical potential for H+ (ΔµH+) is rather high, the electrical nature of the ADP/ATP and aspartate/glutamate carriers provides a powerful means of ejecting ATP and aspartate against the concentration gradient from the mitochondrial matrix to the cytosol. In cases of H+ symport or H+ exchange, the transmembrane pH gradient regulates the distribution of anionic and cationic solutes across the membrane. For example, with a higher pH inside, the carrier-mediated, H+-compensated uptake of Pi or glutamate is stimulated, as well as the efflux of cationic solutes such as the export of ornithine.
The kinetic properties of several MCs and the modulation of their activities by pH, ΔpH, membrane potential, phospholipids and other parameters have been elucidated in detail in mitochondria and especially after functional reconstitution in liposomes. The majority of MCs catalyse strict solute exchange reactions. Those mediating H+-compensated unidirectional substrate flux may also fall into the above category, because at least the phosphate carrier has been shown to function in a phosphate/OH− antiport mode . Kinetic studies varying both the internal and external substrate concentrations have shown that, with the exception of the carnitine carrier, all the MCs analysed so far function according to a simultaneous (sequential) mechanism, implying that one internal and one external substrate molecule form a ternary complex with the carrier before translocation occurs. The carnitine carrier, in contrast, follows a ping-pong mechanism indicating only binary carrier-substrate complexes. As this carrier mediates uniport besides exchange of substrates, it is likely that the activation energy barrier for the reorientation of the unloaded carrier form is much lower for the carnitine carrier than for exchange carriers. As the uniport function is essential in a transport system like the carnitine carrier, the common sequential mechanism has apparently been changed into a ping-pong type of mechanism.
Recombinant expression of the MCs in yeast and in E. coli has enabled site-directed mutagenesis for structure-function studies. The mutation of Cys to Ser, one at a time or all together, evidenced the absence of essential cysteines in several carriers although transport could be inhibited by SH reagents. Thus, the cysteine reagents may cause steric hindrance or conformational changes. Further structure-function studies will be reported below for some MCs.
Description of each member of the SLC25 family with emphasis on the physiological and pathological implications
SLC25—the mitochondrial carrier family (adPEO autosomal dominant progressive external ophthalmoplegia, HHH hyperornithinaemia, hyperammonaemia and homocitrullinuria syndrome, PEP phosphoenolpyruvate, dNDP deoxynucleoside diphosphate, dNTP deoxynucleoside triphosphate, NDP nucleoside diphosphate, ddNTP dideoxynucleoside triphosphate)
Human gene name
Transport type/coupling ions*
Tissue distribution and cellular/subcellular expression
Link to disease#
Human gene locus
Sequence accession ID
Splice variants and their specific features
CIC (citrate carrier)
CTP, tricarboxylate carrier
Citrate, malate, PEP
Liver, kidney, pancreas (also in brain, lung, heart)/inner mitochondrial membrane
ORC2 (ornithine carrier 2)
Ornithine, citrulline, lysine, arginine, histidine
E/ornithine/citrulline plus H+; ornithine/H+
Liver, testis, spleen, lung, pancreas, small intestine, brain, kidney/inner mitochondrial membrane
PiC (phosphate carrier)
C/phosphate plus H+ E/phosphate/OH−
Isoform A: heart, skeletal muscle and diaphragm; isoform B: liver, kidney, brain, thymus, lung, heart, skeletal muscle, diaphragm/inner mitochondrial membrane
NM_005888 isoform A NM_002635 isoform B
Isoforms A and B generated by alternative splicing of exon IIIA or exon IIIB
AAC1 (ADP/ATP carrier 1)
ANT 1, T1, PEO2, PEO3, adenine nucleotide translocator 1
Heart, skeletal muscle, much less in brain, pancreas, prostate, kidney, lung, thymus/inner mitochondrial membrane
adPEOG, Sengers syndromeG
AAC2 (ADP/ATP carrier 2)
ANT 2, T2, adenine nucleotide translocator 2
Brain, lung, kidney, pancreas, heart, skeletal muscle, spleen/inner mitochondrial membrane
AAC3 (ADP/ATP carrier 3)
ANT3, T3, adenine nucleotide translocator 3
Brain, lung, kidney, liver, pancreas, heart, skeletal muscle, spleen, thymus/inner mitochondrial membrane
UCP1 (uncoupling protein 1)
Thermogenin, proton carrier
Brown adipose tissue/inner mitochondrial membrane
UCP2 (uncoupling protein 2)
Proton carrier, UCPH
Brain, lung, kidney, spleen, heart/inner mitochondrial membrane
Obesity, type 2 diabetes
UCP3 (uncoupling protein 3)
Skeletal muscle, lung/inner mitochondrial membrane
UCP3S, this variant (short) lacks 37 amino acids at its C terminus compared to UCP3L (long). UCP3S is generated when a cleavage and polyadenylation signal located in the last intron of the gene prematurely terminates message elongation. Compared with UCP3L, UCP3S lacks the putative transmembrane domain VI and a putative purine nucleotide-binding domain.
DIC (dicarboxylate carrier)
Malate, phosphate, succinate, sulphate, thiosulphate
Liver, kidney, heart, brain, lung, pancreas/inner mitochondrial membrane
OGC (oxoglutarate carrier)
Heart, skeletal muscle, liver, kidney, brain, pancreas/inner mitochondrial membrane
AGC1 (aspartate/glutamate carrier 1)
E/aspartate/glutamate plus H+
Brain, heart, skeletal muscle, lung, pancreas, kidney, but not in liver/inner mitochondrial membrane
AGC2 (aspartate/glutamate carrier 2)
E/aspartate/glutamate plus H+
Liver, kidney, pancreas, heart, skeletal muscle, brain/inner mitochondrial membrane
Citrullinaemia type II (CTLN2)G
UCP5 (uncoupling protein 5)
BMCP1, brain mitochondrial carrier protein
H+ (?) homologous to UCP1/2/3
Widely expressed, with highest levels in brain and testis
AF155809 (long) AF155811 (short) AF155810 (short + insertion)
BCMP1S, this variant (short) lacks 3 amino acids (23–25, VSG) compared with the transcript variant BMCP1L (long). BCMP1SI, this variant (short + insertion) is like BCMP1S but has a 31 amino acid insertion between putative transmembrane segments III and IV.
ORC1 (ornithine carrier 1)
Ornithine, citrulline, lysine, arginine
E/ornithine/citrulline plus H+; ornithine/H+
Liver, pancreas, lung, testis, small intestine, spleen, kidney, brain, heart/inner mitochondrial membrane
GDC (Graves’ disease carrier)
CoA or CoA precursor (?)
Liver, kidney, thyroid, lung, heart, skeletal muscle, brain/inner mitochondrial membrane
ANC (peroxisomal adenine nucleotide carrier)
ATP, ADP, AMP
Brain, kidney, lung, heart, liver, pancreas/peroxisomal membrane
GC2 (glutamate carrier 2)
C/glutamate plus H+, E/glutamate/OH−
Brain, testis, heart, pancreas, kidney, lung/inner mitochondrial membrane
DNC (deoxynucleotide carrier)
dNDPs, dNTPs, NDPs, ATP, ddNTPs
Brain, testis, lung, kidney, liver, spleen, skeletal muscle, heart/inner mitochondrial membrane
Amish microcephaly (MCPHA)G
3 isoforms generated by alternative splicing in the 5′-UTR
CAC (carnitine/acylcarnitine carrier)
CACT, carnitine carrier
E/carnitine/acylcarnitines F (at slow rate)
Heart, skeletal muscle, liver (also in lung, kidney, brain, pancreas, placenta)/inner mitochondrial membrane
ODC (oxoadipate carrier)
Kidney, gall bladder, colon, liver, placenta, testis, lung, spleen, skeletal muscle, brain, heart/inner mitochondrial membrane
GC1 (glutamate carrier 1)
C/glutamate plus H+, E/glutamate/OH−
Pancreas, brain, liver, testis, spleen, kidney, heart, lung, small intestine/inner mitochondrial membrane
UCP4 (uncoupling protein 4)
H+ (?) homologous to UCP 1/2/3
Brain/inner mitochondrial membrane
Citrate carrier CIC (SLC25A1)
CIC, also known as the tricarboxylate carrier, catalyses electroneutral exchange of a tricarboxylate (e.g. citrate, isocitrate) for either another tricarboxylate, a dicarboxylate (e.g. malate) or phosphoenolpyruvate (PEP) (see Table 1). The carrier is inhibited by the impermeable substrate analogue 1,2,3-benzenetricarboxylate. Citrate/malate exchange is electroneutral because the carrier accepts solely the single protonated form of citrate (H-citrate2−) and the unprotonated form of malate (malate2−). CIC has been purified and reconstituted, kinetically characterized, cloned and over-expressed in E. coli ( and references therein). The cDNA sequences of man, cow, rat and yeast are known. The properties of CIC TMS IV and its role in the substrate translocation mechanism have been investigated thoroughly by Kaplan and colleagues . Each of the CIC residues from 173 to 194 were replaced one at a time with cysteine. The single-Cys proteins were over-expressed in E. coli, functionally reconstituted into liposomes and found to be all active, except R181C and R189C. Replacement of either Arg with Lys, or replacement of either Arg with Cys, followed by chemical modification of Cys with (2-aminoethyl)-methanethiosulphonate hydrobromide [MTSEA, a methanethiosulphonate (MTS) reagent carrying a positive charge], partially rescued activity, indicating that positive charges at positions 181 and 189 are essential for CIC function. In further studies the accessibility of engineered single-Cys residues to MTS reagents and the accessibility of a spin label, attached to each engineered Cys, to paramagnetic perturbants were determined, showing that TMS IV is an α-helix and that the face of the helix containing positions 181 and 189 is water accessible and probably represents a portion of the citrate translocation pathway.
CIC transport activity, which is high in the liver and low in the heart and brain, is affected by hormonal and nutritional factors. It is enhanced in hyperthyroidism and cancer and is reduced significantly during starvation and type-1 diabetes in a manner that can be corrected by administration of exogenous insulin (see  and references therein). The human CIC gene has been localized to chromosome 22q11.21 within a region implicated in DiGeorge syndrome (DGS), velo-cardio-facial syndrome (VCFS) and a subtype of schizophrenia . It is hemizygous in nearly all DGS and VCFS patients and may therefore contribute to these syndromes and, in particular, to the mental deficiency of these patients.
Ornithine carriers ORC1 and ORC2 (SLC25A15 and SLC25A2, respectively)
The ornithine carrier has two isoforms in man, ORC1 and ORC2 (see Table 1). In early studies the ORC, also known as the ornithine/citrulline carrier, was purified and reconstituted. In post-genomic studies, the S. cerevisiae ORC gene was the first nucleotide sequence found to encode a mitochondrial ornithine carrier . This sequence was then used to identify the human orthologue ORC1 . Recently, human ORC1 and ORC2 have been over-expressed and characterized in a reconstituted system . Both isoforms catalyse the transport of ornithine, lysine, arginine and citrulline (by substrate exchange or, to a lesser extent, by exchange of basic amino acids for H+) and are inhibited by spermine and spermidine. However, they differ in many respects. Firstly, whereas ORC1 does not transport histidine and exhibits a high preference for the l-stereoisomers, ORC2 transports histidine and both l- and d-stereoisomers equally well. Secondly, ORC2 has a higher affinity for lysine and arginine, and a lower affinity for ornithine and citrulline, than isoform 1. Thirdly, the specific activity of ORC2 is lower than that of ORC1, and finally, ORC1 is expressed at higher levels than ORC2 in all the tissues examined, and ORC2 is virtually absent in brain, heart and kidney.
ORC fulfils the important function of exchanging cytosolic ornithine and intramitochondrial citrulline, and is therefore the essential component of the urea cycle that links the activities of the cycle’s enzymes located in the cytosol and in the mitochondrial matrix (Fig. 3E; see  and references therein). This conclusion has been strengthened by the demonstration that the SLC25A15 gene coding ORC1 is altered in patients affected by the HHH syndrome (OMIM 603861) , which is characterized by hyperornithinaemia, hyperammonaemia and homocitrullinuria. So far, more than 50 patients with this autosomal recessive disorder have been identified. Most of them show episodes of coma when given high-protein diets, growth retardation and periods of lethargy, ataxia and myoclonic seizures.
Co-expression of ORC2 and ORC1 in the liver, the presence of mutations leading to loss of activity in the ORC1 cDNAs of HHH patients and the absence of mutations in the ORC2 cDNAs of the same patients may account for the generally milder phenotype of the HHH patients, compared with that caused by defects in any of the urea cycle enzymes. On the other hand, ORC2 cannot replace the function of ORC1 in the urea cycle completely, as it has a lower affinity for ornithine and citrulline and is less expressed in liver. Several HHH patients have benefited from high-level ornithine administration; blood ammonia decreases and nitrogen tolerance increases, indicating that ornithine is transported into mitochondria by ORC2, leading to improved urea cycle functioning. In the HHH syndrome, as in all the urea cycle disorders, a low-protein diet is appropriate.
As well as in ureogenesis, ORC1 and ORC2 play a role in cell metabolism under various physiological conditions. The net import of lysine, arginine and histidine into the mitochondria is necessary for the synthesis of intramitochondrially translated proteins and that of ornithine for the degradation of excess arginine. When the dietary content of arginine is high, the ornithine formed by arginine hydrolysis can be removed by ornithine aminotransferase localized in the mitochondrial matrix, the expression of which is highly regulated by the dietary protein content. Hepatic ornithine aminotransferase is localized in the pericentral, glutamine synthetase-containing hepatocytes, and not in the hepatocytes containing the urea cycle enzymes. This means that the ornithine/citrulline exchange activity is carried out in mitochondria of periportal hepatocytes, whereas the ornithine/H+ exchange occurs in mitochondria of the pericentral hepatocytes (see  and references therein). The cellular distribution of ORC1 and ORC2 in the liver is not yet known. Efflux of ornithine from the mitochondria may occur in other metabolic processes, such as the biosynthesis of polyamines that are produced from ornithine in the cytosol. Under conditions of low arginine content in the diet and/or in the tissues where the activity of arginase is negligible, the ornithine produced intramitochondrially from glutamate has to be exported to the cytosol to accomplish polyamine biosynthesis. In this context, it is interesting that ORC1 and ORC2 are both inhibited by spermine and spermidine. In addition, ornithine is also produced in intestine mitochondria from glutamine and may supply extra urea cycle intermediates for the liver.
Phosphate carrier PiC (SLC25A3)
Only one gene for the PiC has been detected in man (see Table 1), cow and rat. This gene is divided into nine exons and contains two exons, named IIIA and IIIB, which are closely related and are spliced alternatively. The alternative splicing mechanism affects amino acids 4–45 of the mature PiC (13 residues in the human isoforms) localized mainly in the first putative transmembrane α-helix of the protein. The presence of the two PiC isoforms (A and B) has been shown both at the transcript and protein levels . PiC-A is present only in muscles, whereas PiC-B is ubiquitous. Heart and liver bovine mitochondria contained 69 and 0 pmol PiC-A/mg protein, and 10 and 8 pmol PiC-B/mg protein, respectively. The functional differences of the bovine isoforms have been elucidated following their expression in E. coli, purification and reconstitution. The recombinant isoforms A and B both catalyse the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibit similar properties of substrate specificity and inhibitor sensitivity, which are those characteristic of the PiC (as known from intact mitochondria). However, they differ considerably in their kinetic parameters. The Km of PiC-A for Pi on the external membrane surface is threefold that of PiC-B (about 2.2 and 0.78 mM, respectively). Km on the internal surface is much higher (about 8.5 and 6.5 mM for PiC-A and PiC-B, respectively). The maximum transport rate of PiC-A is about one-third that of PiC-B.
The yeast PiC has been the target of a number of mutations involving negatively charged residues . H32 in TMS I, E126 and E137 in TMS III and D39 and D236 at the matrix ends of the first and fifth TMS are indispensable for normal growth in yeast and transport function. The six charged residues (H32, E126 and E137 of both subunits) are thought to form the proton cotransport channel within the homodimer. An inhibitory disulphide formation under oxidizing conditions between the two Cys-28 residues (also located in TMS I) of two PiC monomers is in agreement with this model.
The main physiological role of the PiC is to catalyse the transport of phosphate into the mitochondrial matrix, either by proton cotransport or in exchange for hydroxyl ions, at the expense of the pH gradient across the membrane (Fig. 3F). This uptake of Pi into mitochondria is essential for the oxidative phosphorylation of ADP to ATP. Inside the mitochondria Pi also fulfils other functions: it participates in other phosphate-requiring reactions (e.g. that catalysed by succinyl-CoA synthetase), and allows the uptake of other metabolites by exchanging with them via other SLC25 members (e.g. the dicarboxylate carrier) and recycling on its own carrier.
The tissue specificity of the two PiC isoforms resembles that of the isoforms of the ADP/ATP carrier (AAC, see below) and other mitochondrial proteins involved in oxidative phosphorylation. The common feature of these proteins is the presence of at least one heart-type isoform, which is expressed abundantly only in muscles, and one liver-type isoform, which is expressed ubiquitously. The differences in the kinetic properties of the two PiC isoforms can account for the differential reliance on oxidative phosphorylation of the tissues in which they are present. The ubiquitous PiC-B isoform might match the basic energy requirement of the tissues and isoform PiC-A might become operative in case of energetic stress, i.e. during contraction of striated muscle fibres. Thus, during muscle contraction the capacity of isoform B, which has a higher affinity for Pi, is overwhelmed, and isoform A with its lower substrate affinity will then be brought into operation by the increased concentrations of cytosolic Pi.
ADP/ATP carriers AAC1, AAC2 and AAC3 (SLC25A4, SLC25A5 and SLC25A6, respectively)
Three isoforms of the AAC have been found in man (see Table 1) and several other organisms. AAC1 is expressed mainly in heart and skeletal muscle, AAC2 is expressed ubiquitously at a level depending on the respiratory activity of the tissues and AAC3 is expressed very weakly in the main tissues and abundantly in highly proliferative cells. The expression of AAC1 and AAC2 is regulated by the cell’s energy requirements, while that of all three isoforms is modulated by the state of cell differentiation, although differently. Recently, the functional differences of the three human AAC isoforms have been investigated employing heterologous expression in a yeast mutant lacking the three endogenous ADP/ATP carriers . Each of the human AAC isoforms is able to restore growth on glycerol, although AAC3 more efficiently. The Km of AAC3 for ADP (8.4 µM) is higher than those of AAC1 and AAC2 (about 3 µM), and the Vmax of AAC3 is about twofold higher than those of AAC1 and AAC2.
AAC1, the most abundant protein in mitochondria, was the first MC to be purified, reconstituted and sequenced. It has high specificity for ADP and ATP, as it does not tolerate any significant structural changes in the base, ribose or phosphate parts of the molecule. It is inhibited by two highly specific and effective inhibitors: carboxyatractyloside (CATR), which binds on the cytosolic side of the protein, and bongkrekic acid (BKA), which enters the mitochondria and binds on the opposite side. At least two different conformations of the carrier exist, both in the native membrane and in the solubilized state. These two states have been proven by their different reactivity to antibodies, proteases and other reagents. After binding of CATR or BKA, the equilibrium between the two conformations is shifted toward the formation of a very stable CATR-carrier (CATR-conformation, c-state) or BKA-carrier (BKA-conformation, m-state). In the absence of CATR and BKA, ADP or ATP can also induce a rapid transition between the two states, suggesting that this transition may occur in the catalytic transport cycle [22, 24]. Under conditions of oxidative phosphorylation (with Δψ positive outside) the ADP/ATP carrier exchanges cytosolic ADP3− for mitochondrial ATP4−. This exchange, which is probably the most active carrier-mediated transport in eukaryotic cells, fulfils the important function of linking the energy metabolism compartimentalized between the mitochondrial matrix (where ATP is synthesized from ADP and phosphate) and the cytosol (where ATP is consumed) (Fig. 3F).
In the yeast isoform 2, three types of charged residues have been probed by mutagenic charge neutralization: positively charged residues located within TMSs (K38A, R96A, R204L and R294A) and within the matrix loops (K48A, R152A, K179I, K182I, R252I, R253I and R254I); and negatively charged residues at the end of TMS I, III and V (E45G, D149S, D249S). By comparing the in vivo (yeast aerobic growth) and in vitro effects (reconstituted transport), these residues were assigned to transport or structure maintenance to varying degrees. Using a regain-of-function approach in yeast, in second-site mutations negatively charged residues were affected, which evidenced ion pair interactions R294-E45, K38-E45, R152-E45, R152-D149, and D149-R252 and indicated an arrangement in which TMS I and VI are in vicinal and skewed position ( and references therein).
Adult AAC1 knock-out mice exhibit a severe intolerance to exercise and lactic acidosis . Histological studies show ragged-red muscle fibres, cardiac hypertrophy and intensive proliferation of mitochondria in heart and skeletal muscles. In the adult the absence of AAC1 is thus probably not compensated by increased expression of the other two isoforms, whereas AAC2 and AAC3 are sufficient for the animals’ survival.
Deficiency of the ADP/ATP carrier has been reported in some patients with myopathy or cardiomyopathy, although the underlying mechanisms and pathognostic significance are obscure. More recently, mutations in the SLC25A4 gene coding AAC1 have been found in various families or in sporadic cases presenting autosomal dominant progressive external ophthalmoplegia (adPEO) (OMIM 157640) [42, 43]. This is an adult-onset mitochondrial disorder characterized by progressive external ophthalmoplegia, ptosis, exercise intolerance and large-scale mitochondrial DNA (mtDNA) deletions. Furthermore, a deficiency of AAC1 in skeletal and heart muscle has been shown, both immunologically and by transport assays, in patients affected by Sengers syndrome (OMIM 103220). Since the SLC25A4 gene is not altered, the carrier deficiency may be due to an impairment of AAC1 transcription or of its import into the mitochondria . Regardless of the genetic defect underlying Sengers syndrome, AAC1 protein deficiency may be the cause of some symptoms (exercise intolerance, hypertrophic cardiomyopathy and lactic acidaemia), as well as of the tissue specificity and of the characteristic morphological features. This is the first example showing a specific MC protein, but not its gene, to be involved crucially in an inherited mitochondrial disease.
Finally, the ADP/ATP carrier is involved in the Ca2+-induced formation of the mitochondrial transition pore (MTP), which plays a major role in apoptosis by inducing the release of cytochrome c. The specific ADP/ATP carrier ligands ADP and BKA inhibit, whereas atractyloside (ATR) induces the formation of the MTP. This pore can also be induced by prooxidants at low Ca2+ and seems to be a major target for oxygen radical damage.
Uncoupling proteins UCP1, UCP2, UCP3, UCP4 and UCP5 (SLC25A7, SLC25A8, SLC25A9, SLC25A27 and SLC25A14, respectively)
The UCPs are a subfamily of mitochondrial carriers comprising UCP1 and four other related proteins (UCP2–5, Table 1). UCP3 and UCP5 exist as two splice variants (short and long forms). UCP1 is specific to brown adipose tissue; UCP2 is ubiquitous and expressed highly in organs that are involved in immune defence or are rich in macrophages; UCP3 is essentially specific to skeletal muscle; UCP4 and UCP5 (originally described as brain mitochondrial carrier protein-1, BMCP1) are expressed predominantly in brain. The expression of UCPs is generally up-regulated by several factors such as fatty acids, prostaglandins, tumour necrosis factor α, other “pyrogenic” cytokines, triiodothyronine, adrenaline and leptin. There is strong evidence that UCP1, UCP2 and UCP3 are H+ transporters and that this transport is stimulated by fatty acids and inhibited by purine nucleotides. Recently, activation of H+ transport through the UCPs by superoxide has also been reported . The mechanism of the protonophoric activity of UCPs is still controversial. Klingenberg has suggested the existence of an H+ translocation pathway within the UCP structure . Using the H+ donor and acceptor function of the carboxyl group, fatty acids (FA) facilitate H+ translocation in conjunction with endogenous carboxyl residues within a translocation pathway. An alternative explanation, advanced by Garlid and Jezek , suggests that UCPs catalyse H+ transport by fatty acid cycling. In this model UCPs are assumed to transport FA anions. In a counter flow, protonated FA can readily flip-flop through the membrane. As a result of this UCP-dependent cycle, H+ is translocated across the membrane.
There is evidence for a direct involvement of charged residues in H+ transport pathway through UCP1 . D27 within TMS I and the H145/H147 unit within loop B are thought to function as proton donor/acceptors, respectively, in the channel and the matrix side entrance of the H+ transport pathway. Further mutagenesis studies on charged residues in TMS II (R83 and E129), IV (R182), VI (R276) and in TMS V at the cytosolic interface (H214) have suggested that the three arginines are part of the purine nucleotide binding site and that H214 and E190 act as pH sensors for controlling the access of the nucleotide phosphate moiety to its binding cleft. Deletion of F267-K268-G269 in matrix loop C, as well as analogous deletions in loops A and B, cause loss of nucleotide regulation, suggesting a gating function of the three hydrophilic matrix loops in the regulation of UCP by purine nucleotides.
The main physiological role of UCPs is to uncouple the mitochondria, i.e. to dissipate the electrochemical proton gradient generated by the respiratory chain across the i.m.m., by increasing its proton conductance (Fig. 3F). Beneficial effects of this basic function would be the regulation of body temperature and the control of oxygen radical production. UCP1 has long been known to be essential for the non-shivering thermogenesis necessary for newborn mammals, cold-adapted rodents and hibernators to cope with a low ambient temperature. In addition, recent studies have suggested that UCP2 and UCP3 are involved in pathological conditions in humans such as obesity, type-2 diabetes and atherosclerosis. UCP1 knock-out mice are cold sensitive but not obese. Mice lacking UCP2 or UCP3 are not cold sensitive, show no gain in body weight, but over-produce reactive oxygen species. UCP2 and UCP3 regulate the secretion of insulin, since UCP2-deficient mice hypersecrete insulin and mice over-expressing UCP3 in skeletal muscle exhibit lower levels of insulin, hyperphagia without increase in body weight and fat accumulation. Furthermore, in the skeletal muscle of UCP3 knock-out mice ATP synthesis is increased (although less than expected) without any change in the Krebs cycle rate, in agreement with an uncoupling activity in vivo (see  for a review).
Dicarboxylate carrier DIC (SLC25A10)
DIC (see Table 1) catalyses the electroneutral exchange of certain dicarboxylates (e.g. malate and succinate), inorganic phosphate and inorganic sulphur-containing compounds (e.g. sulphite, sulphate and thiosulphate). It is inhibited by impermeable substrate analogues such as butylmalonate. Kinetically, the carrier follows a simultaneous mechanism. DIC is, however, more complex than other mitochondrial antiporters. On each membrane side of the protein there are two separate binding sites, one specific for phosphate (thiosulphate, arsenate and phenylphosphate), the other specific for dicarboxylates (sulphate, sulphite and butylmalonate). All four binding sites must be associated with the same translocation pathway through the carrier protein, since the sequential antiport mechanism holds true for the phosphate/malate heteroexchange as well as for the malate/malate or phosphate/phosphate homoexchanges. Km values for malate and Pi are about 0.4 and 1.5 mM, respectively, both in mitochondria and in liposomes reconstituted with purified native protein or with recombinant proteins from S. cerevisiae, C. elegans and rat ( and references therein).
The DIC is involved in gluconeogenesis from pyruvate (and amino acids) and ureogenesis. As shown in Fig. 3G, pyruvate is converted in the matrix into OAA and then into malate; this dicarboxylate is exported by the DIC to the cytosol where it reduces NAD+ to NADH plus H+ and is converted into OAA and then into PEP, which is used by the gluconeogenetic pathway. In the urea cycle fumarate is produced in the cytosol by argininosuccinate lyase (ASL) (see Fig. 3E), it is converted into malate and the latter enters the mitochondrial matrix via the DIC. The DIC is also involved in the metabolism of sulphur compounds. In particular, it facilitates the entry of thiosulphate into mitochondria, where rhodanase and thiosulphate reductase are found, the former exclusively in the mitochondria. Another important function of the DIC is an anaplerotic one, i.e. it supplies substrates of the Krebs cycle to the mitochondrial matrix, as indicated by its presence in brain and heart, i.e. tissues in which ureogenesis and gluconeogenesis are absent. Studies on yeast knock-out cells have shown that the primary function of the DIC is to transport cytosolic dicarboxylates into the mitochondria and not to direct carbon flux to gluconeogenesis .
Oxoglutarate carrier OGC (SLC25A11)
OGC, also known as the oxoglutarate/malate carrier, catalyses the transport of oxoglutarate in electroneutral exchange for some other dicarboxylates, of which malate is bound with the highest affinity (Table 1). It is inhibited by impermeable substrate analogues such as phtalonic acid. It has been isolated from the heart and liver and reconstituted functionally into liposomes. In proteoliposomes OGC has been shown to exist as a homodimer and to function according to a simultaneous (sequential) antiport mechanism. These results have been interpreted by assuming two separate and coordinated substrate translocation pathways, one in each monomer. The amino acid sequence of the OGC has been determined by cDNA sequencing and there is only one gene for this protein in cow, man and rat. The bovine OGC protein contains three cysteines: Cys184 located in TMS IV and Cys221 and Cys224 in TMS V. Mercurials and maleimides interact only with Cys184 of the purified and reconstituted OGC, whereas Cys221 and Cys224 are linked by a disulphide bridge. Furthermore, there is evidence that the two Cys184 residues of the two monomers are close to each other in the dimer structure (see  for a review). OGC was the first MC to be over-expressed in E. coli and refolded to a reconstitutively active state . This actually was the first time that a eukaryotic membrane protein had been expressed in E. coli and renatured.
Using the E. coli expression system, R90 and R98 in TMS II, R190 in TMS IV and R288 in TMS VI, mutated individually into Leu, were found to be essential for activity. A positive charge either from Arg or from Lys sufficed for transport activity at positions 90 and 288 but not at 98 and 190, where the guanidinium moiety of Arg was indispensable. Furthermore, a combination of Cys-scanning mutagenesis, chemical modification and spin-labelling studies identified a water-accessible face of OGC helix IV (containing the residues R190 and Q198 essential for the activity as well as the engineered single-Cys residues protected by oxoglutarate against reaction with SH reagents), and this face was suggested to line part of the substrate translocation pathway through this protein [49, 50]. Unlike other anion MCs, the OGC does not contain any negatively charged residue in the TMSs, excluding the possibility of interhelical stabilization by charge pairs. Therefore, either negatively charged residues located on one of the loops need to penetrate the core of the protein, or the negatively charged substrates are necessary to complement the positively charged amino acid residues.
OGC plays an important role in the malate-aspartate shuttle (Fig. 3H) and is also involved in the oxoglutarate-isocitrate shuttle (Fig. 3B), nitrogen metabolism and gluconeogenesis from lactate if the carbon skeleton for gluconeogenesis is provided by oxoglutarate exported from the mitochondria by the OGC.
Aspartate/glutamate carriers AGC1 and AGC2 (SCL25A12 and SLC25A13, respectively)
The recently identified human aspartate/glutamate carrier  has two isoforms AGC1 and AGC2 (see Table 1). These isoforms belong to a subfamily of Ca2+-binding MCs with a characteristic bipartite structure. Their C-terminal domains have the common sequence features of the MCF and their N-terminal domains harbour four EF-hand Ca2+-binding motifs. Like all the MC monomers, they traverse the i.m.m. with six transmembrane segments located in the C-terminal domains, whereas the N-terminal domains are exposed to the cytoplasmic surface of the i.m.m. AGC1 is found mainly in excitable tissues, whereas AGC2 is expressed abundantly in liver where AGC1 is absent.
In the reconstituted system, recombinant AGC1 and AGC2 transport aspartate, glutamate and cysteinesulphinate by an obligatory 1:1 exchange. Km values for external aspartate and glutamate are about 0.05 and 0.2 mM, respectively, i.e. virtually identical to those determined with natural AGC. Aspartate and cysteinesulphinate are transported as anions, whereas glutamate is cotransported with an H+. Therefore, the aspartate/glutamate and the cysteinesulphinate/glutamate exchanges are electrogenic, while the aspartate/cysteinesulphinate and all the homologous exchanges are not. For example, the aspartateOUT/glutamateIN and the glutamateOUT/aspartateIN exchanges are stimulated and decreased, respectively, when the membrane potential is positive inside. In energized mitochondria, the physiological direction of the exchange involves efflux of aspartate and entry of glutamate. The C-terminal domains of AGC1 and AGC2 account entirely for their activities and transport properties, whereas their N-terminal domains are responsible for their modulation by Ca2+.
The activity of AGC1 and AGC2 in transfected mammalian cells is stimulated by Ca2+ on the external side of the i.m.m. . This stimulation is not affected by ruthenium red, a specific inhibitor of the entry of Ca2+ into the mitochondria. Moreover, in Chinese hamster ovary (CHO) cells over-expressing AGC1 and AGC2 and triggered by Ca2+-mobilizing agonists, the synthesis of mitochondrial ATP is more pronounced than in control cells or in cells over-expressing the carrier C-terminal domains alone . In contrast, the intramitochondrial Ca2+ content is the same in control cells and in cells expressing the different AGC1 and AGC2 variants. These results demonstrate that AGC is regulated by cytosolic Ca2+ acting on the external side of the i.m.m.
AGC plays an important role in the malate-aspartate shuttle (Fig. 3H). This cycle transfers the reducing equivalents of NADH plus H+ from cytosol to mitochondria and is therefore indispensable for glycolysis. The main physiological alternative is the glycerol-3-phosphate cycle, which, however, is poorly active in some tissues such as the human liver. As well as for the malate-aspartate cycle, AGC is essential for the supply of aspartate from the mitochondria to the cytosol. It should be noted that the amount of aspartate taken up by liver cells from the blood is very low. Cytosolic aspartate is required, firstly, for urea synthesis from ammonia and from alanine and precisely for the reaction catalysed by argininosuccinate synthetase (ASS) (see Fig. 3E), secondly, for purine and pyrimidine syntheses, thirdly, for protein synthesis and, finally, for gluconeogenesis from lactate. Another function of the AGC is to import cysteinesulphinate (an intermediate of cysteine degradation to sulphate) in exchange for aspartate produced in the matrix by transamination with oxaloacetate.
Defects in the gene SLC25A13 coding AGC2 (the only AGC isoform present in liver) cause type-II citrullinaemia (CTLN2, OMIM 215700) . This autosomal recessive disease is characterized by hyperammonaemia and neuropsychiatric symptoms, often leading to rapid death. The onset is sudden, usually between the ages of 20 and 40. AGC2 deficiency can account for most of the symptoms of CTLN2. The lack of aspartate in the cytosol causes citrullinaemia, hyperammonaemia and, in some patients, hypoproteinaemia. Citrullinaemia and hyperammonaemia are also due to a unique feature of the disease, i.e. the liver-specific deficiency of ASS with no alterations to either its gene or mRNA. It is unclear how a defective AGC2 brings about the hepatic deficiency of ASS. It may be that critical aspartate levels are required to make ASS active, or that defects in AGC2 destabilize the enzyme. In addition, inhibition of the malate-aspartate shuttle increases the cytosolic NADH/NAD+ ratio. Consequently, glycolysis, alcohol metabolism and gluconeogenesis from reduced substrates are inhibited. Patients dislike carbohydrates, cannot drink alcohol, which often provokes the onset of the disease, and prefer high-protein diets, beans and peanuts (that contain high levels of aspartate and asparagine). Some patients with a high-carbohydrate diet show a marked increase in triglycerides. It is likely that in these patients the operation of the malate-citrate shuttle (Fig. 3D), which may in part compensate the deficiency of the malate-aspartate shuttle by transferring reducing equivalents to the mitochondria, produces acetyl-CoA in the cytosol and stimulates fatty acid synthesis. Furthermore, the accumulation of glycerol-3-phosphate in the cytosol, due to the low activity of the glycerophosphate cycle in human liver, favours the synthesis of triglycerides. Liver transplantation has proven to be a very effective treatment for CTLN2. Now that the function of AGC2 has been elucidated, the aim of future treatment should be to supply aspartate to liver cytosol and reduce the cytosolic NADH/NAD+ ratio. Hormones and peroxisomal proliferators might be used to activate the glycerophosphate NADH plus H+ shuttle.
Graves’ disease carrier GDC (SLC25A16)
The name Graves’ disease carrier derives from the fact that the GDC was cloned from a human thyroid cDNA expression library with the aid of sera of patients with Graves’ disease. This autoimmune disorder is characterized by the production of autoantibodies to the thyroid stimulating hormone receptor. However, GDC is most likely not involved directly in Graves’ disease, as the recombinant human GDC does not react with sera from patients and is, therefore, not a major antigen of the disease . Furthermore, GDC has also been cloned in the cow and found to be expressed not only in the thyroid but also in other tissues (see Table 1) . The yeast GDC (encoded by LEU5) and its human orthologue (encoded by SLC25A16) are thought to catalyse the import of either CoA or a precursor of CoA into the mitochondria . This conclusion is based on the following observations. Firstly, cells lacking LEU5 have reduced levels of CoA in their mitochondria; secondly, the activities of intramitochondrial CoA-dependent enzymes, such as α-isopropylmalate synthase, δ-aminolevulinate synthase and citrate synthase are decreased and, thirdly, after removal of the peroxisomal citrate synthase gene, ΔLeu5 cells are completely unable to synthesize citrate and do not grow on the non-fermentable carbon source glycerol. Unfortunately, except for the one involved in the first step, the locations of the enzymes participating in CoA biosynthesis are not yet known with certainty. Furthermore, direct transport assays on reconstituted yeast and/or human GDC are necessary to determine which substrate(s) are transported.
Adenine nucleotide carrier from peroxisomes ANC (SLC25A17)
Recently, ANC, encoded by SLC25A17 (see Table 1), has been proven to be the human homologue  of the yeast protein Ant1p, shown previously to be an adenine nucleotide transporter in the peroxisomal membrane . The transport properties of Ant1p, encoded by the ANT1 gene, which is essential for the growth of yeast on fatty acids, have been thoroughly investigated after expression and reconstitution into liposomes. It transports ATP, ADP, AMP and, less efficiently, the corresponding deoxynucleotides. The other nucleotides are not transported by Ant1p. Ant1p differs from the mitochondrial ADP/ATP carrier because it transports AMP and is unaffected by the ADP/ATP carrier inhibitors CATR and BKA. Furthermore, the Ant1p shares only 13–16% identity with the yeast ADP/ATP carrier isoforms. Since in yeast the β-oxidation of fatty acids is restricted to peroxisomes, the physiological role of Ant1p is to transport cytosolic ATP into the peroxisomal lumen (where it is required for the activation of fatty acids to acyl-CoA) in exchange for AMP generated in the same activation reaction. In man, ANC is probably required for the activation of branched-chain and very-long-chain fatty acids that are oxidized in peroxisomes (and not in mitochondria). It should be noted that Ant1p was the first functionally identified member of the MCF found to be located in a non-mitochondrial membrane, and the first peroxisomal protein proven to perform a transport function.
Glutamate carriers GC1 and GC2 (SLC25A22 and SLC25A18, respectively)
The human genes SLC25A18 and SLC25A22 (Table 1) were chosen as candidates for the glutamate carrier after screening eukaryotic databases with the sequences of the human aspartate/glutamate carrier isoforms. Their coding sequences (GC1 and GC2) 63% identical to each other, were expressed in E. coli and identified as isoforms of the glutamate carrier on the basis of their transport properties in reconstituted liposomes . Both isoforms catalyse the transport of l-glutamate either with H+ or in exchange for OH−, and do not accept structurally related compounds such as l-aspartate, l-glutamine and l-homocysteinesulphinate. However, they differ markedly in their kinetic parameters. GC1 has a very high Km for glutamate (4–5 mM), whereas the Km value of GC2 is low (about 0.2 mM), and the Vmax of GC1 is higher than that of GC2.
The main physiological role of GC isoforms is to import glutamate from the cytosol, where it is produced by transamination of amino acids with oxoglutarate, to the mitochondrial matrix, where glutamate dehydrogenase (GDH) is exclusively located (Fig. 3I). Although the Vmax for glutamate transport in intact mitochondria is lower than those of the other anion transporters, the rate is high enough to feed the urea cycle, even if glutamate were the only ammonium source for the mitochondrial synthesis of carbamoylphosphate. Since glutamate is cotransported with an H+ by GC and, therefore, its distribution across the mitochondrial membrane is dependent on ΔpH, the entry of glutamate is favoured in energized mitochondria. However, when ammonia rather than glutamate is the major nitrogen source for urea synthesis, or where glutamate is generated intramitochondrially, for example by proline oxidation, the GC may operate in the reverse direction to limit intramitochondrial accumulation of glutamate. Furthermore, prevention of glutamate efflux from mitochondria by cytosolic H+ in kidney tubular cells may play an important role in the kidney response to metabolic acidosis by contributing to increased flux through GDH and hence to increased ammonia production (and excretion) from glutamine . In the light of the differences in the kinetic parameters of the two isoforms and their ubiquitous distribution, it is likely that GC2 matches the basic requirement of all tissues especially with respect to amino acid degradation, and GC1 becomes operative to accommodate the higher demands associated with specific metabolic functions such as ureogenesis and/or with special metabolic conditions, for example after protein-rich diets. GC1 is more abundant than GC2 in all tissues (except brain) and particularly in liver. The impressive expression of GC1 in pancreas might be related to the proposed function of glutamate in insulin secretion as a glucose-derived intracellular messenger . It is thought that glutamate produced by GDH is exported by the GC from the mitochondria to the cytosol where it would act by sensitising the exocytotic process to Ca2+.
Deoxynucleotide carrier DNC (SLC25A19)
DNC (see Table 1) was first identified in man. Phylogenetic analysis of the sequences of all putative C. elegans MCs and of mammalian MCs of known function, disclosed a seven-protein subfamily, related to the ADP/ATP carrier, in C. elegans. By extending the sequence of a human expressed sequence tag (EST) encoding a protein fragment related to this subfamily, the sequence of a new carrier was completed and then identified, from its transport properties and kinetic characteristics, as the DNC . Thus, the recombinant and reconstituted DNC transports deoxynucleoside diphosphates (dNDPs) and less efficiently deoxynucleoside triphosphates (dNTPs), in exchange for ATP or ADP. It does not transport deoxynucleoside monophosphates, nucleoside monophosphates, nucleosides, purines and pyrimidines. Since ribonucleotide reductase is located only in the cytosol of eukaryotic cells, the function of the DNC is to supply deoxynucleotides for mtDNA synthesis (Fig. 3J). Once in the mitochondria, dNDPs are converted into the corresponding triphosphates and incorporated into the mtDNA by DNA γ-polymerase. Interestingly, the DNC can transport dideoxynucleotides more efficiently than the corresponding deoxy analogues. This property suggests that the DNC is involved directly in the cytotoxicity of antiviral nucleoside analogues such as 2′,3′-dideoxycytidine, 2′,3′-dideoxyinosine and 3′-azido-3′-deoxythymidine (AZT). Cytoplasmic kinases convert dideoxynucleosides into their mono-, di- and triphosphate derivatives. The latter two products are transported by the DNC into mitochondria where they inhibit the synthesis of mtDNA by competing for the active site of DNA γ-polymerase and by chain termination. It should be noted that the antiviral nucleotide analogues interfere strongly with the action of viral reverse transcriptases and mitochondrial DNA γ-polymerase, but have a very low affinity for nuclear DNA polymerases. In the future, it should be possible to assess the potential toxicity of nucleotide analogues and develop less toxic antiviral compounds by studying their transport in liposomes reconstituted with the human DNC.
DNC deficiency causes Amish microcephaly (MCPHA, OMIM 607196). This disorder has been observed in Old Order Amish families and segregates as an autosomal recessive disorder with an unusually high incidence of at least 1 in 500 births. Affected individuals are characterized by head circumferences 6–12 standard deviations smaller than the population mean, immature brain similar to that of a 20-week fetus and premature death. All MCPHA patients studied so far exhibited a homozygous point mutation 530G→C in DNC coding sequence, which results in a Gly→Ala change at residue 177 and loss of transport activity .
Carnitine/acylcarnitine carrier CAC (SLC25A20)
CAC (see Table 1) is the key component in the carnitine cycle, which shuttles long-chain fatty acids from the outside to the inside of the mitochondria in three steps: (a) transfer of acyl groups from acyl-CoA to carnitine by the outer mitochondrial membrane carnitine palmitoyltransferase (CPT I), (b) translocation by the CAC of acylcarnitines across the i.m.m. in exchange for free carnitine and (c) transfer of acyl groups from acylcarnitines to CoA inside the mitochondria by CPT II (see Fig. 3K). More precisely, the CAC catalyses the electroneutral exchange of cytosolic acylcarnitine for mitochondrial carnitine (Fig. 3K). This allows the import of fatty acyl moieties into the mitochondria, where they are oxidized by the enzymes of the β-oxidation pathway. CAC-mediated transport is therefore an essential step in long-chain fatty acid β-oxidation, which provides the major source of energy during prolonged fasting as well as for cardiac and skeletal muscle during exercise. Besides the carnitine/acylcarnitine exchange, CAC can facilitate the unidirectional transport of substrates across the membrane, although less efficiently. Physiologically, a net flux of carnitine may occur into carnitine-depleted mitochondria to equilibrate the matrix level of carnitine with that present in the cytosol. CAC has been purified and reconstituted, kinetically characterized, cloned and over-expressed in E. coli and yeast. It has a higher affinity for long-chain than for short-chain acylcarnitines and the lowest affinity for free carnitine (see  and references therein). It is inhibited by sulphobetaines. The cDNA sequences of man, rat and yeast are known. At variance with the mammalian CAC, the yeast orthologue transports acetylcarnitine and, to a far lesser extent, medium- and long-chain derivatives, consistent with the fact that in yeast fatty acid β-oxidation is restricted to peroxisomes.
More than 30 patients with CAC deficiency (OMIM 212138) have been identified to date. The overall β-oxidation rate of long-chain fatty acids and the activity of the CAC in cultured skin fibroblasts are drastically reduced, whereas the activities of the β-oxidation enzymes as well as of CPT I and CPT II are normal. Two phenotypes for this autosomal recessive disorder can be distinguished: severe, neonatal onset with cardiomyopathy (no survivors) and a milder phenotype with hypoglycaemia but no cardiomyopathy. As in the other fatty acid-oxidation disorders, the main features of this disease are episodes of life-threatening coma provoked by fasting, hypoketosis, hypoglycaemia, hyperammonaemia, variable dicarboxylic aciduria, fatty hepatomegaly with abnormal liver function, various cardiac symptoms and skeletal muscle weakness. Hypoglycaemia during fasting is due to rapid depletion of hepatic glycogen and hampered gluconeogenesis. Hypoketosis results from deficient fatty acid transport and consequent lower production of acetyl-CoA. Hyperammonaemia is caused by the limited availability of N-acetylglutamate (derived from acetyl-CoA), essential for the urea cycle enzyme carbamoylphosphate synthase. Hepatomegaly and cardiomyopathy are mainly due to fatty accumulation. CAC deficiency can be distinguished from the other fatty acid-oxidation defects by elevated plasma long-chain acylcarnitines, paucity of fatty acid metabolites in urine, consistent with a defect in the carnitine cycle rather than in the β-oxidation pathway, and a normal ketone response after administration of short-chain triglycerides. Short-chain fatty acids can in fact enter the mitochondria via a still unknown mechanism independently of the CAC. The numerous mutations found in the patients’ CAC gene (SLC25A20)  cause loss of transport activity, as shown by transport assays in the reconstituted system and by complementation of S. cerevisiae or Aspergillus nidulans deleted strains (see  and references therein). In some patients, coding sequence mutations have been shown to increase the amount of aberrant mRNA splicing and exon skipping, causing absence of normal transcripts. Care should be taken to prevent long periods of fasting. In case of coma episodes, intravenous glucose is appropriate to revive the patients and stimulate insulin secretion that will inhibit fatty acid oxidation and adipose-tissue lipolysis. The diet should be low in long-chain fatty acid and high in carbohydrates and medium-chain fatty acids.
Oxodicarboxylate carrier ODC (SLC25A21)
The identification of the human ODC (Table 1) was based on two previously identified isoforms, ODC1 and ODC2, encoded in the genome of S. cerevisiae, that transport C5–C7 oxodicarboxylates across the i.m.m. by a strict counter-exchange mechanism. Orthologues from C. elegans and D. melanogaster were used to bridge between yeast and man. The transport characteristics of the recombinant, reconstituted human ODC differ markedly from those of the bovine OGC. The latter has greatest affinities for C4- and C5-oxodicarboxylates and dicarboxylates, whereas the human ODC prefers the C5–C7 homologues. Furthermore, unlike the OGC, the human ODC does not transport malate, succinate and maleate, but it transports 2-aminoadipate and citrate, although with a low efficiency. The best substrates for the human ODC are 2-oxoadipate and 2-oxoglutarate . Therefore, the main physiological function of the human ODC is probably to catalyse the uptake of 2-oxoadipate into the mitochondria (where oxoadipate dehydrogenase is located), thus playing a central role in the catabolism of lysine, hydroxylysine and tryptophan (Fig. 3L). The probable counter-substrate for the ODC-mediated uptake of 2-oxoadipate is 2-oxoglutarate. The efflux of 2-oxoglutarate is required by lysine-oxoglutarate reductase that, in the first step of lysine catabolism, converts lysine and 2-oxoglutarate into saccharopine. Another possible role for the human ODC may be to catalyse the uptake of 2-aminoadipate into the mitochondrial matrix when this amino acid is not rapidly transaminated to 2-oxoadipate in the cytosol, for example after diets rich in amino acids which cause a decrease in 2-oxoglutarate content in the cytosol and consequently inhibit 2-aminoadipate aminotransferase. In this respect it is worth mentioning that transaminases interconverting oxoadipate and aminoadipate are present both in the cytosol and inside the mitochondria.
The physiological role of the ODC suggests its possible involvement in 2-oxoadipate acidaemia (OMIM 204750), which is accompanied by accumulation and excretion of large amounts of 2-oxoadipate, 2-aminoadipate and 2-hydroxyadipate in urine, and by the clinical symptoms of mental retardation, hypotonia, motor and developmental delay, cerebellar ataxia and learning disability (see  for references). The molecular defect(s) responsible for this disease have not been characterized. Fibroblasts of patients with this condition are unable to oxidize 2-amino(1-14C)adipic and 2-oxo(1-14C)adipic acid to 14CO2 to any significant extent. It has thus been suggested that the disease may be due to defective 2-oxoadipate dehydrogenase, but no such defect has ever been demonstrated. The alternative possibility that defective ODC might provide the basis for this human metabolic disease can now be investigated.
Missing SLC25 (MC) family members
In addition to the SLC25 members discussed above, there are many other genes encoding MCF members of unknown function. The S. cerevisiae genome contains 35 putative MCF members and the Arabidopsis thaliana genome 58 putative members. Among the “missing” MCs, it should be noted that important transport activities observed in intact mitochondria (such as pyruvate-H+ symport, transport of S-adenosylmethionine, exchange between ATP-Mg and Pi, transport of glutamine) have yet to be associated with specific proteins. It is likely that, in the near future, the over-expression/functional reconstitution strategy, devised and employed extensively in our laboratory, will lead to a further substantial extension of the SLC25 members.
Other mitochondrial transporters
As far as is currently known, SLC25 (MCF) members transport a variety of solutes across the i.m.m., including nucleotides, coenzymes and cationic substrates. This membrane also shuttles inorganic cations between matrix and cytosol and it cannot be excluded in principle that the proteins responsible for these fluxes are also members of the MCF. The following “inorganic cation” transporters and/or channels have been investigated extensively in studies with intact mitochondria: the Ca2+ uniporter that catalyses the uptake of Ca2+ into mitochondria and is driven by the electrical membrane potential; the Na+/Ca2+ and Na+/H+ antiporters that catalyse the efflux of Ca2+ and Na+, respectively; the K+ channel that catalyses influx of K+, is inhibited by ATP and sulphonylureas and activated by agents known to open the KATP channel of the plasma membranes; and the K+/H+ antiporter that regulates K+ efflux at rates that balance K+ influx. The main function of the mitochondrial K+ cycle is to regulate the volume of the mitochondria and hence maintain the integrity of their inner membrane which is essential for oxidative phosphorylation and other processes. The physiological role of the mitochondrial Ca2+ cycle is to regulate the matrix Ca2+ increase that activates three matrix dehydrogenases and ultimately makes more ATP available for cellular work. In addition, a Mg2+ uniport and a Mg2+/H+ antiporter catalysing influx and efflux of Mg2+, respectively, across the i.m.m. have been proposed to regulate the homeostasis of Mg2+ and some enzyme activities. Some of the metal ion transporters have been purified , but their sequences and genes are not yet known. Quite recently, the yeast Mrs2p has been demonstrated to be an essential component of the major electrophoretic Mg2+ influx system in mitochondria . This protein is unrelated to the SLC25 members and belongs to the Mg2+ transport protein family comprising CorA in bacteria and Alr1p in the plasma membrane of lower eukaryotes. In the light of this important finding it may be hypothesized that the metal ion transporters/channels will not fall within the MCF.
In man, at least four ATP binding cassette (ABC) transporters are present in the i.m.m. (see  for a review). Two of them, ABC7 and MT-ABC3, are homologous to the yeast Atm1p, whereas the other two transporters (M-ABC1 and M-ABC2) are homologous to the yeast Mdl1p/Mdl2p. ABC7 and MT-ABC3 have an important role in mitochondrial iron homeostasis and in the Fe/S cluster formation. Mutations in ABC7 are responsible for the X-linked sideroblastic anaemia and cerebellar ataxia (OMIM 301310). MT-ABC3 maps in the vicinity of the locus for lethal neonatal metabolic syndrome (OMIM 603358), a disorder of mitochondrial function associated with altered iron metabolism. The function of M-ABC1 and M-ABC2 has not yet known. However, their murine homologue (ABC-me) probably transports intermediates of haem biosynthesis across the i.m.m. in erythroid tissues .
In summary, at least four autosomal recessive diseases are caused by defects in specific mitochondrial carrier genes. Mutations in the gene SLC25A13 coding isoform 2 of the aspartate/glutamate carrier (the only isoform present in liver) cause type II citrullinaemia (OMIM 215700). The hyperornithine-hyperammone-homocitrulline-mia (HHH) syndrome (OMIM 238970) is caused by alterations of the ornithine carrier (isoform 1, gene SLC25A15) that catalyses the exchange between cytosolic ornithine and mitochondrial citrulline, an important step in the urea cycle. Defects in the gene SLC25A20 coding the carnitine/acylcarnitine carrier are responsible for the Stanley syndrome (OMIM 212138). Amish microcephaly (OMIM 607196) is associated with mutant deoxynucleotide carrier (gene SLC25A19). In addition, one of the three distinct loci involved in autosomal dominant progressive external ophthalmoplegia (adPEO) (OMIM 157640) includes the heart- and skeletal muscle-specific isoform of the ADP/ATP carrier (gene SLC25A4). A deficiency of the ADP/ATP carrier protein in skeletal and heart muscle has been shown, both immunologically and by transport assays, in patients affected by Sengers syndrome (OMIM 103220). Hemizygosity of the citrate carrier in the DiGeorge and velo-cardio-facial syndromes is thought to contribute to the mental deficiency of the patients. Finally, although the molecular defect(s) responsible for 2-oxoadipate acidaemia (OMIM 204750) have not yet been characterized, this disease has been suggested to be due to defective oxodicarboxylate carrier (or to defective oxoadipate dehydrogenase).
Research in the author’s laboratory was supported by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR), the Italian Consiglio Nazionale delle Ricerche (CNR), Centre of Excellence “Genomics: genes involved in pathopysiological processes in the biomedical and agricultural fields” (CEGBA), and the European Social Fund.