The flux of a variety of metabolites, nucleotides and coenzymes across the inner membrane of mitochondria is catalysed by a nuclear-coded superfamily of secondary transport proteins called MCs (mitochondrial carriers). The importance of MCs is demonstrated by their wide distribution in all eukaryotes, their role in numerous metabolic pathways and cell functions, and the identification of several diseases caused by alterations of their genes. MCs can easily be recognized in databases thanks to their striking sequence features. Until now, 22 MC subfamilies, which are well conserved throughout evolution, have been functionally characterized, mainly by transport assays upon heterologous gene expression, purification and reconstitution into liposomes. Given the significant sequence conservation, it is thought that all MCs use the same basic transport mechanism, although they exhibit different modes of transport and driving forces and their substrates vary in nature and size. Based on substrate specificity, sequence conservation and carrier homology models, progress has recently been made in understanding the transport mechanism of MCs by new insights concerning the existence of a substrate-binding site in the carrier cavity, of cytosolic and matrix gates and conserved proline and glycine residues in each of the six transmembrane α-helices. These structural properties are believed to play an important role in the conformational changes required for substrate translocation.
Mitochondria are essential not only for the metabolic pathways that take place within these organelles, but also for many others occurring mainly outside the mitochondrial matrix. Because of enzyme compartmentalization, many metabolites produced outside the mitochondria must enter; for example, ADP and phosphate (Pi) for oxidative phosphorylation, substrates of the tricarboxylic acid cycle, fatty acid oxidation, mitochondrial replication and repair of DNA, mitochondrial RNA and protein synthesis, and amino acid degradation, coproporphyrinogen III and iron for haem biosynthesis, group donors (S-adenosylmethionine and folate) and coenzymes [NAD+, FAD, ThPP (thiamine pyrophosphate) and coenzyme A]. Likewise, other metabolites produced inside the mitochondrial matrix must exit the organelles; for instance, ATP, citrate, malate, phosphopyruvate, δ-aminolevulinic acid (the product of the first step in haem biosynthesis), citrulline and ketone bodies (acetoacetate). Besides metabolites, a variety of cations such as Ca2+, Mg2+, Na+ and K+ must cross the inner mitochondrial membrane to regulate enzyme activity and the volume of mitochondria.
The mitochondrion is surrounded by an outer and inner membrane. While the outer membrane is permeable to solutes with a molecular mass ≤ 4–5 kDa, the inner membrane is very impermeable. Only some uncharged molecules such as O2 and CO2 pass through. The passage of hydrophilic compounds across the inner mitochondrial membrane is catalysed mainly by a superfamily of nuclear-coded proteins known as the MCF [MC (mitochondrial carrier) family]. Many MCs have isoforms encoded by different genes. So far, the mammalian Pi carrier (known as PiC) is the only known member of the MCF with two isoforms generated by alternative splicing; however, other isoforms of this type may exist. The distribution of MCs in tissues varies considerably; some are present in virtually all tissues, whereas others are tissue-specific, reflecting their importance for special functions. The substrates transported by MCs are mostly negatively charged, but some are positively charged or zwitterions at physiological pH values and greatly vary in structure and size, from H+ to NAD+. Furthermore, some carriers exert flux control on metabolic pathways. All family members have common structural features; their primary structures consist of three tandemly repeated homologous domains of approximately 100 amino acids in length, and each repeat contains two hydrophobic segments (spanning the membrane as α-helices) and a characteristic sequence motif PX[D/E]XX[K/R]X[K/R] (20–30 residues) [D/E]GXXXX[W/Y/F][K/R]G. Notably, the Ca2+-binding aspartate/glutamate and ATP-Mg/Pi carriers have an additional extensive N-terminal domain which fulfils a regulatory (non-transport) function. MC sequence features are different from those of any other known transporter family. They have probably arisen after mitochondria had appeared in eukaryotes. Sequence conservation and intron position suggest that they may have evolved from two-tandem gene duplications of an ancestral gene encoding the 100 amino acid repeat, and the repeat itself may have evolved by duplication of a DNA sequence encoding a single transmembrane segment. Interestingly, the MCF is not restricted to mitochondria, since some members are localized in other cell organelles, such as peroxisomes and chloroplasts.
The present chapter attempts to summarize what is currently known about the biochemical and molecular characterization of MCs. For detailed reviews of prior literature on MCs, the reader is referred to references [1–14].
The function of mitochondrial carriers
The MCF is defined by the sequence features of its members: a tripartite structure, a three-fold repeated signature motif and six transmembrane α-helices. The term MCF was established after identifying its first members which were all purified from mitochondria. In the post-genomic era, with the availability of DNA databases, the MCF was extended by searching for MCs of unknown function. 58 MCs are encoded by the genome of Arabidopsis thaliana, 53 by that of Homo sapiens and 35 by the Saccharomyces cerevisiae genome. Indeed, the MCF is the largest of the known solute carrier families.
Figure 1 shows all MCs of S. cerevisiae and H. sapiens deduced from genomic analysis. Approximately half of these carriers have now been characterized in terms of substrate specificity and kinetic parameters by transport assays upon gene expression, purification and reconstitution into liposomes. This procedure was first employed for the bacterial overproduction and functional reconstitution of the bovine oxoglutarate carrier, which was indeed the first eukaryotic membrane protein to be expressed in Escherichia coli and refolded . In addition, for some of the carriers characterized biochemically, subcellular localization, tissue distribution and their physiological role in cell metabolism and specialized cell functions have been determined. Table 1 lists the main MC subfamilies identified so far according to their substrate specificity. Notably, some substrates, and even certain defining substrates, are transported by more than one subfamily. Generally, MC subfamilies are widespread in the eukaryotic kingdom indicating that they play a role that has been conserved throughout the evolutionary process. There are, however, some exceptions; for example, the GTP/GDP carrier subfamily seems to be specific to fungi and protista. Moreover, MC subfamilies are characterized by specific triplets (Figure 3 and the section below on ‘Structure’) besides the substrates transported.
Modes of transport and driving forces
MCs transport metabolites across the inner mitochondrial membrane in a highly controlled manner, since it is essential for the H+ electrochemical potential gradient (generated by the respiratory chain) to be preserved across this membrane. Many MC subfamilies catalyse an obligatory 1:1 exchange (antiport) reaction between substrates (Figure 2). Some carry out unidirectional substrate transport (uniport) as the exclusive transport mode [e.g. H+ transport by the UCP (uncoupling protein)] and others a slow uniport besides antiport (e.g. the carnitine-acylcarnitine carrier).
Concerning the electrical nature of their transport reactions, MCs are either electrophoretic (electrogenic) or electroneutral (Figure 2). To date, three well-characterized subfamilies are known to be electrophoretic. The ADP/ATP carrier and aspartate/glutamate carrier subfamilies catalyse exchanges across the mitochondrial membrane that result in charge imbalances because they transport ADP3− for ATP4− and glutamate− plus an H+ for aspartate−, respectively. The third subfamily is the UCP mentioned above. Electroneutral balance can be achieved by co-transport (symport) and countertransport of solutes and by uniport of electroneutral metabolites (Figure 2). In some cases electroneutrality is imposed by simultaneous carrier-mediated H+ movement (Figure 2). The carrier subfamilies for Pi and glutamate, and in yeast for oxaloacetate, mediate the transport of anions together with an equivalent amount of H+ (anion/H+ symport). Moreover, the human ornithine carrier can transport ornithine+ against citrulline plus an H+, the yeast GTP/GDP carrier exchanges GTP4− plus an H+ against GDP3−, and the tricarboxylate (citrate) carrier citrate2− plus an H+ against malate2−. Other subfamilies catalyse an exchange of anions or cations. The oxoglutarate carrier, for instance, exchanges oxoglutarate2− for malate2− and the ornithine carrier ornithine+ for lysine+, arginine+ or an H+.
As their driving force MCs utilize either the electrical or the chemical component, or both, of the H+ electrochemical potential gradient across the inner mitochondrial membrane and/or the concentration gradient of the solutes (Figure 2). Because the electrical component of the protonmotive force is rather high, the electrical nature of the ADP/ATP and aspartate/glutamate carriers provides a powerful means of ejecting ATP4− and aspartate− against the concentration gradient from the mitochondrial matrix to the cytosol. In the case 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 Pi2− (by the PiC) or of glutamate− (by the glutamate carrier) is stimulated, as is also the efflux of cationic solutes such as export of ornithine+ in exchange for an H+ (by the ornithine carrier).
A breakthrough was achieved in 2003 when the atomic structure of the bovine carboxyatractyloside-inhibited ADP/ATP carrier was solved to 2.2 Å (1 Å = 0.1 nm) . Basically, this structure is composed of six transmembrane α-helices (H1–H6) lining a funnel-shaped cavity (occupied by the inhibitor) which is open towards the cytosol and closed on the matrix side by a salt-bridge network. This network is formed by the charged residues of the first part of the three signature motifs, PX[D/E]XX[R/K], which are located at the C-terminus of the odd transmembrane α-helices. The crystal structure of the ADP/ATP carrier–carboxyatractyloside complex has proven previous data and hypotheses, i.e. that the above-mentioned network constitutes the closed gate of monomeric MCs in the ‘c’ (cytosolic)-state , that carriers possess a three-fold pseudosymmetry based on the three-fold sequence repeats  and on electron microscopy observations  and display six transmembrane α-helices with the N- and C-termini exposed toward the cytosolic side of the membrane ( and references therein). Moreover, the three-dimensional structure of the ADP/ATP carrier greatly aided the interpretation of experimental results (e.g. site-directed mutagenesis ) and stimulated further research (e.g. in the monomeric/dimeric state of MCs ).
Unfortunately, the difficulties in crystallizing uninhibited MCs have prevented further progress in defining their structure and, in particular, their conformation in the ‘m’ (matrix)-state. However, important findings have been recently obtained by multiple sequence alignment analysis of MCs of known function (substrate specificity) as an extension of the early and fundamental discovery of the three-fold repeated signature motif in the primary structure of the ADP/ATP carrier . The first of these recent findings regards the existence of a common, or a similarly located, substrate-binding site . Thus Robinson and Kunji  proposed three contact points, corresponding to residues of the three even-numbered transmembrane α-helices, that protrude into the cavity at approximately the midpoint of the membrane one-and-a-half helix turns above the matrix gate. For example, point II on helix 4 is defined by G[IVLMT] in nucleotide carriers, by R[QHNTV] in carboxylic acid carriers and by R[D/E] in amino acid carriers. Furthermore, on the basis of inter-repeat multiple sequence alignment of MCs of known function, Robinson et al.  found that specific triplets protruding into the carrier cavity are distinct features of the various MC subfamilies. These triplets are either asymmetrical or symmetrical. The asymmetrical ones (i.e. aligned residues of each carrier formed by different amino acids) are important for substrate binding. The amino acid pair located at each contact point (I, II and III) are typical asymmetrical triplets (numbers 80 and 81 in Figure 3). Other asymmetrical triplets are, for instance, 84 and 88 in the ATP-Mg/Pi carrier subfamily and 84, 85 and 88 in the aspartate/glutamate and glutamate carrier subfamilies. In analysing the symmetrical triplets (i.e. formed by three identical amino acids), Robinson et al.  revealed the existence of two triplets (93 and 96, which are the charged residues of the motif [F/Y][D/E]XX[R/K]; Figure 3) located at the C-terminus of the even-numbered transmembrane α-helices. These triplets would form a salt-bridge network on the cytosolic side which would close the carrier in the m-state constituting the cytosolic gate of MCs.
Interhelical multiple sequence alignment showed the presence of well-conserved proline and glycine residues in both odd- and even-numbered transmembrane α-helices (see triplets 28 and 83 for proline and 19 and 28 for glycine; Figure 3). The proline and glycine residues of the even helices are aligned with the proline and glycine residues of the odd helices in an antiparallel fashion (Figure 2 of ). Of note, in both the odd and even helices, a proline or a glycine residue is located above (one helix turn) and below (one-and-a-half helix turns) the residues of the common substrate-binding site. In addition, the proline residues of the odd helices and the proline residues of the even helices are located above (one-half helix turn) and below (two-and-a-half helix turns) the matrix and cytosolic gates, respectively. It has been proposed that the glycine and proline residues of the even helices and the glycine residues of the odd helices act as hinges to open or close the carrier on the matrix or cytosolic side , analogous to what has been asserted for the proline residues of the odd helices .
As schematically represented in Figure 4, MCs possess a central cavity, in which the substrate is bound, and two gates alternately open on the cytosolic or the matrix side. When the matrix gate is closed and the c-gate is opened (c-state), the substrate is released toward the cytosol and another substrate enters. As the substrate binds to the carrier, the protein rearranges until the transition state is reached in which a maximum of interactions between the protein and the substrate take place, in agreement with the ‘induced transition fit’ of carrier catalysis . In the transition state (i) the substrate is bound at the centre of the carrier to residues located at the level of the common binding site and to others above and below, according to its size and shape, and (ii) the carrier is almost entirely closed on either side of the membrane (Figure 4). The binding energy of the optimum fit interactions between the carrier and the substrate in the transition state triggers additional structural changes leading to the matrix conformation (in which the c-gate is closed and the m-gate is opened) (Figure 4). At this stage the substrate, which entered the carrier from the cytosolic side, exits into the matrix and the cycle continues with the entry of another substrate from the matrix (Figure 4). In summary, during the transition from the c- to the m-state, on the matrix side the even and odd transmembrane helices move apart and the matrix gate breaks; on the cytosolic side the helices come together to close the c-gate. The opposite occurs during the transition from the m- to the c-state.
The conformational changes occurring during the transition from the c- to the m-state, and vice versa, are still unknown. Recently, we have proposed that the conformational changes are caused by a tilt of the even and odd helices and by the ability of proline and glycine residues of both the even and odd helices (mentioned in the ‘Structure’ section) to kink or swivel towards the cavity axis . Specifically, after the substrate enters the carrier in the c-state and is bound in the transition state, a tilt of the even and odd helices takes place, which would be seen from the cytosol as occurring clockwise from the c- to the m-state and counter-clockwise during the m- to c-state. Consequently, the [F/Y][D/E]XX[K/R] portions of the even helices are brought together by a swivel or kink at the level of the proline residues of these helices (and the c-gate closes) while the N-termini of the odd helices are rotated behind the c-gate by the kink or swivel of the glycine residues of these helices. Analogously, after the substrate enters the carrier in the m-state and is bound in the transition state, the PX[D/E]XX[K/R] segments are brought together by a kink of the proline residues in the odd helices (forming the m-gate), while the N-termini of the even helices are rotated behind the m-gate, as seen in the crystal structure of the carboxyatractyloside- inhibited ADP/ATP carrier . The above-reported mechanism describes the MC-mediated antiport mode of transport. However, some carriers are able to undergo a reversible transition between the c-state and the m-state in the absence of substrate. Therefore they catalyse uniport, besides antiport, although at a lower rate. This means that the activation energy barrier of the transition between the two states of these carriers is much lower than that of strict 1:1 exchange carriers.
Regulation of mitochondrial carrier activity
MCs are generally present in minute amounts and must ensure a sufficient rate of flux to fulfil the needs of the respective metabolic pathways. Thus their activity has to be adapted to different tissues under various physiological conditions and in varied metabolic and energetic states. Carrier activity can be regulated in a number of ways. (i) Modulation of driving forces, kinetic parameters and concentrations of the substrate transported, counter-substrate and competing substrates. The relevance of the kinetic parameters of carrier isoforms can be exemplified by the rate of phosphate import into mitochondria (controlling the rate of ATP production by oxidative phosphorylation). Thus during muscle contraction, the capacity of the ubiquitous PiC-B, which has a higher affinity for Pi, is overwhelmed and the muscle-/heart-specific PiC-A, with its lower substrate affinity, becomes operative with increased concentrations of cytosolic Pi. (ii) Interaction with allosteric inhibitors or activators. The ornithine carrier, for example, is inhibited by spermine and spermidine and stimulated by malate and phosphate, and the aspartate/glutamate and ATP-Mg/Pi carriers are activated by Ca2+. In this mechanism of Ca2+ signal transduction, Ca2+ exerts metabolic effects by reacting with the N-terminal domains of these transporters without entering the organelles. (iii) Modulation of carrier gene expression at the transcriptional or translational level as shown, for example, for the citrate carrier.
In recent years, the rapid advance in the identification of MC function has led to the disclosure of several diseases associated with defective carriers ( for a review; [26,27]). These disorders are rare errors of metabolism caused by alterations of nuclear genes encoding MCs. Their symptoms correlate with the metabolism affected and its significance in tissues. The 11 known MC-related diseases are listed in Table 2; they are inherited in an autosomal recessive manner, except for adPEO (autosomal dominant progressive external ophthalmoplegia).
To obtain information on the structure and function of MC proteins, extensive site-directed mutagenesis of several carriers has been performed. The results indicate that a relatively limited number of amino acid substitutions affect transport activity. Notably, nearly all amino acid substitutions causing loss of function and the missense mutations found in patients with MC-related diseases are located in areas of carrier structure that are key for their function, i.e. carrier cavity surface corresponding to the substrate-binding site, matrix and cytosolic gates, and proline-glycine areas of the transmembrane α-helices .
Carrier import into mitochondria
MCs are synthesized on cytosolic ribosomes and must be imported into mitochondria. Based on data obtained using the ADP/ATP carrier, a general model of MC import has been proposed . Unlike most nuclear-coded mitochondrial proteins, MCF members contain targeting information in their mature sequence. Several short targeting sequences are distributed over the MCF sequence, and it is currently believed that recognition by the mitochondrial import machinery involves the simultaneous binding of several signals . A few MCs possess cleavable presequences which are not essential for targeting. Of late, it has been found that the presequences of the Pi and citrate carriers improve import through different mechanisms, by providing a binding site for the chaperone Hsc70 (heat-shock cognate 70 stress protein) (Pi carrier) and by acting as an internal chaperone (citrate carrier) .
Despite the substantial effort of many laboratories and the significant progress achieved in the field, as described herein, further research is still needed to complete our understanding of mitochondrial metabolite transport.
One obvious goal in the study of MCs is the identification of the substrate specificity of family members that have remained functionally unidentified. Notably, many important transport activities across the mitochondrial membrane, such as of pyruvate, acetoacetate, α-oxo acids of branched-chain amino acids, glutamine, choline, γ-aminobutyric acid, N-acetyl glutamate and several cations, have yet to be associated with specific proteins; it may be that proteins other than MCs are responsible for some of these activities. This is very likely to be the case for cations, since Mg2+ has been found to be translocated by a member of the Mg2+ transporter family comprising CorA in bacteria and Alr1p in the plasma membrane of lower eukaryotes. Another important aspect of MCs that warrants further in-depth investigation is their physiological role in cell metabolism and special functions. The recent findings that citrate carrier plays a role in insulin secretion and histone acetylation demonstrate that this area of research is expanding and may reveal relevant data.
The determination of the crystal structure of the ADP/ATP carrier (roughly corresponding to the c-conformation) and the exciting novel insights into the MC structural properties reported herein allow us to build a realistic schematic model of the MC-catalysed transport mechanism. However, the molecular details of substrate translocation through MCs will be elucidated when the difficulties in crystallizing the highly hydrophobic MC proteins are overcome and the three-dimensional structure of uninhibited carriers in various conformations (including the m-state) becomes available.
The ongoing functional identification of other MCF members will help detect other carrier-related diseases and to comprehend the molecular basis of their symptoms. Understanding their pathogenetic mechanism will probably lead to new therapeutic approaches. For example, patients affected by carnitine-acylcarnitine carrier deficiency with some residual carrier activity and a mild phenotype might benefit from treatment with statins and/or fibrates acting via stimulation of carrier gene expression. Another interesting finding concerns the potential significance of aspartate/glutamate carrier gene polymorphisms in autism. Therefore the possible role of MC gene polymorphisms in complex disorders is a promising area for future research. Lastly, carriers that are specific to fungi and/or protista (e.g. the GTP/GDP carrier subfamily) may be suitable targets for novel drugs effective against pathogenic fungi and protista of H. sapiens (e.g. Candida albicans and Trypanosoma brucei) and plants (e.g. Gibberella zeae).
• MCs are nuclear-coded proteins that transport numerous metabolites across the mitochondrial matrix. To date, 22 MC subfamilies have been characterized according to their substrate specificity.
• Important recent findings concern the existence of a similarly located substrate-binding site, a cytosolic gate (besides the known matrix gate) and of conserved proline and glycine residues in the odd and even transmembrane α-helices.
• The conformational changes occurring after substrate binding and leading to substrate translocation involve a tilt of even and odd transmembrane α-helices and a kink/swivel of the helix proline and glycine pairs that are strategically located between the substrate-binding site in the centre of the carrier and each of the two gates.
• MC activity is regulated in different ways to meet the demands of the respective metabolic pathways.
• Several diseases are caused by alterations of nuclear genes encoding specific MCs.
We thank the Ministero dell’Università e della Ricerca (MIUR), the Center of Excellence in Genomics (CEGBA) and the Italian Human ProteomeNet no. RBRN07BMCT_009 for supporting our work.This chapter was received on 24 October 2009 and accepted on 23 November 2009.
- © The Authors Journal compilation © 2010 Biochemical Society