2.A.29 The Mitochondrial Carrier (MC) Family

Permeases of the MC family (the human SLC25 family) possess six transmembrane α-helical spanners. The proteins are of fairly uniform size of about 300 residues. They arose by tandem intragenic triplication in which a genetic element encoding two spanners gave rise to one encoding six spanners (Palmieri 2012; Wang et al. 2016). This event may have occurred less than 2 billion years ago when mitochondria first developed their specialized endosymbiotic functions within eukaryotic cells. Members of the family are found exclusively in eukaryotic organelles although they are nuclearly encoded. Most are found in mitochondria, but some are found in peroxisomes of animals, in hydrogenosomes of anaerobic fungi, and in amyloplasts of plants. Members of the MC family are functional and structural monomers although early reports indicated that they are dimers (Bamber et al., 2006, 2007). Many of them preferentially catalyze the exchange of one solute for another (antiport). Fifteen paralogues of the MC family are encoded within the genome of Saccharomyces cerevisiae. Fifty have been identified in humans. 58 in A. thaliana and 35 in S. cerevisiae. The functions of many of the human homologues are unknown, but most of the yeast homologues have been functionally identified. Functional aspects have been reviewed by Palmieri (2004), Palmieri et al. (2006) and Plamieri and Pierri (2010).  Diseases caused by defects of mitochondrial carriers are reviewed by Palmieri et al. (2008) and by Gutiérrez-Aguilar and Baines 2013. Residues involved in substrate binding in the middle of the transporter and gating have been identified and analyzed (Monné et al. 2013).  The physiology and pathology of MCs has been reviewed (Palmieri and Monné 2016). Several of the 53 human mitochondrial SLC25 carriers are associated with genetic diseases (Rochette et al. 2020). In fact, mitochondrial transporter expression patterns distinguish tumors from normal tissues and identify cancer subtypes with different survival and metabolism (Wohlrab et al. 2022). Abnormal energy metabolism in mitochondria can be related to symptomatic severity in nemaline myopathy and may constitute a contributor to phenotypic variability (Tinklenberg et al. 2023).

Members of the mitochondrial carrier family are involved in transporting keto acids, amino acids, nucleotides, inorganic ions and co-factors across the mitochondrial inner membrane. The transporters are thought to share the same structural fold, which consists of six trans-membrane alpha-helices and three matrix helices, arranged with threefold pseudo-symmetry. There are 53 MC homologues in humans. During the transport cycle two salt bridge networks on either side of the central cavity might regulate access to a single substrate binding site in an alternating fashion. In the case of proton-substrate symporters, the substrate binding sites contain negatively charged residues that are proposed to be involved in proton transport (Kunji and Robinson, 2010). Wang et al. 2016 haved reviewed the structures and transport mechanisms of these porters. Some of the residues in the charged residue positions of the PX[DE]XX[KR] motifs are important for reasons other than forming salt bridges, probably for playing specific roles related to the substrate interaction-mediated conformational changes leading to the M-gate opening/closing (Miniero et al. 2022). Several intron positions are present in numerous MC sequences at the same specific points, of which some are 3-fold symmetry related (Monné et al. 2023).

The high resolution 3-D structure of the human homologue, the bovine ATP/ADP antiporter (TC #2.A.29.1.1), has been solved by x-ray crystallography to 2.2 Å resolution (Pebay-Peyroula et al., 2003; Klingenberg et al., 2008). The carrier was crystalized in complexation with the inhibitor, carboxyatractyloside. The six TMSs (with the N- and C-termini normally facing the cytoplasmic side of the membrane and the three hairpin loops of the repeat sequences facing the matrix) form a compact barrel domain which shows a deep cone-shaped depression at the surface facing the intermembrane space. At its base was found the signature sequence of these nucleotide carriers (R R R M M M). The cavity has a maximal diameter of 20 Å and a depth of 30 Å. The fold of the three repeat elements is very similar. Each odd-numbered helix exhibits a sharp kink, due to a conserved prolyl residue located in the conserved P X(D/E) X X (K/R) motif, characteristic of all mitochondrial carriers. The even-numbered helices pass straight through the membrane without a kink. The structure reveals large hydrophilic surfaces in the interior of the conical pit, due to the weak hydrophobicities of these proteins. A positive electrostatic surface potential on the matrix side and at the bottom of the pit provides the force for anionic substrate binding. Two lipid molecules, both cardiolipin molecules, are tightly bound to the carrier.

The mitochondrial uncoupling protein 2 structure has been determined by NMR molecular fragment searching (Berardi et al., 2011). UCP2 closely resembles the bovine ADP/ATP carrier, but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4 (Berardi et al., 2011). The expression patterns and functions of different UCP homologs have been reviewed (Monteiro et al. 2021). Mitochondrial uncoupling proteins UCP1 and UCP2 execute their mitochondrial uncoupling function through different molecular mechanisms. Non-shivering thermogenesis by UCP1 is mediated through a transmembrane dissipation of the proton motive force to create heat during sympathetic stimulation. UCP2, on the other hand, modulates through the interaction with methylated MICU1 (TC# 8.A.44.1.1) the permeability of the cristae junction, which acts as an isolator for the cristae-located mitochondrial membrane potential. Oflaz et al. 2023 discussed and compared the molecular mechanism of UCP1 in brown adipose tissue and UCP2 in aged and cancer non-excitable cells that contribute to mitochondrial uncoupling, and the synergistic effects of both UCPs with the mitochondrial Ca²⁺ uptake machinery.

The transport substrates of MC family members may bind to the bottom of the cavity, and translocation results in a transient transition from a 'pit' to a 'channel' conformation (Kunji and Robinson, 2006; Robinson and Kunji, 2006). The inhibitor, carboxyatractyloside, probably binds where ADP binds, in the pit on the outer surface, thus blocking the transport cycle. Another inhibitor, bongkrekic acid, is believed to stabilize a second conformation, with the pit facing the matrix. In this conformation, the inhibitor may bind to the ATP-binding site. Functional and structural roles for residues in the TMSs have been proposed (Cappello et al., 2006, 2007). The mitochondrial carrier signature, Px[D/E]xx[K/R], of carriers is probably involved both in the biogenesis and in the transport activity of these proteins (Zara et al., 2007). A homologue has been identified in the mimivirus genome and shown to be a transporter for dATP and dTTP (Monné et al., 2007).

One of the MC family members, the uncoupling protein, UCP1 (TC# 2.A.29.3.1), functions to dissipate the proton motive force, thereby generating heat. This protein has been shown to be capable of transporting fatty acids, long chain alkylsulfonates and chloride. It is believed to allow transport of protons down their electrochemical gradient in a cyclic, fatty acid-dependent process by first exporting fatty acyl anions and then allow the free diffusion of the protonated fatty acid across the bilayer into the mitochondrion. UNC1 is therfore probably an anion translocator that may not require that transport occurs by an antiport mechanism. The fatty acid behaves as a cycling protonophore (Garlid et al., 2000). UNC1 uses coenzyme Q (ubiquinone) as a cofactor (Echtay et al., 2000). Like many other MC family members, uncoupling proteins are found in the mitochondria of plants as well as animals. Various compounds such as the reactive aldehyde (produced under oxidative stress conditions), 4-hydroxy-2-nonenal, as well as trans-retinal and other 2-alkenals activate uncoupling via UCP1-3 (TC #2.A.29.3.1) as well as the ATP/ADP antiporter (TC #2.A.29.1.1) (Echtay et al., 2003).

Mitochondrial uncoupling protein 1 (UCP1) is responsible for nonshivering thermogenesis in brown adipose tissue (BAT). Upon activation by long-chain fatty acids (LCFAs), UCP1 increases the conductance of the inner mitochondrial membrane (IMM) to make BAT mitochondria generate heat rather than ATP. UCP1 transports H⁺. UCP1 is an LCFA anion/H⁺ symporter (Fedorenko et al. 2012), but the LCFA anions cannot dissociate from UCP1 due to hydrophobic interactions established by their hydrophobic tails, and UCP1 effectively operates as an H⁺ carrier activated by LCFA. A similar LCFA-dependent mechanism of transmembrane H⁺ transport may be employed by other UCP members and be responsible for mitochondrial uncoupling and regulation of metabolic efficiency in various organisms and tissues.

Mitochondrial transporters have 3 homologous repeats and a structure with pseudosymmetry. Each repeat is folded into 2 transmembrane α-helices linked by a short α-helix on the matrix side and contains the signature motif PX[DE]XX[RK]. The proline residues kink the odd-numbered transmembrane α-helices, and the charged residues form a salt-bridge network connecting the C-terminal ends of the transmembrane α-helices, closing the transporter on the matrix side. During the transport cycle, the carriers form states in which the substrate-binding state of the carrier is open to the mitochrondrial intermembrane space and matrix, respectively. According to the single binding center-gating pore mechanism, interconversion of the 2 conformational states via a transition intermediate leads to substrate translocation. In the cytoplasmic state, a central substrate-binding site has been identified by applying chemical and distance constraints to comparative models. The substrates bind to 3 major sites on the even-numbered α-helices, which are related by symmetry and located approximately in the middle of the membrane. Yeast ADP/ATP carriers function as monomers (Bamberg et al., 2007).

Residues that are important for the transport mechanism are likely to be symmetrical, whereas residues involved in substrate binding will be asymmetrical reflecting the asymmetry of the substrates. By scoring the symmetry of residues in the sequence repeats, Robinson et al. (2008) identified the substrate-binding sites and salt bridge networks that are important for transport. The symmetry analyses provides an assessment of the role of residues and provides clues to the chemical identities of substrates of uncharacterized transporters. Downy mildew (Peronospora belbahrii, Peronospora effusa, and Peronospora tabacina, which are specialized pathogens of basil, spinach, and tobacco, respectively)-causing plant pathogens are biotrophic oomycetes that transport essential nutrients from their hosts to grow. Gene transcripts encoding the ADP/ATP translocase and the mitochondrial phosphate carrier protein were the most abundant mRNAs detected in each Peronospora species (Johnson et al. 2023).

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced; it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a few mitosomal proteins are encoded in the complete genome of the microsporidian, Encephalitozoon cuniculi, no proteins of the mitochondrial carrier family were identified. However, the microsporidian, Antonospora locustae, has a protein that is heterologously targeted to mitochondria in Saccharomyces cerevisiae (Williams et al., 2008). The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but it has high specificity for ATP and ADP when expressed in E. coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.

BID, a proapoptotic BCL-2 family member, plays an essential role in the tumor necrosis factor alpha (TNF-alpha)/Fas death receptor pathway in vivo. Activation of the TNF-R1 receptor results in the cleavage of BID into truncated BID (tBID), which translocates to the mitochondria and induces the activation of BAX or BAK. In TNF-alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex. Grinberg et al. (2005) described the biochemical purification of this complex and the identification of mitochondrial carrier homolog 2 (Mtch2; TC# 2.A.29.25.2) as part of this complex. Mtch2 is similar to members of the mitochondrial carrier family. Mtch2 is an integral outer membrane protein exposed on the surface of mitochondria. Mtch2 resides in a protein complex of ca. 185 kDa, and the addition of TNF-alpha to these cells leads to the recruitment of tBID and BAX to this complex. Thus, Mtch2 is a mitochondrial target of tBID. The Mtch2-resident complex probably participates in the mitochondrial apoptotic program (Grinberg et al., 2005; Gross, 2005). 

The ADP/ATP carrier is electrogenic (electrophoretic), the GTP/GDP carrier is dependent on the pH gradient, the aspartate/glutamate carrier is dependent on both, and the oxoglutarate/malate carrier is independent of them (Monné and Palmieri 2014). The bovine ADP/ATP carrier consists of a six-transmembrane alpha-helix bundle with a pseudo-threefold symmetry and a closed matrix gate. By using this structure as a template in homology modeling, residues engaged in substrate binding and the formation of a cytoplasmic gate in MCs have been proposed. The functional importance of the residues of the binding site, the matrix, and the cytoplasmic gates is supported by transport activities of different MCs with single point mutations. Cumulative evidence has been used to postulate a general transport mechanism for MCs (Monné and Palmieri 2014).

The ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway (Xiao et al. 2021).

Mitochondrial heat production is crucial for the maintenance of body temperature, regulation of the pace of metabolism, and prevention of oxidative damage. Mitochondria produce heat as the result of H⁺ leak across their inner membrane. Bertholet and Kirichok 2022 provided an assessment of the current field of mitochondrial H⁺ leak and thermogenesis, with a focus on the molecular mechanisms involved and regulation of uncoupling protein 1 and the ADP/ATP carrier, the two proteins that mediate mitochondrial H⁺ leak.

The generalized transport reaction for carriers of the MC family is:

S1 (out) + S2 (in) ⇌ S1 (in) + S2 (out)