The SLC25 Mitochondrial Carrier Family: Structure and Mechanism

Mitochondrial carriers, belonging to the SLC25 family of transport proteins, are essential for the functioning of eukaryotes because they transport nutrients across the mitochondrial inner membrane for energy conversion and maintenance of the cell.The recently determined structure of a mitochondrial ADP/ATP carrier in the inhibited matrix-state, combined with earlier structures in the inhibited cytoplasmic state, provides the first description of the transport mechanism for an SLC25 protein.Sequence elements, conserved across the mitochondrial carrier family, play important roles in the mechanism, pointing to a common transport mechanism across SLC25 proteins.Mitochondrial carriers are the most dynamic transport proteins yet characterized, undergoing large conformational changes, allowing them to transport some of the largest biochemical compounds without significant proton leak. Members of the mitochondrial carrier family (SLC25) provide the transport steps for amino acids, carboxylic acids, fatty acids, cofactors, inorganic ions, and nucleotides across the mitochondrial inner membrane and are crucial for many cellular processes. Here, we use new insights into the transport mechanism of the mitochondrial ADP/ATP carrier to examine the structure and function of other mitochondrial carriers. They all have a single substrate-binding site and two gates, which are present on either side of the membrane and involve salt-bridge networks. Transport is likely to occur by a common mechanism, in which the coordinated movement of six structural elements leads to the alternating opening and closing of the matrix or cytoplasmic side of the carriers. Members of the mitochondrial carrier family (SLC25) provide the transport steps for amino acids, carboxylic acids, fatty acids, cofactors, inorganic ions, and nucleotides across the mitochondrial inner membrane and are crucial for many cellular processes. Here, we use new insights into the transport mechanism of the mitochondrial ADP/ATP carrier to examine the structure and function of other mitochondrial carriers. They all have a single substrate-binding site and two gates, which are present on either side of the membrane and involve salt-bridge networks. Transport is likely to occur by a common mechanism, in which the coordinated movement of six structural elements leads to the alternating opening and closing of the matrix or cytoplasmic side of the carriers. With 53 members, the mitochondrial carrier family (solute carrier family 25, SLC25) is the largest solute transporter family in humans. They transport solutes across the impermeable inner membrane of mitochondria for important cellular processes, such as oxidative phosphorylation of fats and sugars, amino acid catabolism and interconversion, synthesis of iron sulfur clusters and heme, macromolecular synthesis, and heat production (Figure 1). Approximately one-third of human mitochondrial carriers are currently orphan transporters, with no known substrate. Most operate as strict counter-exchangers of chemically related substrates [antiporters (see Glossary)], but some display unidirectional (uniporters) or substrate–proton (symporters) transport activities. In this review, we focus on the role and properties of some of the best characterized members of the family, which will be introduced first. Nucleotide transporters include the mitochondrial ADP/ATP carrier, also called adenine nucleotide translocase or translocator (ANT), which imports ADP into the mitochondrial matrix, where it can be converted to ATP by ATP synthase, and exports the newly synthesized ATP to the cytosol, where it fuels the metabolic energy-requiring processes that are vital for cell survival [1Palmieri F. Klingenberg M. On the possible role of structural protein in the binding and translocation of adenine nucleotides in mitochondria.Biochim. Biophys. Acta. 1967; 131: 582-585Crossref PubMed Scopus (3) Google Scholar, 2Aquila H. et al.Complete amino acid sequence of the ADP/ATP carrier from beef heart mitochondria.Hoppe Seylers Z. Physiol. Chem. 1982; 363: 345-349Crossref PubMed Scopus (174) Google Scholar, 3Klingenberg M. The ADP and ATP transport in mitochondria and its carrier.Biochim. Biophys. 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Calcium-regulated mitochondrial ATP-Mg/Pi carriers evolved from a fusion of an EF-hand regulatory domain with a mitochondrial ADP/ATP carrier-like domain.IUBMB Life. 2018; 70: 1222-1232Crossref PubMed Scopus (8) Google Scholar]. There are three human isoforms, APC1 (SLC25A24), APC2 (SLC25A23), and APC3 (SLC25A25) [8Fiermonte G. et al.Identification of the mitochondrial ATP-Mg/Pi transporter. Bacterial expression, reconstitution, functional characterization, and tissue distribution.J. Biol. Chem. 2004; 279: 30722-30730Crossref PubMed Scopus (144) Google Scholar], which have the three-domain structure [11Harborne S.P. et al.Calcium-induced conformational changes in the regulatory domain of the human mitochondrial ATP-Mg/Pi carrier.Biochim. Biophys. 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Most of the structural information for the SLC25 family comes from studies of the mitochondrial ADP/ATP carrier; this is due to the high natural abundance of this protein and the availability of inhibitors that trap the carrier in specific conformations. Atractyloside (ATR) and the chemically related carboxyatractyloside (CATR) lock the carrier in a cytoplasmic state (c-state) in which the substrate-binding site is accessible to the intermembrane space, which is confluent with the cytosol [42Vignais P.V. et al.Gummiferin, an inhibitor of the adenine-nucleotide translocation. Study of its binding properties to mitochondria.FEBS Lett. 1971; 17: 281-288Crossref PubMed Scopus (47) Google Scholar, 43Vignais P.V. et al.Adenosine diphosphate translocation in mitochondria. Nature of the receptor site for carboxyatractyloside (gummiferin).Biochemistry. 1973; 12: 1508-1519Crossref PubMed Scopus (114) Google Scholar, 44Luciani S. et al.Effects of carboxyatractyloside a structural analogue of atractyloside on mitochondrial oxidative phosphorylation.Life Sci II. 1971; 10: 961-968Crossref PubMed Scopus (44) Google Scholar]. Bongkrekic acid (BKA) and its isomer isobongkrekic acid lock the carrier in a matrix state (m-state) with the substrate-binding site accessible to the matrix [45Henderson P.J. Lardy H.A. Bongkrekic acid. An inhibitor of the adenine nucleotide translocase of mitochondria.J. Biol. Chem. 1970; 245: 1319-1326Abstract Full Text PDF PubMed Google Scholar,46Lauquin G.J. et al.Isobongkrekic acid, a new inhibitor of mitochondrial ADP-ATP transport: radioactive labeling and chemical and biological properties.Biochemistry. 1976; 15: 2323-2327Crossref PubMed Scopus (28) Google Scholar]. The first structural information was obtained by electron crystallography of 2D crystals of the Saccharomyces cerevisiae ADP/ATP carrier ScAac3p, trapped in the c-state by ATR [47Kunji E.R.S. Harding M. Projection structure of the atractyloside-inhibited mitochondrial ADP/ATP carrier of Saccharomyces cerevisiae.J. Biol. Chem. 2003; 278: 36985-36988Crossref PubMed Scopus (86) Google Scholar]. The projection maps showed that the carrier was monomeric with threefold pseudo-symmetry and had a central substrate translocation pathway. The first atomic structure of the bovine ADP/ATP carrier inhibited by CATR was determined by X-ray crystallography (PDB ID: 1OKC and 2C3E) [48Pebay-Peyroula E. et al.Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.Nature. 2003; 426: 39-44Crossref PubMed Scopus (726) Google Scholar]. The structure revealed a barrel-shaped protein composed of three domains related by threefold pseudo-symmetry. Each domain is composed of an odd-numbered transmembrane α-helix (H1, H3, or H5), a loop containing a short matrix α-helix (h12, h34, or h56) that lies parallel to the membrane plane, and an even-numbered transmembrane α-helix (H2, H4, or H6). This structural fold was subsequently confirmed by structures of the yeast ADP/ATP carrier isoforms ScAac2p (PDB ID: 4C9G and 4C9H) and ScAac3p (PDB ID: 4C9J and 4C9Q), also trapped in the c-state by CATR (Figure 2A,C) [49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar]. The odd-numbered transmembrane α-helices have pronounced kinks, located at the proline residues of the highly conserved signature motif Px[DE]xx[KR] (P kink in Figure 2), giving them a pronounced L-shape, which helps to block access to the central cavity from the mitochondrial matrix in the c-state. Over 40% of ADP/ATP carrier sequences, including both ScAac2p and ScAac3p, have a serine substitution for the proline of the signature motif on H3, which forms a hydrogen bond to its own backbone amide group, mimicking the disruption proline causes to α-helical backbone hydrogen bonding [49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar]. The charged residues of the signature motifs form interdomain salt-bridges [48Pebay-Peyroula E. et al.Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.Nature. 2003; 426: 39-44Crossref PubMed Scopus (726) Google Scholar, 49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar, 50Nury H. et al.Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.FEBS Lett. 2005; 579: 6031-6036Crossref PubMed Scopus (151) Google Scholar], now called the matrix salt-bridge network [51Robinson A.J. et al.The mechanism of transport by mitochondrial carriers based on analysis of symmetry.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17766-17771Crossref PubMed Scopus (147) Google Scholar] (Figure 2), as predicted earlier by genetic analysis [52Nelson D.R. et al.Highly conserved charge-pair networks in the mitochondrial carrier family.J. Mol. Biol. 1998; 277: 285-308Crossref PubMed Scopus (159) Google Scholar]. Residues of the salt-bridge between domains 1 and 3 interact with a proximal glutamine residue (Px[DE]xx[KR]xxxQ), which hydrogen bonds to both salt-bridge residues, forming a brace that stabilizes the matrix network (Q brace in Figure 2) [49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar]. The glutamine residues are highly conserved, and one to three Q braces are typically found in SLC25 members, bracing the salt bridges of the matrix network. CATR inhibits the ADP/ATP carrier by binding tightly in the central cavity, forming multiple salt-bridges and hydrogen bonds with protein residues, blocking the translocation pathway (Figure 2A) [48Pebay-Peyroula E. et al.Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.Nature. 2003; 426: 39-44Crossref PubMed Scopus (726) Google Scholar,49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar]. Three cardiolipin molecules are tightly bound to the carrier, bridging the matrix helices and the even-numbered transmembrane helices (Figure 2) [49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar,50Nury H. et al.Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.FEBS Lett. 2005; 579: 6031-6036Crossref PubMed Scopus (151) Google Scholar]. The cardiolipin molecules play an important role in stabilizing their proximal interdomain interfaces and are tightly bound to the protein [53Beyer K. Klingenberg M. ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance.Biochemistry. 1985; 24: 3821-3826Crossref PubMed Scopus (258) Google Scholar] by hydrogen bonds [48Pebay-Peyroula E. et al.Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.Nature. 2003; 426: 39-44Crossref PubMed Scopus (726) Google Scholar] and by electrostatic interactions with helix dipoles [49Ruprecht J.J. et al.Structures of yeast mitochondrial ADP/ATP carriers support a domain-based alternating-access transport mechanism.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E426-E434Crossref PubMed Scopus (121) Google Scholar]. Recently, the first structure of a mitochondrial carrier in the m-state has been solved: the ADP/ATP carrier from the thermotolerant fungus Thermothelomyces thermophila (TtAac), inhibited by BKA (PDB ID: 6GCI; Figure 2B,D) [54Ruprecht J.J. et al.The molecular mechanism of transport by the mitochondrial ADP/ATP carrier.Cell. 2019; 176: 435-447Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. The m-state structure shows the characteristic three-domain architecture, but with the domains rotated compared with the c-state, opening up the central cavity to the mitochondrial matrix and closing it to the intermembrane space. As a consequence, the matrix network and Q brace are disrupted (Figure 2D). On the intermembrane side, the transmembrane helices are positioned close together, allowing the charged residues of the [YF][DE]xx[KR] motifs on the even-numbered α-helices to form the interdomain cytoplasmic salt-bridge network (Figure 2D) [51Robinson A.J. et al.The mechanism of transport by mitochondrial carriers based on analysis of symmetry.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17766-17771Crossref PubMed Scopus (147) Google Scholar,54Ruprecht J.J. et al.The molecular mechanism of transport by the mitochondrial ADP/ATP carrier.Cell. 2019; 176: 435-447Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. This network is stabilized by hydrogen bonds with the hydroxyl groups of the tyrosines of the motif, forming a tyrosine brace (Y brace in Figure 2). Most SLC25 family members have one to three Y braces. Comparison of the available c- and m-state structures indicates that both CATR and BKA induce subtle perturbations in the protein structure, which likely contribute to their inhibition mechanism [54Ruprecht J.J. et al.The molecular mechanism of transport by the mitochondrial ADP/ATP carrier.Cell. 2019; 176: 435-447Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. Nevertheless, the structural information now available for both states has significantly advanced our understanding of how these proteins operate at the molecular level [5Ruprecht J.J. Kunji E.R.S. Structural changes in the transport cycle of the mitochondrial ADP/ATP carrier.Curr. Opin. Struct. Biol. 2019; 57: 135-144Crossref PubMed Scopus (24) Google Scholar,54Ruprecht J.J. et al.The molecular mechanism of transport by the mitochondrial ADP/ATP carrier.Cell. 2019; 176: 435-447Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar]. The structural features seen in the ADP/ATP carriers are likely to be conserved throughout the mitochondrial carrier family, with few exceptions. SLC25 proteins can transport substrates with exquisite specificity, as exemplified by the ADP/ATP carrier which transports only ADP or ATP or their deoxy variants, but not any other adenine or guanine nucleotides [55Pfaff E. Klingenberg M. Adenine nucleotide translocation of mitochondria. 1. Specificity and control.Eur. J. Biochem. 1968; 6: 66-79Crossref PubMed Scopus (301) Google Scholar, 56Mifsud J. et al.The substrate specificity of the human ADP/ATP carrier AAC1.Mol. Membr. Biol. 2013; 30: 160-168Crossref PubMed Scopus (33) Google Scholar, 57Majd H. et al.Screening of candidate substrates and coupling ions of transporters by thermostability shift assays.Elife. 2018; 7: e38821Crossref PubMed Scopus (20) Google Scholar]. To understand how they achieve this, it is essential to know where substrates bind and how they interact with the protein. Unfortunately, the structures of the ADP/ATP carriers were solved using inhibitors that are chemically distinct from substrates and thus were unable to reveal the substrate-binding site directly. Diverse bioinformatic and modeling approaches have been undertaken to try to pin-point the substrate-binding site. In one approach, homology models were probed with chemical and distance constraints to identify conserved residues that were capable of discriminating between keto acid and amino acid substrates, and adenine nucleotides [58Kunji E.R.S. Robinson A.J. The conserved substrate binding site of mitochondrial carriers.Biochim. Biophys. Acta. 2006; 1757: 1237-1248Crossref PubMed Scopus (94) Google Scholar,59Robinson A.J. Kunji E.R.S. Mitochondrial carriers in the cytoplasmic state have a common substrate binding site.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2617-2622Crossref PubMed Scopus (171) Google Scholar]. A common substrate-binding site in the central cavity was identified, which includes residues on each of the even-numbered transmembrane helices, called the contact points (shown as black spheres with Roman numerals in Figure 3) [58Kunji E.R.S. Robinson A.J. The conserved substrate binding site of mitochondrial carriers.Biochim. Biophys. Acta. 2006; 1757: 1237-1248Crossref PubMed Scopus (94) Google Scholar,59Robinson A.J. Kunji E.R.S. Mitochondrial carriers in the cytoplasmic state have a common substrate binding site.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2617-2622Crossref PubMed Scopus (171) Google Scholar]. Importantly, this analysis indicated that contact point II (on H4) plays the key role of discriminating between different classes of substrate (e.g., keto or amino acids), contact point I (on H2) can discriminate between different substrates within the same class, and contact point III (on H6) is nearly always a positively charged residue and therefore does not confer specificity [58Kunji E.R.S. Robinson A.J. The conserved substrate binding site of mitochondrial carriers.Biochim. Biophys. Acta. 2006; 1757: 1237-1248Crossref PubMed Scopus (94) Google Scholar,59Robinson A.J. Kunji E.R.S. Mitochondrial carriers in the cytoplasmic state have a common substrate binding site.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2617-2622Crossref PubMed Scopus (171) Google Scholar]. A second approach was based upon the striking difference between the threefold pseudo-symmetry of the carriers and the asymmetric nature of the substrates they transport [51Robinson A.J. et al.The mechanism of transport by mitochondrial carriers based on analysis of symmetry.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 17766-17771Crossref PubMed Scopus (147) Google Scholar]. Carriers must have evolved asymmetric substrate-binding site residues to match their asymmetric substrates. From sequence information alone, a score was devised to reflect both the conservation and degree of s