The SLC25 Carrier Family: Important Transport Proteins in Mitochondrial Physiology and Pathology

Abstract

Members of the mitochondrial carrier family (SLC25) transport a variety of compounds across the inner membrane of mitochondria. These transport steps provide building blocks for the cell and link the pathways of the mitochondrial matrix and cytosol. An increasing number of diseases and pathologies has been associated with their dysfunction. In this review, the molecular basis of these diseases is explained based on our current understanding of their transport mechanism.

Keywords: bioenergetics; impaired transport mechanism; mitochondrial disease; mitochondrial physiology; pathological mutations.

Figure 1. The role of the human mitochondrial carrier family (SLC25) in metabolism and mitochondrial function

Schematic representation of the mammalian mitochondrion. Shown in green are the electron transfer chain complexes. Bottom to top: glycerophosphate dehydrogenase, fatty acid-dehydrogenase-electron transfer flavoprotein, dihydroorotate dehydrogenase, and complex I to IV with cytochrome c. Shown in blue is the dimer of ATP synthase, and in red/orange the mitochondrial pyruvate carrier heterodimer (MPC). Shown in purple and yellow are unidentified and identified mitochondrial carriers, respectively. AAC1–4, ADP/ATP carriers; AGC1–2, aspartate/glutamate carriers; APC1–4, ATP-Mg/Pi carriers; BAC, basic amino acid carrier; CAC, carnitine-acylcarnitine carrier; CIC, citrate carrier; DIC, dicarboxylate carrier; GC1–2, glutamate carriers; GLYC, glycine carrier; MTFRN1–2, mitoferrins; ODC, oxoadipate carrier; OGC, oxoglutarate carrier; ORC1–2, ornithine carriers; PIC, phosphate carrier; SAMC, S-adenosylmethionine carrier; TPC, thiamine pyrophosphate carrier; UCP1, uncoupling protein; UCP2, uncoupling-like protein 2; CS, cytosol; IS, intermembrane space; OM, outer membrane; IM, inner membrane; MM, mitochondrial matrix; Q, ubiquinone; Pi, phosphate; ALA, aminolevulinic acid.

Figure 2. Structures of the mitochondrial ADP/ATP carrier and the calcium-regulated ATP-Mg/phosphate carrier and aspartate/glutamate carrier

Structures of three different mitochondrial carriers, based on PDB entries 1OKC (128) and 4C9Q (141) for the carrier domains and 4P5W (157) and 4ZCU (59) for the calcium-regulatory domains. A: the mitochondrial ADP/ATP carrier. B: the mitochondrial ATP-Mg/Pi carrier consists of three domains: 1) NH₂-terminal calcium-regulatory domain with four EF-hands (EF1–4), each binding calcium, 2) amphipathic helix, and 3) COOH-terminal carrier domain. C: the aspartate/glutamate carrier also has a three-domain structure, but with a different order: 1) NH₂-terminal calcium-regulatory domain, 2) carrier domain, and 3) COOH-terminal amphipathic helix. The NH₂-terminal domain has eight EF-hand folds, but only EF-hand 2 is capable of binding calcium, which together with EF-hands 1 and 3 forms a calcium-responsive mobile unit. EF-hands 4–8 have evolved to form a static dimerization interface. The structures are shown in a cartoon representation, colored from the NH₂-terminus in blue to the COOH-terminus in red. Also shown are the canonical substrates as well as calcium ions (green), magnesium ion (chartreuse), and protons (white) in sphere representations. IS, intermembrane space; OM, outer membrane; IM, inner membrane; MM, mitochondrial matrix.

Figure 3. Mitochondrial carriers have a threefold pseudo-symmetrical structure

A: aligned amino acid sequences of the three repeats of the human ADP/ATP carrier AAC1 (ANT1). Symmetrically conserved residues are shown in the consensus sequence and as bars, when present in at least two out of three repeats. B and C: comparative model of the human ADP/ATP carrier, based on PDB:1OKC (128), viewed from the membrane and the intermembrane space, respectively. Shown also is the threefold pseudosymmetrical axis, symbolized by an equilateral triangle. Odd-numbered (H1, H3, H5), matrix (h12, h34, h56), linker (l12, l34, l56), and even-numbered helices (H2, H4, H6) are shown in primary colors for the core elements and in gray for the gate elements (142). The black spheres with roman numerals show the positions of the three contact points of the substrate binding site (94, 136). The example, triplet 8–113–210, is indicated by a rectangle across the three repeats in A and by cyan spheres in B. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 4. Alternating access transport mechanism for the mitochondrial ADP/ATP carrier

Lateral view from the membrane of the mitochondrial ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). Shown are the cytoplasmic state of the bovine ADP/ATP carrier (PDB:1OKC) (128) and the matrix state of the ADP/ATP carrier of Thermothelomyces thermophila (PDB:6GCI) (142). The water-accessible surfaces are shown in light blue. Also indicated are the three main functional features: cytoplasmic gate, substrate binding site, and matrix gate. The black spheres with roman numerals are the contact points of the substrate binding site (94, 136). Shown in green are residues that most likely bind the adenine nucleotide substrates. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 5. Structural changes in the transport cycle of the human mitochondrial ADP/ATP carrier

View from the intermembrane space (A) and lateral view from the membrane of the human ADP/ATP carrier (B) in the cytoplasmic state (left) and matrix state (right). For the import of ADP, conformational changes involve the simultaneous outward rotation of the core elements, shown in primary colors, and inward rotation of the gate elements, shown in gray, in a coordinated way (142). For the export of ATP, the converse happens. Substrate binding increases the probability of the state interconversion (156). The structural models are based on the structures of the cytoplasmic state of the bovine ADP/ATP carrier (PDB:1OKC) (128) and the matrix state of the ADP/ATP carrier of Thermothelomyces thermophila (PDB:6GCI) (142). The black spheres with roman numerals are the contact points of the substrate binding site (94, 136). IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 6. Pathogenic mutations observed in disease variants of mitochondrial carriers

Aligned triplets of the 16 mitochondrial carriers associated with developmental, metabolic, and neuromuscular diseases. The mutations that have a severe effect on function are shown in red boxes, whereas those that have milder effects are in yellow. Mutations in blue boxes have been identified by genetic analysis, but their effect has not been studied experimentally. At the bottom are the three residue numbers that form a triplet in the human ADP/ATP carrier (SLC25A4). At the top are the conserved structural and functional features of mitochondrial carriers. The triplet is labeled by the one-letter code of the most conserved residue or by the most common property: π, small amino acids; Φ, hydrophobic amino acids;ξ, hydrophilic amino acids; Ω, aromatic amino acids; or by X for any amino acid. The black spheres with roman numerals are the contact points of the substrate binding site (94, 136). H6 in the ADP/ATP carrier is one residue shorter than other carriers and lacks a residue in triplet 90. The matrix loops (indicted by the black bar), as well as the cytoplasmic loops and NH₂ and COOH terminus have been omitted.

Figure 7. Small amino acid residues on the odd-numbered helices

View from the intermembrane space (A) and lateral view from the membrane (B) of the human ADP/ATP carrier in the cytoplasmic state (left) and the matrix state (right). The structural models are based on the structures of the cytoplasmic state of the bovine ADP/ATP carrier (128) and the matrix state of the ADP/ATP carrier of Thermothelomyces thermophila (142). The carrier is shown in surface representation and the helices in cartoon representation with the core elements in primary colors and the gate elements in gray. Glycine or small residues in the interface with the preceding helix are shown in pink, whereas those in the interface with the following helix are shown in magenta. The sequence motif is πGπxπGxxπxxxπ, where G stands for glycine and π for small amino acids (–144). When the carrier changes from the cytoplasmic state to the matrix state, the inter-helical distances become smaller on the cytoplasmic side of the carrier to facilitate the formation of the cytoplasmic network, requiring small residues in the helical interfaces. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 8. Key amino acid residues of the matrix gate

View from the intermembrane space (A) and lateral view from the membrane (B) of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. The key residues shown belong to the sequence motif Px[DE]xx[RK]xxxQ on the odd-numbered helices. The proline residues (orange) are found at the pronounced kink in the odd-numbered helices, bringing the negatively charged (red) and positively charged (blue) residues together to form the matrix network in the cytoplasmic state. Underneath one of the salt bridges is a glutamine residue (green) that forms hydrogen bonds with both residues (glutamine brace), but in other carriers one, two, or three glutamine braces can be found (FIGURE 6) (141). IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 9. Detailed view of one of the three binding sites for cardiolipin

One cardiolipin molecule is shown in ball-and-stick representation (cdl802, PDB: 2C3E), which is bound on the surface of the carrier and spans the inter-domain interface. The carrier is shown in cartoon representation with transmembrane H4 (yellow and gray) and matrix helix h56 (red) enhanced. The two phosphate groups of cardiolipin, which are linked by a glycerol moiety, form hydrogen bonds with the amide groups (128) and interact with the positively charged ends of the helix dipoles of the NH₂-terminal ends of the matrix helices (cardiolipin binding site I) and the even-numbered helices (cardiolipin binding site II) (24, 141, 142). The four fatty acid chains of cardiolipin interact with the surface of the carrier in a non-specific way via van der Waals interactions (41). Residues in the conserved cardiolipin binding site I and II are shown as green and purple sticks, respectively. The interface between domain 2 and 3 is shown as a dashed line.

Figure 10. Amino acid residues involved in cardiolipin binding

View from the intermembrane space (A) and lateral view from the membrane (B) of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. There are three highly conserved binding sites for cardiolipin in the mitochondrial ADP/ATP carrier. The residues of cardiolipin binding site I belong to the [YF]xG motif (green), whereas those of cardiolipin binding site II belong to the [YWF][RK]G motif (purple) (128, 141, 142). IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 11. Amino acid residues important for the domain structures on the matrix side

View from the intermembrane space (A) and lateral view (B) from the membrane of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. The positively charged residue of the E-R link I (blue), which is located in the matrix gate area, interacts with the negatively charged residue of the E-R link II, which is located on the matrix helices. The NH₂-terminal and the fourth residues of the linker helices are most commonly glycine residues (magenta), although the latter can be replaced by other small residues. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 12. Amino acid residues of the substrate binding site

View from the intermembrane space (A) and lateral view (B) from the membrane of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. The black spheres with roman numerals are the contact points of the substrate binding site, which are involved in binding of the substrates (94, 136). Other residues in this site (green) may either bind substrate directly or may allow the binding. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 13. Small amino acid residues on the even-numbered helices

View from the intermembrane space (A) and lateral view (B) from the membrane of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. The small residues (chartreuse) in the interhelical interfaces with the odd-number helices facilitate conformational changes. Some residues are larger, such as the phenylalanine on H6, because their side chains face the membrane in both conformations. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.

Figure 14. Amino acid residues of the cytoplasmic gate

View from the intermembrane space (A) and lateral view (B) from the membrane of the human ADP/ATP carrier in the cytoplasmic state (left) and matrix state (right). The structures are described in the legend to FIGURE 7. The key residues belong to the sequence motif ξ[FY]xx[YF][DE]xx[RK]. The negatively charged (red) and positively charged (blue) residues together form the cytoplasmic network in the matrix state. Underneath are aromatic residues (orange), which are part of the hydrophobic plug that closes the cytoplasmic gate. The aromatic residue preceding the negatively charged residue is the tyrosine brace (Y-brace) (–144). Preceding the aromatic residues are hydrophilic residues (ξ), which form the ceiling of the substrate binding site. IS, intermembrane space; IM, inner membrane; MM, mitochondrial matrix.