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At least 19 recordsLinked to original sources
PubMed2026

Evolutionary history and recombination in the mitochondrial carrier SLC25 superfamily analyzed by similarities in the exon and transmembrane α-helix sequences.

Mitochondrial carriers (MCs), which constitute a superfamily also called the solute carrier family 25 (SLC25), are characterized by conserved signature motif sequences and a six-transmembrane α-helical transporter domain. They transport a wide variety of substrates ranging from protons, inorganic ions, citric acid cycle intermediates, and amino acids to nucleotides and cofactors. The superfamily members can be divided into subfamilies, each with a distinct substrate specificity. In an attempt to understand how different subfamilies have evolved, we analyzed the protein sequences of the exons (with conserved boundaries) and the six transmembrane α-helices of MCs from highly diverged organisms. The results show that some MC subfamilies have all exons and transmembrane α-helices most similar to a closely related subfamily, which is consistent with a scenario of gene duplication and mutational divergence from a last common ancestor. However, several MC subfamilies appear to be mosaics of exons and transmembrane α-helices most similar to different and distant subfamilies, which in some cases could be explained by recombination between the superfamily genes during evolution. It seems that this latter mechanism could have played a role in the formation of new subfamilies with different substrate specificities by the combination of MC transporter domain segments that had been optimized previously for binding specific portions of the substrates. This study presents novel evolutionary relationships between MC subfamilies and may provide clues for how protein superfamilies have expanded and how to investigate their evolution.

Evolution, Molecular↗
PubMed2026

Structural Basis for Stepwise Substrate Transport and Disease Phenotypic Heterogeneity of the Mitochondrial ADP/ATP Carrier.

The mitochondrial ADP/ATP carrier (AAC) is essential for cellular energy metabolism and responsible for exchanging ADP for ATP across the inner mitochondrial membrane. However, the precise molecular determinants of substrate binding and the mechanisms underlying the phenotypic heterogeneity of AAC-related diseases remain poorly understood. Here, we combined AlphaFold3 predictions, molecular dynamics simulations, and experimental validation to identify and characterize a previously unrecognized ADP-binding site in AAC (site S2), which is distinct from the canonical bottom site (site S1). AlphaFold3 predictions on AAC variants with disrupted site S1 consistently placed ADP at site S2, interacting with residues R188, K92, and K96, a finding that was independently corroborated by our prior MD simulations. Systematic mutagenesis and functional analysis revealed distinct roles for site S2 residues: R188 serves as the primary phosphate-specific anchor; K92 and K96 facilitate initial recruitment and stabilization; and the aromatic ladder (Y187/Y191/F192/Y195) assists the conformational transition of ADP from anti to syn, a critical step enabling downward translocation. Functional characterization demonstrated that mutations in site S2 significantly impaired ADP transport and oxidative phosphorylation. Notably, the spatial distribution of AAC disease mutations correlates with this bipartite architecture: mild PEOA2-associated mutations cluster near site S2 and perturb the local conformation without abolishing binding, whereas lethal mutations cluster near site S1 and disrupt both ADP binding and structural integrity of the m-gate. Our findings provide new mechanistic insights into stepwise substrate transport and potential therapeutic targets for AAC-related diseases.

Mitochondrial ADP, ATP Translocases↗
PubMed1978

Studies on the orientations of the mitochondrial redox carriers. II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated, oriented mitochondrial multilayers.

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c1, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the "tightly bound" mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90 degrees.

Animals↗
PubMed1978

Studies on the orientations of the mitochondrial redox carriers. I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane.

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45 degrees between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2-1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a3. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90 degrees.

Animals↗
PubMed1979

Cell-free synthesis of the mitochondrial ADP/ATP carrier protein of Neurospora crassa.

ADP/ATP carrier protein was synthesized in heterologous cell-free systems programmed with Neurospora poly(A)-containing RNA and homologous cell-free systems from Neurospora. The apparent molecular weight of the product obtained in vitro was the same as that of the authentic mitochondrial protein. The primary translation product obtained in reticulocyte lysates starts with formylmethionine when formylated initiator methionyl-tRNA (fMet-tRNAfMet) was present. The product synthesized in vitro was released from the ribosomes into the postribosomal supernatant. The evidence presented indicates that the ADP/ATP carrier is synthesized as a polypeptide with the same molecular weight as the mature monomeric protein and does not carry an additional sequence.

Animals↗
PubMed1975

Respiratory control in isolated perfused rat heart. Role of the equilibrium relations between the mitochondrial electron carriers and the adenylate system.

The effects of KCl-induced cardiac arrest on the redox state of the fluorescent flavoproteins and nicotinamide nucleotides and on that of cytochromes c and a were studied by surface fluorometric and reflectance spectrophotometric methods. These changes were compared with measurements of the concentrations of the adenylate system, creatine phosphate, some intermediates of the tricarboxylic acid cycle and reactants of the glutamate dehydrogenase system. KCl-induced cardiac arrest caused reduction of the fluorescent flavoproteins and nicotinamide nucleotides, oxidation of cytochromes c and a, inhibition of oxygen consumption and an increase in the ATP/(ADP X Pi) ratio. The increase in the latter was due mainly to a decrease in the concentration of Pi and an equivalent increase in creatine phosphate. The cytochromes c and a were maintained at equal redox potential and changed in parallel. When the redox state of the mitochondrial NAD couple was calculated from the glutamate dehydrogenase equilibrium, the free energy change (deltaG) corresponding to the potential difference between the NAD couple and cytochrome c was 115.8 kj/mol in the beating heart and 122.2 kj/mol in the arrested heart. The deltaG values of ATP hydrolysis calculated from the concentrations of ATP, Pi and ADP, corrected for bound ADP, were 111.1 kj/2 mol and 115.4 kj/2 mol in the beating and arrested heart respectively. The accumulation of citrate and the direction of the redox changes in the respiratory carriers indicate that the tricarboxylic acid cycle flux is controlled by the respiratory chain. The data also show a near equilibrium between the electron carriers and the adenylate system and suggest that the equilibrium hypothesis of mitochondrial respiratory control is applicable to intact myocardial tissue.

Adenosine Diphosphate↗
PubMed1979

Studies of the orientation of the mitochondrial redox carriers. III. Orientation of the gx and gy axes of the hemes of cytochrome oxidase with respect to the plane of the membrane in oriented membrane multilayers.

The EPR absorption properties of the hemes of cytochrome oxidase and their liganded derivatives were examined in oriented multilayers from isolated oxidase, mitochondrial membranes and membrane fragments of a bacterium, Paracoccus denitrificans. The hemes of the oxidase in all the systems investigated were oriented normal to the plane of the multilayers. The directions of the g signals corresponding to the gx and gy axes of the g tensor were found to be different in low-spin ferric heme in fully oxidized oxidase and in half-reduced liganded oxidase. It is suggested that this different orientation of gx and gy in fully oxidized oxidase and half-reduced liganded oxidase arises because the respective EPR signals belong to two different hemes, those of cytochrome a and a3.

Animals↗
PubMed1976

Stimulation of ammonia production and excretion in the rabbit by inorganic phosphate. Study of control mechanisms.

The purpose of this study was to clarify the mechanism (s) responsible for regulation of ammonia production and excretion in the rabbit. The normally low ammonia excretion rate during acute metabolic acidosis was stimulated acutely and increased approximately ninefold after infusion of sodium phosphate, but remained low if sodium sulphate or Tris was substituted for phosphate. Ammonia production was increased significantly by phosphate in rabbit renal cortex slices and in isolated renal cortex mitochondria. In isolated mitochondria, mersalyl, an inhibitor of both the phosphate/hydroxyl and phosphate/dicarboxylate mitochondrial carriers, inhibited the phosphate-induced stimulation, indicating that phosphate must enter the mitochondrion for stimulation. A malate/phosphate exchange seemed to be involved since N-ethylmaleimide, an inhibitor of the phosphate/hydroxyl exchange, did not inhibit phosphate-stimulated ammonia production, whereas there was inhibition by 2-n-butylmalonate, a competitive inhibitor of the dicarboxylate carrier. Phosphate itself was not essential since malonate stimulated ammoniagenesis in the absence of added phosphate. Similarly, citrate stimulated ammoniagenesis in isolated mitochondria in the absence of inorganic phosphate provided that it induced L-malate exit on the citrate transporter associated with inhibition of citrate oxidation by fluoroacetate. Similar results were also seen in mitochondria from rat renal cortex. A fall in mitochondrial alpha-ketoglutarate level resulted in an increase in ammonia production. This could be achieved directly with malonate or indirectly via L-malate exit. Simultaneous measurements of glutamate showed that the rate of ammonia production was reciprocally related to the glutamate content. We conclude that phosphate-induced stimulation of ammoniagenesis in the rabbit kidney is mediated by removal of glutamate, the feedback inhibitor of phosphate-dependent glutaminase. Glutamate removal is linked to phosphate-induced dicarboxylate exit across the mitochondrial membrane.

Acidosis↗
PubMed1977

Spin-labeled acyl atractyloside as a probe of the mitochondrial adenosine diphosphate carrier. Asymmetry of the carrier and direct lipid environment.

A number of spin-labeled acyl derivatives of atractyloside, (m,n)acyl-ATR (general formula: CH3- (CH2)mCX(CH2)nCOO-ATR, where X is an o-azolidine ring containing a nitroxide), have been synthesized. As shown by electron spin resonance (ESR) spectra of spin-labeled acyl-ATR, the nitroxide placed on the acyl chain interacts with the diterpene residue of the atractyloside moiety when incorporated in liposomes. Spin-labeled acyl-ATRs were used to probe the ADP carrier in heart mitochondria. They inhibit ADP transport with the same efficiency as unlabeled acyl-ATRs. The inhibition is a mixed competitive and noncompetitive inhibition. The inhibitor constant is close to 10(-7) M. The long chain acyl-ATRs (10,3)- (7,6)-, (7,8)-, and (5,10)acyl-ATRs) and also the short chain (0,2)acyl-ATR, when added at low concentrations to heart mitochondria, give rise to more immobilized ESR spectra than when added to liposomes. Immobilization is stronger for the first three molecules of the series. The (1,14)acyl-ATR, which possesses a nitroxide almost at the end of the acyl chain near the terminal methyl, gives rise to a spectrum corresponding to a high degree of fluidity. Upon addition of atractyloside or of other specific ligands, spin-labeled long-chain acyl-ATRs bound to the ADP carrier are displaced from their binding site toward the lipid phase of the mitochondrial membrane and the short chain (0,2)acyl-ATR is released into the aqueous phase. Spin-labeled long-chain acyl-ATRs do not show any evidence of binding to a protein when incubated with "inside out" submitochondrial particles, in spite of the fact that these particles are able to transport ADP. These results are discussed with respect to the size and the asymmetry of the ADP carrier in the mitochondrial membrane and the mechanism of ADP transport.

Adenosine Diphosphate↗
PubMed1975

Magnetic resonance studies on the mitochondrial divalent cation carrier.

Measurements of water proton spin relaxation enhancements (epsilon) can be used to discriminate high-affinity binding of Mn-2+ or Gd-3+ to biological membranes, from low-affinity binding. In rat liver mitochondria, epsilon b values of approx. 11 are observed upon binding of Mn-2+ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn-2+ uptake by liver mitochondria results in the subsequent decay of epsilon. Comparison of epsilon with the initial velocity of Mn-2+ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 degrees C and 40 degrees C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn-2+ to the divalent cation pump. Binding of Mn-2+ to blowfly flight muscle also results in substantial epsilon, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in epsilon due to uptake occurs after Mn-2+ is bound. Lanthanide ions are also bound and transported by mitochondria. Addition of Gd-3+ to pigeon heart or rat liver mitochondria results in epsilon b approximately equal to 5-6, which decays with similar kinetics in both systems. The uptake velocity of Gd-3+ in rat liver mitochondria is about 1/6 the rate with which Mn-2+ is transported. Lanthanides also diminish epsilon due to the addition of Mn-2+, and greatly retard the Mn-2+ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses epsilon upon addition of Mn-2+ or Gd-3+ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn-2+, but does not affect epsilon significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of "de-energization". Measurements of epsilon at several NMR frequencies indicate a correlation time (tau b) for carrier-bound Mn-2+ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 degrees C and 30 degrees C. The 13 Kcal/mole activation energy for tau b suggests that the 11 ns time constant at room temperature represents the movement of the Mn-11-carrier comples. On the other hand, tau b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn-2+ binds to a small arm of the carrier which moves independent

Animals↗