Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.

Adenosine 5' triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.

Identification of new highly selective inhibitors of the human ADP/ATP carriers by molecular docking and in vitro transport assays.

Mitochondrial carriers are proteins that shuttle a variety of metabolites, nucleotides and coenzymes across the inner mitochondrial membrane. The mitochondrial ADP/ATP carriers (AACs) specifically translocate the ATP synthesized within mitochondria to the cytosol in exchange for the cytosolic ADP, playing a key role in energy production, in promoting cell viability and regulating mitochondrial permeability transition pore opening. In Homo sapiens four genes code for AACs with different tissue distribution and expression patterns. Since AACs are dysregulated in several cancer types, the employment of known and new AAC inhibitors might be crucial for inducing mitochondrial-mediated apoptosis in cancer cells. Albeit carboxyatractyloside (CATR) and bongkrekic acid (BKA) are known to be powerful and highly selective AAC inhibitors, able to induce mitochondrial dysfunction at molecular level and poisoning at physiological level, we estimated here for the first time their affinity for the human recombinant AAC2 by in vitro transport assays. We found that the inhibition constants of CATR and BKA are 4 nM and 2.0 µM, respectively. For finding new AAC inhibitors we also performed a docking-based virtual screening of an in-house developed chemical library and we identified about 100 ligands showing high affinity for the AAC2 binding region. By testing 13 commercially available molecules, out of the 100 predicted candidates, we found that 2 of them, namely suramin and chebulinic acid, are competitive AAC2 inhibitors with inhibition constants 0.3 µM and 2.1 µM, respectively. We also demonstrated that chebulinic acid and suramin are "highly selective" AAC2 inhibitors, since they poorly inhibit other human mitochondrial carriers (namely ORC1, APC1 and AGC1).

Enhanced oxidative stress sensitizes the mitochondrial permeability transition pore to opening in heart from Zucker Fa/fa rats with type 2 diabetes.

AIMS: Obesity and diabetes mellitus type 2 (DM2) frequently coexist and increase the propensity of cardiovascular dysfunction by numerous mechanisms. Chief among them are oxidative stress and Ca(2+) dysregulation, and both are inducers of the mitochondrial permeability transition pore (MPTP). Nevertheless, it is unknown whether MPTP formation is triggered in DM2 animals, and thereby contributing to cardiac dysfunction. We assessed MPTP sensitivity and reactive oxygen species production in cardiac mitochondria, as well as cytosolic Ca(2+) handling in ventricular myocytes from rats with DM2. MAIN METHODS: Male Zucker Fa/fa rats (Fa/fa) 32weeks old presenting DM2, concentric hypertrophy, and diastolic dysfunction were used. MPTP formation was evaluated in isolated mitochondria and Ca(2+) handling in ventricular myocytes, by spectrophotometric and confocal microscope techniques, respectively. KEY FINDINGS: We found that the systolic Ca(2+) transient relaxation was ~40% slower, while mitochondrial H2O2 production increased by ~6-fold. MPTP opening in isolated mitochondria from Fa/fa (mFa/fa) was more sensitive to Ca(2+) than in mitochondria from lean rats (mLean), and correlated with increased thiol group exposure. The mFa/fa showed decreased oxidative phosphorylation capacity. The ATP content decreased in myocytes, while the PCr/ATP ratio remained unchanged and caspase 9 activity largely increased in myocytes from Fa/fa animals. SIGNIFICANCE: Our results showed that oxidative stress and diastolic Ca(2+) dysregulation increased MPTP sensitivity leading to mitochondrial dysfunction and apoptosis. Mitochondrial dysfunction could compromise ATP synthesis, and lower ATP could be linked to decreased SERCA2 activity, which might underlie diastolic dysfunction. Prolonged Ca(2+) transients might further exacerbate mitochondrial dysfunction.

Titration of lysine residues on adenine nucleotide translocase by fluorescamine induces permeability transition.

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

[Atractylis gummifera: from poisoning to the analytic methods]. / Atractylis gummifera : de l'intoxication aux méthodes analytiques.

Atractylis L gummifera is a plant that causes every year serious and often deadly poisonings. In Morocco, 153 cases of poisoning have been recorded between January 1980 and June 1995 by the Moroccan Antipoison Centre. The ignorance by the clinicians, the fast evolution and the frequency of these poisonings are the origin of diagnosis problems. The solution of those problems is to detect atractyloside and carboxyatractyloside in the biologic liquids. Since several decades, some toxicological analytical methods were established in view of an assay. The aim of our paper is to describe the poisoning by this plant and to review the methods of toxicological analysis used from the colorimetric technique until the news recent chromatographic methods.

High efficiency of energy flux controls within mitochondrial interactosome in cardiac intracellular energetic units.

The aim of our study was to analyze a distribution of metabolic flux controls of all mitochondrial complexes of ATP-Synthasome and mitochondrial creatine kinase (MtCK) in situ in permeabilized cardiac cells. For this we used their specific inhibitors to measure flux control coefficients (C(vi)(JATP)) in two different systems: A) direct stimulation of respiration by ADP and B) activation of respiration by coupled MtCK reaction in the presence of MgATP and creatine. In isolated mitochondria the C(vi)(JATP) were for system A: Complex I - 0.19, Complex III - 0.06, Complex IV 0.18, adenine nucleotide translocase (ANT) - 0.11, ATP synthase - 0.01, Pi carrier - 0.20, and the sum of C(vi)(JATP) was 0.75. In the presence of 10mM creatine (system B) the C(vi)(JATP) were 0.38 for ANT and 0.80 for MtCK. In the permeabilized cardiomyocytes inhibitors had to be added in much higher final concentration, and the following values of C(vi)(JATP) were determined for condition A and B, respectively: Complex I - 0.20 and 0.64, Complex III - 0.41 and 0.40, Complex IV - 0.40 and 0.49, ANT - 0.20 and 0.92, ATP synthase - 0.065 and 0.38, Pi carrier - 0.06 and 0.06, MtCK 0.95. The sum of C(vi)(JATP) was 1.33 and 3.84, respectively. Thus, C(vi)(JATP) were specifically increased under conditions B only for steps involved in ADP turnover and for Complex I in permeabilized cardiomyocytes within Mitochondrial Interactosome, a supercomplex consisting of MtCK, ATP-Synthasome, voltage dependent anion channel associated with tubulin ßII which restricts permeability of the mitochondrial outer membrane.

Probing the interactions of carboxy-atractyloside and atractyloside with the yeast mitochondrial ADP/ATP carrier.

Mitochondrial ADP/ATP carriers are inhibited by two natural compounds, atractyloside (ATR) or carboxy-atractyloside (CATR), which differ by one carboxylate group. The interactions of the inhibitors with the carrier were investigated by single-molecule force spectroscopy. Transmembrane alpha helices of the ATR-inhibited carrier displayed heterogeneous mechanical and kinetic properties. Whereas alpha helix H2 showed the most brittle mechanical properties and lowest kinetic stability, alpha helix H5 was mechanically the most flexible and possessed a kinetic stability 9 orders of magnitude greater than that of alpha helix H2. In contrast, CATR-binding substantially increased the kinetic stability of alpha helix H2 and tuned the mechanical flexibility of alpha helices H5 and H6. NMR spectroscopy confirmed that the additional carboxylate group of CATR binds to the sixth alpha helix, indicating that the enhanced stability of H2 is mediated via interactions between CATR and H6.

The effect of N-ethylmaleimide on permeability transition as induced by carboxyatractyloside, agaric acid, and oleate.

In this work, we studied the effect of N-ethylmaleimide on permeability transition. The findings indicate that the amine inhibited the effects of carboxyatractyloside and agaric acid. It is known that these reagents interact with the adenine nucleotide carrier through the cytosolic side. When oleate, which interacts through the matrix side, was used it was found that the amine amplified the effects of oleate on permeability transition. The results also show that N-ethylmaleimide strengthened the inhibition induced by carboxyatractyloside, agaric acid, and oleate on ADP exchange. Furthermore, it was also found that oleate improved the binding of eosin-5-maleimide on the adenine nucleotide translocase.

The ADP and ATP transport in mitochondria and its carrier.

Different from some more specialised short reviews, here a general although not encyclopaedic survey of the function, metabolic role, structure and mechanism of the ADP/ATP transport in mitochondria is presented. The obvious need for an "old fashioned" review comes from the gateway role in metabolism of the ATP transfer to the cytosol from mitochondria. Amidst the labours, 40 or more years ago, of unravelling the role of mitochondrial compartments and of the two membranes, the sequence of steps of how ATP arrives in the cytosol became a major issue. When the dust settled, a picture emerged where ATP is exported across the inner membrane in a 1:1 exchange against ADP and where the selection of ATP versus ADP is controlled by the high membrane potential at the inner membrane, thus uplifting the free energy of ATP in the cytosol over the mitochondrial matrix. Thus the disparate energy and redox states of the two major compartments are bridged by two membrane potential responsive carriers to enable their symbiosis in the eukaryotic cell. The advance to the molecular level by studying the binding of nucleotides and inhibitors was facilitated by the high level of carrier (AAC) binding sites in the mitochondrial membrane. A striking flexibility of nucleotide binding uncovered the reorientation of carrier sites between outer and inner face, assisted by the side specific high affinity inhibitors. The evidence of a single carrier site versus separate sites for substrate and inhibitors was expounded. In an ideal setting principles of transport catalysis were elucidated. The isolation of intact AAC as a first for any transporter enabled the reconstitution of transport for unravelling, independently of mitochondrial complications, the factors controlling the ADP/ATP exchange. Electrical currents measured with the reconstituted AAC demonstrated electrogenic translocation and charge shift of reorienting carrier sites. Aberrant or vital para-functions of AAC in basal uncoupling and in the mitochondrial pore transition were demonstrated in mitochondria and by patch clamp with reconstituted AAC. The first amino acid sequence of AAC and of any eukaryotic carrier furnished a 6-transmembrane helix folding model, and was the basis for mapping the structure by access studies with various probes, and for demonstrating the strong conformation changes demanded by the reorientation mechanism. Mutations served to elucidate the function of residues, including the particular sensitivity of ATP versus ADP transport to deletion of critical positive charge in AAC. After resisting for decades, at last the atomic crystal structure of the stabilised CAT-AAC complex emerged supporting the predicted principle fold of the AAC but showing unexpected features relevant to mechanism. Being a snapshot of an extreme abortive "c-state" the actual mechanism still remains a conjecture.

The Saccharomyces cerevisiae ADP/ATP carrier-iso 1 cytochrome c fusion protein: one-step purification and functional analysis in vitro.

Genetic expression versus plasmidic overexpression of a functional recombinant fusion protein combining the yeast Saccharomyces cerevisiae mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p) has been investigated, with the main aim of increasing the polar surface of the carrier to improve its crystallization properties. The gene encoding the his6-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2 or under the control of the strong yeast PMA1 promoter. In both cases, the chimeric carrier, Anc2-Cyc1(His6)p, was able to restore growth on a non-fermentable carbon source of a yeast strain devoid of functional ADP/ATP carrier, demonstrating its transport activity. Nevertheless, when the expression vector was used, the level of expression of Anc2-Cyc1(His6)p was no greater than that of the chimeric carrier obtained in yeast mitochondria after homologous recombination. Optimal conditions to extract and to purify Anc2-Cyc1(His6)p were determined. A series of detergents was screened for their ability to extract and to preserve in vitro the chimeric carrier. A rapid, single step purification of Anc2-Cyc1(His6)p was developed, using n-dodecyl-beta-d-maltoside (DoDM) as the best detergent to solubilize the chimeric protein. Carboxyatractyloside- (CATR-) and nucleotide-binding sites were preserved in the purified protein. Moreover, the Cyc1p moiety of Anc2-Cyc1(His6)p-CATR complex solubilized in DoDM was still able to interact in vitro with the cytochrome c oxidase (COX), with the same affinity as yeast Cyc1p. Improved production and purification of Anc2-Cyc1(His6)p-CATR complex opens up new possibilities for the use of this protein in crystallographic approaches to the yeast ADP/ATP carrier. Furthermore, Anc2-Cyc1(His6)p may be an useful molecular tool to investigate in vivo interactions between components of the respiratory chain complexes such as COX and the proteins implicated in ATP biogenesis, such as the ATP/ADP carrier.

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart.

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 microM), ATR and CAT (5-100 microM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.

Structural dynamics of the mitochondrial ADP/ATP carrier revealed by molecular dynamics simulation studies.

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier has been recently crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). In the crystal structure, the six-transmembrane helix bundle that defines the nucleotide translocation pathway is closed on the matrix side due to sharp kinks in the odd-numbered helices. The closed conformation is further sealed by the loops protruding into the matrix that interact through an intricate network of charge-pairs. To gain insight into its structural dynamics we performed molecular dynamics (MD) simulation studies of the ADP/ATP carrier with and without its cocrystallized inhibitor. The two trajectories sampled a conformational space around two different configurations characterized by distinct salt-bridge networks with a significant shift from inter- to intrarepeat bonding on the matrix side in the absence of CATR. Analysis of the geometrical parameters defining the transmembrane helices showed that even-numbered helices can undergo a face rotation, whereas odd-numbered helices can undergo a change in the wobble angle with a conserved proline acting as molecular hinge. Our results provide new information on the dynamical properties of the ADP/ATP carrier and for the first time yield a detailed picture of a stable carrier conformation in absence of the inhibitor.

Activation of a novel long-chain free fatty acid generation and export system in mitochondria of diabetic rat hearts.

A number of reports indicate that a long-chain free fatty acid export system may be operating in mitochondria. In this study, we sought evidence of its existence in rat heart mitochondria. To determine its potential role, we also sought evidence of its activation or inhibition in streptozotocin (STZ)-induced diabetic rat heart mitochondria. If confirmed, it could be a novel mechanism for regulation of long-chain fatty acid oxidation (FAO) in mitochondria. To obtain evidence of its existence, we tested whether heart mitochondria presented with palmitoyl-carnitine can generate and export palmitate. We found that intact mitochondria indeed generate and export palmitate. We have also found that the rates of these processes are markedly higher in STZ-diabetic rat heart mitochondria, in which palmitoyl-carnitine oxidation is also increased. Since mitochondrial thioesterase-1 (MTE-1) hydrolyzes acyl-CoA to CoA-SH + free fatty acid, and uncoupling protein-3 (UCP-3), reconstituted in liposomes, transports free fatty acids, we examined whether these proteins are also increased in STZ-diabetic rat heart mitochondria. We found that both of these proteins are indeed increased. Gene expression profile analysis revealed striking expression of mitochondrial long-chain fatty acid transport and oxidation genes, accompanying overexpression of MTE-1 and UCP-3 in STZ-diabetic rat hearts. Our findings provide the first direct evidence for the existence of a long-chain free fatty acid generation and export system in mitochondria and its activation in STZ-diabetic rat hearts in which FAO is enhanced. We suggest that its activation may facilitate, and inhibition may limit, enhancement of FAO.

The transcript levels of two plant mitochondrial uncoupling protein (pUCP)-related genes are not affected by hyperosmotic stress in durum wheat seedlings showing an increased level of pUCP activity.

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

Conformation-dependent swinging of the matrix loop m2 of the mitochondrial Saccharomyces cerevisiae ADP/ATP carrier.

Structure-function relationships of the membrane-embedded Saccharomyces cerevisiae mitochondrial ADP/ATP carrier were investigated through two independent approaches, namely, limited proteolysis and cysteine labeling. Experiments were carried out in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport that bind to two distinct conformers involved in the translocation process. The proteolysis approach allowed us to demonstrate (i) that N- and C-terminal extremities of ADP/ATP carrier are facing the intermembrane space and (ii) that the central region of the carrier corresponding to the matrix loop m2 is accessible to externally added trypsin in a conformation-sensitive manner, being cleaved at the Lys163-Gly164 and Lys178-Thr179 bonds in the carrier-CATR and the carrier-BA complexes, respectively. The cysteine labeling approach was carried out on the S161C mutant of the ADP/ATP carrier. This variant of the carrier is fully active, displaying nucleotide transport kinetic parameters and inhibitor binding properties similar to that of wild-type carrier. Alkylation experiments, carried out on mitochondria with the nonpermeable reagents eosin-5-maleimide and iodoacetamidyl-3,6-dioxaoctanediamine-biotin, showed that Cys 161 is accessible from the outside in the carrier-CATR complex, whereas it is masked in the carrier-BA complex. Taken together, our results indicate that the matrix loop m2 connecting the transmembrane helices H3 to H4 intrudes to some extent into the inner mitochondrial membrane. Its participation in the translocation of ADP/ATP is strongly suggested, based on the finding that its accessibility to reagents added outside mitochondria is modified according to the conformational state of the carrier.

Subunits of the yeast mitochondrial ADP/ATP carrier: cooperation within the dimer.

The mitochondrial ADP/ATP carrier, or Ancp, is a member of the mitochondrial carrier family (MCF). It exchanges ADP and ATP between matrix and intermembrane space. It is postulated from numerous experiments that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, and it is inferred that the active unit is a dimer, too. However, the structure of beef Ancp bound to CATR obtained at high resolution is that of a monomer. To ascertain the dimeric organization of Ancp, we have constructed covalent tandem dimers of which one "subunit" (protomer) is the wild type and the other is inactive for ADP/ATP exchange. We have chosen either the op1 mutant or another member of the MCF, the phosphate carrier (Picp). Activities of the chimeras were first evaluated in vivo. The Ancp/op1 constructs exchange the adenine nucleotides. The Anc/Pic chimeras are considered as bifunctional forms since they exchange ADP and ATP and transport P(i) within the same cells. We have then controlled the fact that the chimeras are stable in vivo and in vitro. Proteinase K digestion showed that both protomers of Ancp/op1 have similar organization in the membrane. Analyses of kinetic properties indicated that protomers of Ancp/op1 chimeras crosstalk during the nucleotide exchange unlike those of Anc/Pic. However, full inhibition of phosphate uptake by CATR, a very specific inhibitor of Ancp, strongly suggests that the native functional unit of Ancp, and thus of Picp, is a dimer.

Ectopic expression of the human adenine nucleotide translocase, isoform 3 (ANT-3). Characterization of ligand binding properties.

The adenine nucleotide translocase (ANT) is a key component in maintaining cellular energy homeostasis, and has also been implicated in formation of the mitochondrial permeability transition pore. Human ANT-3 was cloned from a human heart cDNA library and expressed as a histidine-tagged fusion protein in the mitochondria of the Trichoplusia ni. cell line. Overexpression resulted in a concomitant decrease in the endogenous ANT content, allowing for the characterization of binding of known ANT ligands to the human protein. Binding affinities for bongkrekic acid (BKA), ADP, and atractyloside (ATR) were measured in mitochondria from the human ANT-3 expressing cell line, and compared to similar preparations from bovine heart mitochondria by use of a novel radioiodinated derivative of ATR. Binding to ANT-3 by the high affinity inhibitors BKA and ATR, as well as the lower affinity natural ligand ADP, was similar to that measured in bovine heart mitochondria, and to that previously reported for mammalian heart mitochondria. Characterizations such as these of human ANT isoforms may lead to drug development for enhanced mitochondrial function and cellular viability.

Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states.

To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis. Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA). EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively. When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel. Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix. In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier. These residues correspond to those modified with EMA in the yeast carrier in the c-state. Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation. On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.

Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation.

Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6'-O-fluorescein-atractyloside.

Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6'- O -fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family.