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Hydrogen/deuterium (H/D) exchange coupled to mass spectrometry is nowadays routinely used to probe protein interactions or conformational changes. The method has many advantages, e.g. very low sample consumption, but offers limited spatial resolution. One way to higher resolution leads through the use of different proteases or their combinations. In the present work we describe recombinant production, purification and use of aspartic protease zymogen from Rhizopus chimensis, protease type XVIII (EC 3.4.23.6), commonly referred to as rhizopuspepsinogen (Rpg). The enzyme was expressed in Escherichia coli, refolded and purified to homogeneity. A typical yield was approximately 100 mg of pure enzyme per 1 L of original bacterial culture. The kinetics of protease activation, i.e. removal of the propeptide achieved by autolysis in an acidic environment, was followed by mass spectrometry. The digestion efficiency was tested for the protease in solution as well as for the immobilized enzyme. Apomyoglobin was successfully digested under all conditions tested and the protease displayed very low or no autodigestion. The results outperformed those obtained with commercial protease where the digestion of apomyoglobin was incomplete and accompanied by many contaminating peptides. Taken together, the recombinant protease type XVIII can be considered as a new and highly efficient tool for H/D exchange followed by mass spectrometry.

In providing the cell with ATP generated by oxidative phosphorylation, the mitochondrial ADP/ATP carrier plays a central role in aerobic eukaryotic cells. Combining biochemical, genetic, and structural approaches contributes to understanding the molecular mechanism of this essential transport system, the dysfunction of which is implicated in neuromuscular diseases.

International audience; 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.

International audience; Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p . BA complex.

The mitochondrial adenine nucleotide carrier, or Ancp, plays a key role in the maintenance of the energetic fluxes in eukaryotic cells. Human disorders have been found associated to unusual human ANC gene (HANC) expression but also to direct inactivation of the protein, either by autoantibody binding or by mutation. However, the individual biochemical properties of the three HAncp isoforms have not yet been deciphered. To do so, the three HANC ORF were expressed in yeast under the control of the regulatory sequences of ScANC2. Each of the three HANC was able to restore growth on a nonfermentable carbon source of a yeast mutant strain lacking its three endogenous ANC. Their ADP/ATP exchange properties could then be measured for the first time in isolated mitochondria. HANC3 was the most efficient to restore yeast growth, and HAnc3p presented the highest V(M) (80 nmol ADP min(-1) mg protein(-1)) and K(ADP)(M)(8.4 microM). HAnc1p and HAnc2p presented similar kinetic constants (V(M) approximately 30-40 nmol ADP min(-(1) mg protein(-1) and K(ADP)(M) approximately 2.5-3.7 microM), whose values were consistent with HANC1's and HANC2's lower capacity to restore yeast growth. However, the HANC genes restored growth at a lower level than ScANC2, indicating that HAncp amount may be limiting in vivo. To optimize the HAncp production, we investigated their biogenesis into mitochondria by mutagenesis of two charged amino acids in the N-terminus of HAnc1p. Severe effects were observed with the D3A and D3K mutations that precluded yeast growth. On the contrary, the K10A mutation increased yeast growth complementation and nucleotide exchange rate as compared to the wild type. These results point to the importance of the N-terminal region of HAnc1p for its biogenesis and transport activity in yeast mitochondria.

Polyunsaturated fatty acid (PUFA) deficiency affects respiratory rate both in isolated mitochondria and in hepatocytes, an effect that is normally ascribed to major changes in membrane composition causing, in turn, protonophoriclike effects. In this study, we have compared the properties of hepatocytes isolated from PUFA-deficient rats with those from control animals treated with concentrations of the protonophoric uncoupler 2,4-dinitrophenol (DNP). Despite identical respiratory rate and in situ mitochondrial membrane potential (ΔΨ), mitochondrial and cytosolic ATP/ADP–Pi ratios were significantly higher in PUFA-deficient cells than in control cells treated with DNP. We show that PUFA-deficient cells display an increase of phosphorylation efficiency, a higher mitochondrial ATP/ADP–Pi ratio being maintained despite the lower ΔΨ. This is achieved by (1) decreasing mitochondrial Pi accumulation, (2) increasing ATP synthase activity, and (3) by increasing the flux control coefficient of adenine nucleotide translocation. As a consequence, oxidative phosphorylation efficiency was only slightly affected in PUFA-deficient animals as compared to protonophoric uncoupling (DNP). Thus, the energy waste induced by PUFA deficiency on the processes that generate the proton motive force (pmf) is compensated in vivo by powerful adaptive mechanisms that act on the processes that use the pmf to synthesize ATP.

A functional recombinant mitochondrial ADP/ATP carrier from the yeast Saccharomyces cerevisiae that bears a six-histidine tag at the C-terminus, Anc2(His(6))p, has been engineered to allow its purification by immobilized metal-ion affinity chromatography (IMAC). The tagged carrier was expressed at a level similar to that of unmodified Anc2p as determined by immunodetection and titration of the specific atractyloside binding sites. Anc2(His(6))p, enriched by chromatography on hydroxyapatite of detergent extracts of mitochondria, was still contaminated by mitochondrial proteins and a large amount of ergosterol. It was highly purified after adsorption on Ni-NTA resin and elution by imidazole buffer, with a 90-95% overall yield. Anc2(His(6))p interacted differently with immobilized ions depending on whether it was unliganded or bound to carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors of the ADP/ATP transport, thus indicating that accessibility of the C-terminus is markedly influenced by the conformational state of the carrier. Fluorometric assays demonstrated that purified unliganded Anc2(His(6))p was in a functional state since it underwent CATR- and BA-sensitive and ADP (or ATP)-induced conformational changes. Large-scale purification of Anc2(His(6))p-CATR and Anc2(His(6))p-BA complexes by IMAC will be of major interest for structural analysis of the ADP/ATP carrier.

Under the conditions of oxidative phosphorylation, the mitochondrial ADP/ATP carrier catalyses the one to one exchange of cytosolic ADP against matrix ATP across the inner mitochondrial membrane. The ADP/ATP transport system can be blocked very specifically by two families of inhibitors: atractyloside (ATR) and carboxyatractyloside (CATR) on one hand, and bongkrekic acid (BA) and isobongkrekic acid (isoBA) on the other hand. It is well established that these inhibitors recognise two different conformations of the carrier protein, the CATR- and BA-conformations, which exhibit different chemical, immunochemical and enzymatic reactivities. The reversible transition of the ADP/ATP carrier between the two conformations was studied by fluorometric techniques. This transconversion, which is only triggered by transportable nucleotides, is probably the same as that which occurs during the functioning of ADP/ATP transport system. The fluorometric approach, using the tryptophanyl residues of the yeast carrier as intrinsic fluorescence probes, was combined to a mutagenesis approach to elucidate the ADP/ATP transport mechanism at the molecular level. Finally, recent reports that myopathies might result from defect in ADP/ATP transport led us to develop a method to quantify the carrier protein in muscular biopsies.

We describe here the chemical synthesis of the novel methylanthraniloyl (Mant-) derivative of atractyloside (ATR), which is a specific inhibitor of the mitochondrial ADP/ATP carrier. The spectral properties of Mant-ATR and naphthoyl-ATR (N-ATR) are analyzed. Both derivatives bind to the membrane-bound ADP/ATP carrier at the same sites as ATR and carboxyatractyloside (CATR). When Mant-ATR and N-ATR are displaced by CATR, their fluorescence emissions are decreased and increased, respectively. These fluorescence changes allow the titration of the CATR binding sites and therefore the quantitation of the amount of ADP/ATP carrier protein in a biological preparation. The validity of the fluorometric titration was tested with beef heart mitochondria and confirmed by binding assays using radioactive ATR. The fluorometric method was applied to rabbit skeletal muscle homogenate and the results of titration were confirmed by binding assays of radioactive ATR. The reliability of the fluorometric method was assessed by comparing the amounts of CATR binding sites and the content of heme aa3 in muscle homogenates and in isolated mitochondria from the same homogenates. Because of its high sensitivity, the fluorometric titration of the ADP/ATP carrier requires small amounts of tissue. Mant-ATR and N-ATR can therefore be considered as convenient, reliable, and sensitive probes to quantify the amount of ADP/ATP carrier and detect a putative carrier protein deficiency in biopsy samples from human patients suffering from myopathies with no clear identified etiology.

During the transport process the mitochondrial adenine nucleotide carrier (Ancp) undergoes conformational changes which result in modifications of the intrinsic fluorescence of the carrier. To further study these changes by a fluorometric approach, the three tryptophanyl residues (Trp87, Trp126, and Trp235) of the Saccharomyces cerevisiae Anc2p were individually mutated to their tyrosine counterparts. The resulting mutated genes (two-Trp, one-Trp or Trp-less variants) were integrated at the ANC2 locus. A prerequisite for such studies is that all the engineered carrier molecules are still able to catalyze ADP/ATP exchange. The cellular characteristics of the strains expressing the mutated Anc2p and the biochemical properties of the variant Anc2p in mitochondria were examined. Although Trp87 is absolutely conserved in all 30 available Ancp sequences, none of the tryptophanyl residues is essential to the carrier protein folding and the transport activity. The mutated and wild-type Anc2p were expressed to the same level, as evidenced by both ligand binding and immunochemical analyses. When isolated in the presence of detergent, all the variant Anc2p preparations contained ergosterol in similar amounts (9 mol/mol of 35 kDa Anc2p) but no specific interaction was revealed. Our results show that the tryptophanmutated Anc2p are suitable for fluorescence studies, which are reported in the accompanying paper by Roux et al. [(1996) Biochemistry 35, 16125-16131].

The ADP/ATP carrier is an integral membrane protein located in the mitochondrial inner membrane. It mediates the exchange of cytosolic ADP for ATP generated in the mitochondrial matrix. Here, the purification and crystallization of the bovine ADP/ATP carrier in complex with the inhibitor carboxyatractyloside in the presence of LAPAO, an aminoxide detergent, is reported. High-quality crystals were only obtained when excess detergent was removed to reach a controlled detergent-to-protein ratio.

International audience; ATAD3 is a mitochondrial integral inner membrane ATPase with unknown function. ATAD3 is absent in yeast and protozoan and present in all pluricellular eucaryotes where its expression is essential for development. To date, bacterial-based expression of full-length ATAD3 has been unsuccessful because of very high levels of endogenous degradation. Based on Saccharomyces cerevisiae as a heterogeneous expression system, we engineered a high copy strain expressing human ATAD3A-Myc-HIS at a relative high level (2.5mg/l of yeast extract) without significantly affecting yeast growth. Most of the expressed human ATAD3A-Myc-HIS co-purified with the yeast mitochondrial fraction thus suggesting that targeting to this organelle is preserved in yeast. Like the endogenous protein in human cells, ATAD3A-Myc-HIS expressed in yeast is found resistant to extraction with salt and certain detergents, suggesting membrane insertion. Sarkosyl, C13-DAO, C12-DAO and ONMG efficiently solubilized ATAD3A-Myc-HIS from yeast extracts, but these soluble species did not bind to agarose-nickel matrix. By contrast, urea-denaturated ATAD3A-Myc-HIS bound to agarose-nickel beads and could be renatured and eluted to obtain highly pure ATAD3A-Myc-HIS. As the native protein in vivo, this recombinant, renatured species specifically bound in vitro to S100B and S100A1 in Far-Western assays.

In the process of oxidative phosphorylation, the exchange of cytosolic ADP3- against mitochondrial ATP4- across the inner mitochondrial membrane is mediated by a specific carrier protein. Two different conformations for this carrier have been demonstrated on the basis of interactions with specific inhibitors, namely carboxyatractyloside (CATR) and bongkrekic acid (BA). The two conformations, referred to as CATR and BA conformations, are interconvertible, provided that ADP or ATP are present. The functional ADP/ATP carrier is probably organized as a tetramer. In the presence of CATR or BA the tetramer is split into two dimers combined with either of the two inhibitors. The amino acid sequence of the beef heart carrier monomer (297 residues) contains three repeats of about 100 residues each. Experimental results obtained through different approaches, including photolabeling, immunochemistry, and limited proteolysis, can be interpreted on the basis of a model with five or six transmembrane alpha helices per carrier monomer. Two mobile regions involved in the binding of nucleotides and accessible to proteolytic enzymes have been identified. Each of them may be visualized as consisting of two pairs of short amphipathic alpha helices, which can be juxtaposed to form hydrophilic channels facilitating the nucleotide transport. Mutagenesis in yeast is currently being used to detect strategic amino acids in ADP/ATP transport.

The O 2 − generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocyes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O 2 − is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O 2 − production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b −245 , the major membrane redox component of the O 2 − generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b −245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.

The yeast mitochondrial adenine nucleotide carrier isoform encoded by the ANC2 gene has been specifically expressed in a yeast strain disrupted for the two other genes, ANC1 and ANC3. Isolation of the carrier in a functional form was achieved by utilisation of a mixture of two detergents, dodecylmaltoside and Emulphogen. The intrinsic fluorescence of the Anc2 protein was specifically and rapidly enhanced upon addition of the transportable nucleotides ADP and ATP. Fluorescence enhancement was prevented or reversed by the addition of a stoichiometric amount of CATR. Addition of CATR alone elicited a dose-dependent decrease of fluorescence. The ANC2-specific yeast stain offers the means to study a single ADP/ATP carrier, with a well-defined amino acid sequence, suitable for analysis of substrate- or inhibitor-induced conformational changes.

International audience; Detergents are frequently used for protein isolation and solubilization. Their presence is crucial in membrane protein protocols or in lipid raft proteomics. However, they are usually poorly compatible with mass spectrometry. Several different sample preparation protocols are routinely used, but they are either laborious or suffer from sample losses. Here, we describe our alternative method for nonionic detergent removal. It is based on selective detergent extraction after capture of the sample on a reversed phase cartridge. The extraction is performed by chlorinated solvents and works well for polyoxyethylene based nonionic detergents, but also for polymers like polyethylene and propylene glycol. Detergent removal can be also carried out on the protein level but a special care must be taken with hydrophobic proteins. In such cases, it is preferable to perform detergent removal after proteolysis which digests the protein to peptides and reduces the hydrophobicity. The method can easily be automated and is compatible with hydrogen/deuterium exchange coupled to mass spectrometry.

The mitochondrial ADP/ATP carrier (Ancp) has long been a paradigm for studies of the mitochondrial carrier family due to, among other properties, its natural abundance and the existence of specific inhibitors, namely, carboxyatractyloside (CATR) and bongkrekic acid (BA), which lock the carrier under distinct and stable conformations. Bovine Anc1p isolated in complex with CATR in the presence of an aminoxyde detergent (LAPAO) was crystallized and its 3D structure determined. It is the first mitochondrial carrier structure resolved at high resolution (2.2 A, as reported by Pebay-Peyroula et al. (Nature 426:39-44, 2003)). Analyses revealed a monomer while most of the biochemical studies led to hypothesize Ancp functions as a dimer. To address the structural organization issue, we engineered a mutant of the yeast Ancp that corresponds to a covalent homodimer in view of 3D structure determination. We compare in this chapter the purification yield and quality of the chimera tagged either with six histidines at its C-ter end or nine histidines at its N-ter. We show that, as expected, length and position of the tag are important criteria for qualitative purification. We also discuss the advantages and drawbacks of purifying Ancp either from a natural source or from engineered yeast cells.

ATAD3 is a mitochondrial inner membrane-associated protein that has been predicted to be an ATPase but from which no associated function is known. The topology of ATAD3 in mitochondrial membranes is not clear and subject to controversy. A direct interaction of the N-terminal domain (amino-acids 44-247) with the mtDNA has been described, but the same domain has been reported to be sensitive to limited proteolysis in purified mitochondria. Furthermore, ATAD3 has been found in a large purified nucleoid complex but could not be cross-linked to the nucleoid. To resolve these discrepancies we used two immunological approaches to test whether the N-terminal (amino-acids 40-53) and the C-terminal (amino-acids 572-586) regions of ATAD3 are accessible from the cytosol. Using N-terminal and C-terminal specific anti-peptide antibodies, we carried out back-titration ELISA measurements and immuno-fluorescence analysis on freshly purified human mitochondria. Both approaches showed that the N-terminal region of ATAD3A is accessible to antibodies in purified mitochondria. The N-terminal region of ATAD3A is thus probably in the cytoplasm or in an accessible intermembrane space. On the contrary, the C-terminal region is not accessible to the antibody and is probably located within the matrix. These results demonstrate both that the N-terminal part of ATAD3A is outside the inner membrane and that the C-terminal part is inside the matrix.

International audience; Fenamates like flufenamic acid (FFA) are anti-inflammatory drugs known to alter ion fluxes through the plasma membrane. They are for instance potent blockers of cation and anion channels, and FFA is now commonly used to block currents through TRP channels and receptor-operated channels. However, FFA exerts complex and multifaceted actions on ion transport systems and, in most instances, a molecular understanding of these FFA-dependent modulations is lacking. In addition, FFA is also to known to perturb the homeostasis of Ca(2+). In the present report, we investigated whether the FFA-induced alterations of the Ca(2+) homeostasis could play a role in the FFA-dependent modulation of transmembrane ion fluxes. Experiments performed with the Ca(2+) indicator Fluo-4 on cultured cortical neurons and HEK-293 cells showed that FFA increased the cytosolic concentration of Ca(2+) even in cells kept in a Ca(2+)-free medium or when the endoplasmic reticulum was depleted with thapsigargin. The FFA-dependent Ca(2+) responses were, however, strongly reduced by bongkrekic acid, a specific ligand of the mitochondrial ADP/ATP carrier which, in addition, inhibits the permeability transition pore. Like FCCP, FFA released Ca(2+) from isolated brain mitochondria and indirectly modulates store-operated Ca(2+) channels. We suggest that some of the effects of FFA on plasma membrane ion channels could be explained, at least partially, by its ability to modulate the mitochondrial Ca(2+) homeostasis.

The adenine nucleotide carrier (Ancp) catalyzes the transport of ADP and ATP across the mitochondrial inner membrane, thus playing an essential role in the cellular energy metabolism. Two regions of Anc2p from Saccharomyces cerevisiae are specifically photolabeled using a photoactivable ADP derivative; they are the central matrix loop, m2, and the C-terminal end. To get more insights into the structure-function relationships of the C-terminal region during nucleotide transport, we have developed two independent approaches. In the first we have deleted the last eight amino acids of Anc2p (Anc2pDeltaCter) and demonstrated that the C-terminal end of Anc2p plays an essential role in yeast growth on a non-fermentable carbon source. This resulted from impaired nucleotide binding properties of the Anc2pDeltaCter variant in line with conversion of ADP binding sites from high to low affinity. In the second we probed the ligand-induced conformational changes of Anc2p C-terminal end (i) by assessing its accessibility to anti-C-terminal antibodies and (ii) by measuring intrinsic fluorescence changes of an Anc2p mutant containing only one tryptophan residue located at its C-terminal end (Anc2p3Y-u). We show that the C-terminal region is no further accessible to antibodies when Anc2p binds non-transportable analogues of ADP. Besides, Trp-316 fluorescence is highly increased upon ligand binding, suggesting large conformational changes. Taken together, our results highlight the involvement of the Anc2p C-terminal region in nucleotide recognition, binding, and transport.

The mitochondrial ADP/ATP carrier is the paradigm of the mitochondrial carrier family (MCF), whose members are crucial for cross-talks between mitochondria, where cell energy is mainly produced, and the cytosol, where cell energy is mainly consumed. These carriers share structural and functional characteristics. Resolution of the 3D structure of the beef mitochondrial ADP/ATP carrier, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in substrate binding and transfer from the outside to the inside of mitochondria. To ascertain the role of these residues, namely, Y186, Y190, F191, and Y194, they were mutated into alanine in the yeast mitochondrial ADP/ATP carrier at equivalent positions (Y203, Y207, F208, and Y211). Two residues, Y203 and F208, appeared to be crucial for transport activity but not for substrate binding per se, indicating their involvement in the substrate transfer process through the carrier. Furthermore, it was possible to show that these mutations precluded conformational changes of the matrix loop m2, whose movements were demonstrated to participate in substrate transport by the wild-type carrier. Therefore, these aromatic residues may be involved in substrate gliding, and they may also confer specificity toward adenine nucleotides for the ADP/ATP carrier as compared with the MCF members.

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.

Proteins of the mitochondrial carrier family (MCF) mediate the transport of a large range of compounds, including metabolites and cofactors. They are localized mainly in the inner mitochondrial membrane, except for a few members found in the membranes of peroxisomes. Similarity searches among Dictyostelium discoideum protein sequences identified a total of 31 MCF members. All these are membrane proteins that possess three characteristic repeats of a domain of approximately 100 residues. Among them, three proteins have supplementary structural domains consisting of Ca(2+)-binding motifs made up of 2 or 4 EF-hand units localized on the N-terminal end, facing the mitochondrial intermembrane space. The nature of transported substrates is proposed on the basis of sequence comparison with orthologs characterized biochemically in other organisms, of phylogenetic analysis, and of the conservation of discriminating amino acid residues belonging to the substrate binding sites. Carriers have been grouped in subclasses based on their specificity for the transport of nucleotides, amino acids or keto acids. Furthermore, we have identified an iron carrier of the mitoferrin type, an inorganic phosphate carrier, and three carriers with similarity to uncoupler proteins. This study provides a focus for mitochondrial carrier analysis in Dictyostelium discoideum.

International audience; The oligomerization state of the ADP/ATP carrier is an important issue in understanding the mechanism underlying nucleotide exchange across the inner mitochondrial membrane. The first high resolution structure obtained in the presence of carboxyatractyloside revealed a large cavity formed within a monomer in which the inhibitor is strongly bound. Whereas the protein-protein interactions implicated in the first crystal form are not biologically relevant, the new crystal form described herein, highlights favorable protein-protein interactions. The interactions are mediated by endogenous cardiolipins, which are tightly bound to the protein, two cardiolipins being sandwiched between the monomers on the matrix side. The putative dimerization interface evidenced here is consistent with other structural, biochemical or functional data published so far.

International audience; A recombinant fusion protein combining the mitochondrial ADP/ATP carrier (Anc2p) and the iso-1-cytochrome c (Cyc1p), both from Saccharomyces cerevisiae, has been genetically elaborated with the aim of increasing the polar surface area of the carrier to facilitate its crystallization. The gene encoding the his-tagged fusion protein was expressed in yeast under the control of the regulatory sequences of ScANC2. 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, which demonstrated its transport activity. The kinetic exchange properties of Anc2-Cyc1(His6)p and the wild type his-tagged carrier Anc2(His6)p were very similar. However, Anc2-Cyc1(His6)p restored cell growth less efficiently than Anc2(His6)p which correlates with the lower amount found in mitochondria. Purification of Anc2-Cyc1(His6)p in complex with carboxyatractyloside (CATR), a high affinity inhibitor of ADP/ATP transport, was achieved by combining ion-exchange chromatography and ion-metal affinity chromatography in the presence of LAPAO, an aminoxide detergent. As characterized by absorption in the visible range, heme was found to be present in isolated Anc2-Cyc1(His6)p, giving the protein a red color. Large-scale purification of Anc2-Cyc1(His6)p-CATR complex opens up novel possibilities for the use of crystallographic approaches to the yeast ADP/ATP carrier.

International audience; We isolated yeast Saccharomyces cerevisiae cells transformed with one of the three human adenine nucleotide carrier genes (HANC) that exhibited higher growth capacity than previously observed. The HANC genes were isolated from these clones, and we identified two independent mutations of HANC that led to replacement of valine 181 located in the fourth transmembrane segment by methionine or phenylalanine. Tolerance of this position toward substitution with various amino acids was systematically investigated, and since HANC/V181M was among the most efficient in growth complementation, it was more extensively studied. Here we show that increased growth capacities were associated with higher ADP/ATP exchange activities and not with higher human carrier amount in yeast mitochondria. These results are discussed in the light of the bovine Ancp structure, that shares more than 90% amino acid identity with Hancps, and its interaction with the lipid environment.

Mitochondrial carrier proteins are embedded in the inner mitochondrial membrane and ensure the transport of many important metabolites. The ADP/ATP carrier imports ADP into the mitochondrial matrix in exchange for ATP after synthesis. It is the most studied mitochondrial carrier and its structure was the first to be unraveled at high resolution. The structure reveals six transmembrane helices forming a tightly closed bundle toward the matrix and a funnel-shaped cavity opening toward the intermembrane space. The cavity ends in a narrow pit 10A from the matrix. The analysis of residues located in the cavity hints at the mechanism of binding of adenine nucleotides. Additionally, the presence of conserved proline residues in three sharply kinked helices suggests a translocation mechanism.

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt). Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Two distinct conformations of the mitochondrial ADP/ATP carrier involved in the adenine nucleotide transport are called BA and CATR conformations, as they were distinguished by binding of specific inhibitors bongkrekic acid (BA) and carboxyatractyloside (CATR), respectively. To find out which amino acids are implicated in the transition between these two conformations, which occurs during transport, mutants of the Saccharomyces cerevisiae ADP/ATP carrier Anc2p responsible for resistance of yeast cells to BA were identified and characterized after in vivo chemical or UV mutagenesis. Only four different mutations could be identified in spite of a large number of mutants analyzed. They are located in the Anc2p transmembrane segments I (G30S), II (Y97C), III (L142S), and VI (G298S), and are independently enabling growth of cells in the presence of BA. The variant and wild-type Anc2p were produced practically to the same level in mitochondria, as evidenced by immunochemical analysis and by atractyloside binding experiments. ADP/ATP exchange mediated by Anc2p variants in isolated mitochondria was more efficient than that of the wild-type Anc2p in the presence of BA, confirming that BA resistance of the mutant cells was linked to the functional properties of the modified ADP/ATP carrier. These results suggest that resistance to BA is caused by alternate conformation of Anc2p due to appearance of Ser or Cys at specific positions. Different interactions of these residues with other amino acids and/or BA could prevent formation of stable inactive Anc2p . BA complex.

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.

A novel photoactivatable radioactive ADP derivative, namely, 2-azido-3'-O-naphthoyl-[beta-(32)P]ADP (2-azido-N-[(32)P]ADP), was synthesized with the aim at mapping the substrate binding site(s) of the yeast mitochondrial ADP/ATP carrier. It was used with mitochondria isolated from genetically modified strains of Saccharomyces cerevisiae, producing the native or the His-tagged Anc2p isoform of the carrier. In darkness, 2-azido-N-[(32)P]ADP was reversibly bound to the carrier in mitochondria, without being transported. Upon photoirradiation, only the ADP/ATP carrier was covalently radiolabeled among all mitochondrial proteins. Specificity of labeling was demonstrated since carboxyatractyloside (CATR), a potent inhibitor of ADP/ATP transport, totally prevented the incorporation of the photoprobe. To localize the radioactive region(s), the purified photolabeled carrier was submitted to CNBr or hydroxylamine cleavage. The resulting fragments were characterized and identified by SDS-PAGE, Western blotting, amino acid sequencing, and MALDI-MS and ESI-MS analyses. Two short photolabeled distinct segments, eight and nine residues long, were identified: S183-R191, located in the central part of the ADP/ATP carrier; and I311-K318, belonging to its C-terminal end. Plausible models of organization of the nucleotide binding site(s) of the carrier involving the two regions specifically labeled by 2-azido-N-[(32)P]ADP are proposed.

The mitochondrial adenine nucleotide translocator (ANT) catalyses the exchange of ATP and ADP between the mitochondria and the cytosol. We have cloned and sequenced the gene encoding the Dictyostelium discoideum ANT (DdANT) and analysed its transcriptional regulation. The single copy D. discoideum ant gene encodes a protein of 309 amino acid residues with a predicted molecular mass of 33 469 Da and a pI of 9.85. These values are comparable to those of ANTs from mammals, insects and fungi. The long N-terminal extension characteristic of plant ANT is absent in DdANT. The protein coding region of the D. discoideum ant gene is interrupted by three introns. Polyclonal antibodies directed against the beef heart mitochondrial ANT or its C-terminal peptide recognized the D. discoideum protein. Northern blot analysis revealed that the expression of the D. discoideum ant gene decreased rapidly during the first hours of multicellular development but the amount of protein remained stable throughout differentiation.

Tryptophanyl substitution of the Saccharomyces cerevisiae adenine nucleotide carrier (Anc2p isoform) was not deleterious for the transport activity or the folding of the carrier [preceding paper by Le Saux et al. (1996) Biochemistry 35, 16116-16124]. Conformational changes of the isolated wild-type and Trp-substituted Anc2p variants, induced upon binding of specific substrates [adenosine triphosphate (ATP) or diphosphate (ADP)] or inhibitors [carboxyatractyloside (CATR) or bongkrekic acid (BA)], were studied by measurement of intrinsic fluorescence. Titration of CATR and BA binding sites ended in the same number of sites, namely, 6-7 nmol/mg of wild-type and variant Anc2p. Isolated Anc2p in detergent presented similar emission spectra, suggesting that all tryptophanyl residues were in environments of similar hydrophobicity. Trp87 and Trp126 contributed largely and to a similar extent to the fluorescence enhancement observed in response to ATP binding, while Trp235 contributed negatively and to a small extent to the fluorescence change. Both Trp126 and Trp235, and to a lower extent Trp87, participate in the CATR-induced fluorescence decrease of Anc2p. Responses to BA binding were observed only in the presence of ATP; they consisted of a further fluorescence increase of the Anc2p.ATP complex, which was mainly due to Trp87 and Trp126, Trp235 being much less responsive. The different fluorescence responses of the three Trp residues of Anc2 variants to ATP, CATR, and BA are in agreement with distinct binding sites for these ligands and distinct conformations of the carrier protein recognizing specifically CATR or BA. A mechanistic model is proposed to interpret the transitions between the different conformational states of Anc2p.

As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.

Adenine nucleotide transport in mitochondria is mediated by a specific carrier protein located in the inner mitochondrial membrane. Chemical, immunochemical, enzymatic, fluorometric and genetic approaches have been used to investigate conformational changes of the ADP/ATP carrier that are thought to participate in the transport process.

The main redox component of the O2- generating oxidase complex in neutrophils is believed to be a b-type cytochrome, named cytochrome b558. In the course of purification of cytochrome b558 from rabbit peritoneal neutrophils, another hemoprotein with an apparent molecular mass of 30 kDa, referred to as p-30, was isolated. Although the spectrum of p-30 was virtually identical to that of cytochrome b558, its redox potential, Em,7 = -4 +/- 10 mV, was much less negative than that of cytochrome b558 (-270 +/- 5 mV). The alkaline pyridine hemochrome from purified p-30 was typical of a b-type cytochrome. The 20 N-terminal amino acid residues and some tryptic peptides isolated from p-30 did not show any significant sequence homology to the human phagocyte cytochrome b558 or to mitochondrial and microsomal cytochromes, except for the N-terminal region which displayed some homology to that of rat liver P-450. After subcellular fractionation, p-30 was found to be located in the plasma membrane and the granule fractions, similarly to cytochrome b558. Upon neutrophil activation, part of p-30 was transferred from granules to the plasma membrane.

The folding of the peptide chain of the bovine heart oxoglutarate carrier in the inner mitochondrial membrane and in the membrane of reconstituted proteoliposomes has been investigated by enzymatic and immunochemical approaches using proteinase K and polyclonal site-directed antibodies, respectively. Two peptides corresponding to the amino acid sequences 2-12 (N-terminal peptide) and 303-314 (C-terminal peptide) have been synthesized and coupled to ovalbumin before being used to immunize rabbits. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and by Western blot analysis. Both anti-N-terminal and anti-C-terminal antibodies reacted specifically with the corresponding peptides and with the isolated oxoglutarate carrier, whereas only anti-C-terminal antibodies immunodetected the carrier in mitochondrial lysates and reacted with the membrane-bound carrier in mitoplasts and in freeze-thawed mitochondria. This result indicated that the last 12 C-terminal amino acid residues of the oxoglutarate carrier protein are accessible from the cytosolic side of the inner mitochondrial membrane. Anti-C-terminal antibodies did not recognize the oxoglutarate carrier in reconstituted proteoliposomes unless the membrane was inverted, indicating that the carrier was inserted unidirectionally in proteoliposomes, with an orientation opposite that found in mitochondria. The immunological data were complemented by data from a limited proteolysis study performed on the membrane-bound oxoglutarate carrier in proteoliposomes, using proteinase K. Cleavage of the carrier caused a time-dependent inhibition of the oxoglutarate-oxoglutarate exchange activity of the reconstituted system. Four cleavage sites were identified, between Val-39 and Gln-40, between Tyr-61 and Lys-62, between Phe-169 and Arg-170, and between Arg-182 and Gly-183.(ABSTRACT TRUNCATED AT 250 WORDS)

The presumed NADPH dehydrogenase function of the heterodimeric cytochrome b558 in the neutrophil oxidase complex has been investigated by combined photoaffinity labeling and immunoblot analysis of membrane proteins from bovine neutrophils. The photoaffinity probe was a radiolabeled analog of NADPH, [4-[N-(4-azido-2-nitrophenyl)[3H]amino]butyryl]NADPH ([3H]azido-NADPH), and the antibodies were directed against the C-terminal regions of the two subunits of cytochrome b558. Plasma membrane vesicles obtained by differential centrifugation of bovine neutrophil homogenates were routinely used as a source of NADPH oxidase. They were permeabilized by sodium deoxycholate to facilitate the access of NADPH or its azido analog to the totality of the specific binding sites. In the absence of light, azido-NADPH behaved as a competitive inhibitor of NADPH oxidase with a Ki of 6 microM, and was able to bind to high-affinity specific binding sites with a Kd of 5-6 microM, indicating a higher affinity of the oxidase for the photoprobe than for the substrate NADPH (KM = 30-40 microM). Upon photolabeling, the oxidase was fully inactivated. Following resolution of the membrane proteins by SDS-PAGE, a predominant photolabeled protein band of 80-100 kDa was revealed, which coincided with the large subunit (beta) of cytochrome b558 identified by immunoblot in a parallel gel. The enzymatic deglycosylation of photolabeled neutrophil membranes shifted the masses of both the photolabeled band and the immunoreactive beta subunit from 80-100 to 55-65 kDa in accordance with the glycoprotein nature of the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

The beef heart adenine nucleotide carrier protein (Anc) of the inner mitochondrial membrane can be purified in a form stabilized by binding the inhibitor carboxyatractyloside. The protein is copurified with bound lipid. We show for the first time that phosphatidic acid is one of the lipids bound to Anc. The short spin lattice relaxation time found by 31P MAS/NMR for this lipid indicates that it is tightly bound to the protein. However, this lipid also has a comparatively small chemical shift anisotropy suggesting that it can undergo reorientation in space. The lipid bound to Anc is in a bilayer arrangement, but the phosphatidic acid shows rapid isotropic motion. Phosphatidic acid is also shown to be present in mitochondria, prior to the isolation of Anc. In Triton-solubilized mitochondria, phosphatidic acid, cardiolipin, phosphatidyethanolamine and phosphatidylcholine exhibit resonance lines in the static 31P NMR spectra, but in the purified Anc only the phosphatidyethanolamine and phosphatidylcholine can be detected by this method, even though the other lipids are still present. This demonstrates that the phosphatidic acid and cardiolipin are interacting with the Anc.The thermal denaturation of the Anc was determined by differential scanning calorimetry. The protein denatures at 74°C with a calorimetric enthalpy corresponding to a deeply membrane inserted integral membrane protein and a van't Hoff enthalpy indicative of a dimeric state for the protein.

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

Two peptides corresponding to the amino acid sequences 1–10 (N-terminal peptide) and 303–313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide; they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr ≅14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mt ≅17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152. It can be concluded that the N-terminal and the C-terminal regions of the phosphate carrier both protrude into the cytosol, giving an even number of transmembrane segments, and that the loop containing Arg-140 and Arg-152 protrudes into the matrix space

A rapid filtration technique has been used to measure at room temperature the kinetics of ADP and ATP transport in rat heart mitochondria in the millisecond time range. Transport was stopped by cessation of the nucleotide supply, without the use of a transport inhibitor, thus avoiding any quenching delay. The mitochondria were preincubated for 30 s either in isotonic KCl containing succinate, MgCl2, and Pi (medium P) or in isotonic KCl supplemented only with EDTA and Tris (medium K); they were referred to as energized and resting mitochondria, respectively. The kinetics of [14C]ADP transport in energized mitochondria were apparently monophasic. The plateau value for [14C]ADP uptake reached 4-5 nmol of nucleotide.(mg of protein)-1. Vmax values for [14C]ADP transport of 400-450 nmol exchanged.min-1.(mg of protein)-1 with Km values of the order of 13-15 microM were calculated, consistent with rates of phosphorylation in the presence of succinate of 320-400 nmol of ATP formed.min-1.(mg of protein)-1. The rate of transport of [14C]ATP in energized mitochondria was 5-10 times lower than that of [14C]ADP. Upon uncoupling, the rate of [14C]ATP uptake was enhanced, and that of [14C]ADP uptake was decreased. However, the two rates did not equalize, indicating that transport was not exclusively electrogenic. Transport of [14C]ADP and [14C]ATP by resting mitochondria followed biphasic kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

1. 1. Arylazido analogs of ADP and ATP ( N -4-azido-2-nitrophenyl-aminobutyryl-ADP and N -4-azido-2-nitrophenylaminobutyryl-ATP have been prepared in radioactive form and used in photolabeling experiments to identify the adenine nucleotide carrier in mitochondria and sonic submitochondrial particles. 2. 2. When added in the dark to beef heart mitochondria, azidonitrophenyl-aminobutyryl-ADP binds to the adenine-nucleotide carrier. It is not transported across the membrane to the matrix space, but it inhibits ADP transport in mito-chondria. The inhibition is of a mixed type with a K i value of about 10 μM. 3. 3. The nitrene derivative formed upon photoirradiation of tritiated azidonitrophenylaminobutyryl-ADP or -ATP binds to a polypeptide of apparent molecular weight 30 000 in beef heart mitochondria and 37 000 in Saccharomyces cerevisiae mitochondria. The photolabeling is prevented by preincubation of the mitochondria with atractyloside or carboxyatractyloside. 4. 4. Photoirradiation of sonic submitochondrial particles from beef heart (inside-out particles) with tritiated azidonitrophenylaminobutyryl-ADP or -ATP results in the labeling of the 30 000-dalton polypeptide and also in the labeling of higher molecular weight peptides (50 000–55 000) probably belonging to F 1 -ATPase. Addition of bongkrekic acid specifically decreases the photolabeling of the 30 000-dalton polypeptide. 5. 5. An arylazido derivative of atractyloside ( N -4-azido-2-nitrophenylaminobutyryl atractyloside) binds upon photoirradiation to the 30 000-dalton polypeptide in beef heart mitochondria and to the 37 000-dalton polypeptide in S. cerevisiae mitochondria. 6. 6. Since the adenine nucleotide carrier is readily damaged by ultraviolet light, nitro-arylazido analogs of ADP and ATP or of atractyloside, which are photoactivated in visible light, were used in preference to other azido analogs, which require ultraviolet light for photoactivation. 7. 7. Data presented in this paper support the view that the same mitochondrial protein belonging to the adenine nucleotide transport system is able to bind ADP (or ATP) and atractyloside.

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.

The adenine nucleotide carrier, or Ancp, is an integral protein of the inner mitochondrial membrane. It is established that the inactive Ancp bound to one of its inhibitors (CATR or BA) is a dimer, but different contradictory models were proposed over the past years to describe the organization of the active Ancp. In order to decide in favor of a single model, it is necessary to establish the orientations of the N- and C-termini and thus the parity of the Ancp transmembrane segments (TMS). According to this, we have constructed a gene encoding a covalent tandem dimer of the Saccharomyces cerevisiae Anc2p and we demonstrate that it is stable and active in vivo as well as in vitro. The properties of the isolated dimer are strongly similar to those of the native Anc2p, as seen from nucleotide exchange and inhibitor binding experiments. We can therefore conclude that the native Anc2p has an even number of TMS and that the N- and C-terminal regions are exposed to the same cellular compartment. Furthermore, our results support the idea of a minimal dimeric functional organization of the Ancp in the mitochondrial membrane and we can suggest that TMS 1 of one monomer and TMS 6 of the other monomer in the native dimer are very close to each other.

Publisher Summary This chapter reviews adenine nucleotide (ADP/ATP) transport across the inner membrane of mitochondria, which is catalyzed by a protein and can be identified by its ability to bind specific inhibitory ligands including atractyloside, carboxyatractyloside, and bongkrekic acid. ADP/ATP transport has been found to occur in mitochondria isolated from a number of sources. The ADP/ATP carrier has been recently characterized in the yeast Saccharomyces cerevisiae by covalent photolabeling with 3 H-labeled photoactivable derivatives of atractyloside and ADP. The mitochondrial protein that is able to recognize specifically atractyloside and that may be the ADP/ATP carrier or a part of the carrier has been recently purified from the yeast Saccharomyces cerevisiae . The atractyloside-binding protein is able to bind covalently, upon photoirradiation, all-labeled photoactivable derivatives of atractyloside and of ADP or ATP. Photolabeling by ADP or ATP derivatives is prevented by preincubation of the mitochondria with atractyloside or carboxyatractyloside, which points to the specificity of the ADP or ATP labeling.

The adenine nucleotide carrier (AdN carrier) is a membrane-bound protein (minimum Mr ≃ 32,000) which is responsible for the vectorial exchange between mitochondrial ATP and cytosolic ADP through the inner mitochondrial membrane. During the course of oxidative phosphorylation, the ATP synthesized in mitochondria is exported against ADP generated in cytosol by energy-consuming reactions; this asymmetric exchange is governed by the membrane potential generated by mitochondrial respiration. Two specific inhibitors of the AdN carrier, (carboxy) atractyloside (CATR) and bongkrekic acid (BA), have been used extensively to study the mechanism of ADP/ATP transport. The fact that CATR binds to the carrier from the outside of the mitochondrial membrane and BA from the inside points to the asymmetric orientation of the carrier in the membrane (for review, see Vignais et al. 1985).