Molecular, functional, and pathological aspects of the mitochondrial ADP/ATP carrier.

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.

Relations between structure and function of the mitochondrial ADP/ATP carrier.

Import and export of metabolites through mitochondrial membranes are vital processes that are highly controlled and regulated at the level of the inner membrane. Proteins of the mitochondrial carrier family ( MCF ) are embedded in this membrane, and each member of the family achieves the selective transport of a specific metabolite. Among these, the ADP/ATP carrier transports ADP into the mitochondrial matrix and exports ATP toward the cytosol after its synthesis. Because of its natural abundance, the ADP/ATP carrier is the best characterized within MCF, and a high-resolution structure of one conformation is known. The overall structure is basket shaped and formed by six transmembrane helices that are not only tilted with respect to the membrane, but three of them are also kinked at the level of prolines. The functional mechanisms, nucleotide recognition, and conformational changes for the transport, suggested from the structure, are discussed along with the large body of biochemical and functional results.

Structural basis for lipid-mediated interactions between mitochondrial ADP/ATP carrier monomers.

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.

Fluorometric detection of ADP/ATP carrier deficiency in human muscle.

Defects in mitochondrial energy metabolism lead to severe disorders in humans referred to as mitochondriocytopathies. Most of them have been reported to result from deficiencies of one or more complexes of the respiratory chain and, more rarely, from mitochondrial transmembrane metabolite carrier defects. Dysfunctioning of the ADP/ATP carrier, which catalyses the export of matrix ATP in exchange for cytosolic ADP, has been demonstrated to induce myopathies in mouse and in humans. To screen for ADP/ATP carrier deficiency in patients suffering from mitochondriocytopathy with no defined etiology, we have set up a fluorometric assay to quantify the ADP/ATP carrier in small muscle homogenates, without preliminary isolation of mitochondria. The assay is based on the use of a fluorescent derivative of atractyloside, namely naphthoyl-atractyloside, a highly specific inhibitor of ADP/ATP transport. Here, we describe analysis of healthy and pathological muscle samples, and characterization of ADP/ATP carrier deficiencies in two patients, one displaying an absence of the carrier and the second one containing a limited amount of the carrier with altered binding properties.

The secondary structure of the inhibited mitochondrial ADP/ATP transporter from yeast analyzed by FTIR spectroscopy.

Fourier transform infrared spectroscopy has been applied to the study of the carboxyatractyloside-inhibited mitochondrial ADP/ATP transporter from the yeast Saccharomyces cerevisiae, either solubilized in dodecyl maltoside or reconstituted in phosphatidylcholine liposomes. Its secondary structure has been estimated by means of Fourier self-deconvolution followed by curve fit. A Voigt function was used to fit the components of the deconvoluted spectrum, aiming to account for any distortions introduced by deconvolution. For any of the states analyzed, reconstituted or solubilized, in solution or in dry films, 60-70% of the amino acids are found to adopt alpha-helix plus unordered structures, coherent with the six transmembrane spanning helix model. Moreover, the problem of structure preservation on drying was addressed, and several observations pointed to a maintenance of the protein structure in dry films. Comparison of reconstituted and solubilized samples indicated the presence of both lipid-induced changes in the protein (decrease of the beta-sheets and increase of unordered structures) and protein-induced changes in the lipids (strong hydrogen bonding of lipid C=O groups). To obtain a better discrimination of alpha-helix and unordered structure contributions for the reconstituted form, H/D exchange experiments were performed. Between 35% and 45% of the amino acids were finally assigned to alpha-helix structures, compatible with the existence of five or six transmembrane spanning helices in the transporter. The level of H/D exchange was determined after 15 h of exposure to D(2)O vapor to be 85%, reflecting a high accessibility of the amide hydrogens even for the carboxyatractyloside-inhibited state.

Two distinct regions of the yeast mitochondrial ADP/ATP carrier are photolabeled by a new ADP analogue: 2-azido-3'-O-naphthoyl-[beta-32P]ADP. Identification of the binding segments by mass spectrometry.

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.

Purification of histidine-tagged mitochondrial ADP/ATP carrier: influence of the conformational states of the C-terminal region.

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.

A covalent tandem dimer of the mitochondrial ADP/ATP carrier is functional in vivo.

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.

Expression of the ADP/ATP carrier encoding genes in aerobic yeasts; phenotype of an ADP/ATP carrier deletion mutant of Schizosaccharomyces pombe.

The expression of a key mitochondrial membrane component, the ADP/ATP carrier, was investigated in two aerobic yeast species, Kluyveromyces lactis and Schizosaccharomyces pombe. Although the two species differ very much in their respiratory capacity, the expression of the carrier in both yeast species was decreased under partially anaerobic conditions and was induced by nonfermentable carbon sources. The single ADP/ATP carrier encoding gene was deleted in S. pombe. The null mutant exhibits impaired growth properties, especially when cultivated at reduced oxygen tension, and is unable to grow on a nonfermentable carbon source. Our results suggest that the inability of K. lactis and S. pombe to grow under anaerobic conditions can be related in part to the absence of a functional ADP/ATP carrier due to repression of the corresponding gene expression.

The mitochondrial ADP/ATP carrier: structural, physiological and pathological aspects.

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.

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 1. Tryptophanyl residues of the carrier can be mutated without impairing protein activity.

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].

Conformational changes of the yeast mitochondrial adenosine diphosphate/adenosine triphosphate carrier studied through its intrinsic fluorescence. 2. Assignment of tryptophanyl residues of the carrier to the responses to specific ligands.

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.

Cloning of the gene encoding the mitochondrial adenine nucleotide carrier of Schizosaccharomyces pombe by functional complementation in Saccharomyces cerevisiae.

We describe the isolation and sequencing of both cDNA and genomic clones encoding the mitochondrial ADP/ATP carrier (Anc) of Schizosaccharomyces pombe (Sp). The cDNA clone was isolated from a cDNA library of this fission yeast by complementation of a Saccharomyces cerevisiae (Sc) strain defective in adenine nucleotide carrier. The predicted amino acid (aa) sequence (322 aa) shared similarity with the known Anc sequences. It is more closely related to Neurospora crassa (Nc) Anc than to ScAnc1, 2, or 3 or Kluyveromyces lactis (Kl) Anc. Hybridization experiments with ordered libraries of Sp genomic DNA led to the physical mapping (chromosome II, NotI-B region) and the isolation of the Sp ANC1 gene. We also conclude that a single-copy gene encodes the Sp Anc.

Fluorometric titration of the mitochondrial ADP/ATP carrier protein in muscle homogenate with atractyloside derivatives.

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.

Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane.

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.

Biochemical characterisation of the isolated Anc2 adenine nucleotide carrier from Saccharomyces cerevisiae mitochondria.

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.

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor.

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).

A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae.

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

An Escherichia coli tyrosine transfer RNA is a leucine-specific transfer RNA in the yeast Saccharomyces cerevisiae.

While the Escherichia coli Su-3 (tyrT) tyrosine tRNA suppressor inserts only tyrosine at amber codons in E. coli, we show here that in Saccharomyces cerevisiae this tRNA inserts leucine and no significant amounts of any other amino acid. Thus, the E. coli tyrosine tRNA is functionally a leucine tRNA in yeast cytoplasm. This functional identity may correlate with a structural relationship between the E. coli tyrosine and yeast leucine tRNAs, which are both members of the uncommon type II class of tRNA structures. The results raise the possibility that in evolution a tRNA may be more closely related to a tRNA of different acceptor specificity, but of the same type class, than to one with the same amino acid specificity, but of a different type class.