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.

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.

A Rox1-independent hypoxic pathway in yeast. Antagonistic action of the repressor Ord1 and activator Yap1 for hypoxic expression of the SRP1/TIR1 gene.

Hypoxic SRP1/TIR1 gene expression depends on the absence of haem but is independent of Rox1-mediated repression. We have found a new hypoxic pathway involving an antagonistic interaction between the Ixr1/Ord1 repressor and the Yap1 factor, a transcriptional activator involved in oxidative stress response. Here, we show that Ord1 repressed SRP1 gene expression under normoxia and hypoxia, whereas Yap1 activated it. Ord1 and Yap1 have been shown to bind the SRP1 promoter in a region extending from -299 to -156 bp upstream of the start codon. A typical AP-1 responsive element lying from -247 to -240 bp allows Yap1 binding. Internal deletion of sequences within the SRP1 promoter were introduced. Two regions were characterized at positions -299/-251 and -218/-156 that, once removed, resulted in a constitutive expression of SRP1 in a wild-type strain under normoxic conditions. Deletion of both these two sequences allowed the bypass of YAP1 requirement in a Deltayap1 strain, whereas these two internal deletions did not yield increased expression in a Deltaord1 strain compared with the full-length promoter. Both a single Deltaord1 mutant and a doubly disrupted Deltayap1 Deltaord1 strain yielded normoxic constitutive SRP1 expression and increased hypoxic SRP1 induction, thereby demonstrating that ord1 is epistatic to yap1. Thus, Yap1 is not directly involved in SRP1 induction by hypoxia, but is necessary to counteract the Ord1 effect.

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.

A constitutive role for GPI anchors in Saccharomyces cerevisiae: cell wall targeting.

GPI anchors are widely represented among organisms and have several cellular functions. It has been proposed that in yeast there are two groups of GPI proteins: plasma membrane-resident proteins, such as Gas1p or Yap3p, and cell wall-targeted proteins, such as Tir1p or alpha-agglutinin. A model has been proposed for the plasma membrane retention of proteins from the first group because of a dibasic motif located just upstream of the GPI-anchoring signal. The results we report here are not in agreement with such a model as we show that constructs containing the C-terminal parts of Gas1p and Yap3p are also targeted to the cell wall. We also detect the genuine Gas1p after cell wall treatment with Quantazyme or Glucanex glycanases. In addition, we show that the GPI-anchoring signal from the human placental alkaline phosphatase (PLAP) is not compatible with the yeast machinery unless the human transamidase hGpi8p is co-expressed. In this condition, this human signal is able to target a protein to the cell wall. Moreover, TIR1 proved to be a multicopy suppressor of Deltagas1 mutation. The present findings suggest a constitutive role for GPI anchors in yeast: the cell wall targeting of proteins.

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.

The Saccharomyces cerevisiae LYS7 gene is involved in oxidative stress protection.

Saccharomyces cerevisiae Lys7p was proposed to be the enzyme catalyzing the dehydratation of homocitrate to cis-homoaconitate, the second step of the lysine biosynthetic pathway. In this communication we provide evidence that Lys7p is involved in oxidative stress protection. Cells deleted for the LYS7 gene displayed, in addition to lysine auxotrophy, methionine auxotrophy, sensitivity to superoxide generating drugs and light irradiation, and diminution of calcineurin activity. The SOD1 gene encoding the Cu/Zn-superoxide dismutase was expressed in strains lacking Lys7p, and although Sodlp was produced in normal amounts no detectable enzyme activity was found. In contrast, the mitochondrial Mn-superoxide dismutase activity did not seem to be impaired. lys7 cells exhibited a normal uptake of Cu from growth medium. The Cu/Zn-superoxide dismutase activity was restored by addition of Cu (but not by addition of other metallic cations) to the growth medium or to cellular extracts, suggesting a lack of Cu2+ at the active site. These results render it necessary to reconsider the role of the Lys7p. Its involvement in Cu metabolism and oxidative-stress protection, and the possibility of a human equivalent in amyotrophic lateral sclerosis are discussed.

Pmt1 mannosyl transferase is involved in cell wall incorporation of several proteins in Saccharomyces cerevisiae.

We constructed hybrid proteins containing a plant alpha-galactosidase fused to various C-terminal moieties of the hypoxic Srp1p; this allowed us to identify a cell wall-bound form of Srp1p. We showed that the last 30 amino acids of Srp1p, but not the last 16, contain sufficient information to signal glycosyl-phosphatidylinositol anchor attachment and subsequent cell wall anchorage. The cell wall-bound form was shown to be linked by means of a beta1,6-glucose-containing side-chain. Pmt1p enzyme is known as a protein-O-mannosyltransferase that initiates the O-glycosidic chains on proteins. We found that a pmt1 deletion mutant was highly sensitive to zymolyase and that in this strain the alpha-galactosidase-Srp1 fusion proteins, an alpha-galactosidase-Sed1 hybrid protein and an alpha-galactosidase-alpha-agglutinin hybrid protein were absent from both the membrane and the cell wall fractions. However, the plasma membrane protein Gas1p still receives its glycosyl-phosphatidylinositol anchor in pmt1 cells, and in this mutant strain an alpha-galactosidase-Cwp2 fusion protein was found linked to the cell wall but devoid of beta1,6-glucan side-chain, indicating an alternative mechanism of cell wall anchorage.

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.

The Saccharomyces cerevisiae RTG2 gene is a regulator of aconitase expression under catabolite repression conditions.

The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACOI mRNA level is further reduced when glutamate is supplied with glucose. To further explore regulation of ACOI expression, we have screened for mutations that reduce expression of an ACOI-lacZ fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACOI only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.

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.

Regulation by low temperatures and anaerobiosis of a yeast gene specifying a putative GPI-anchored plasma membrane protein [corrected].

Expression of the yeast Saccharomyces cerevisiae SRP1 (Serine-rich Protein) gene is shown here to be induced both by low temperature and anaerobic growth conditions. We show that anaerobic SRP1 expression is haem-dependent; however, haem influence does not operate through the action of the hypoxic-gene ROX1 repressor. The SRP1 promoter region displaying the stress-responsive elements is restricted to its first 551 bp, upstream of the initiation codon, although an upstream activation site contained in upstream sequences is required for full promoter activity. In addition, we demonstrate that the TIP1 gene, sharing similar nucleotide and polypeptide structure with SRP1, and previously reported to be a cold-shock-inducible gene, is also a hypoxic gene. Srp1 protein production is similarly induced by low temperature and anaerobic growth conditions. This protein, detected in the plasma membrane fraction, is shown to be exposed on the cell surface via a glycosyl-phosphatidylinositol membrane anchoring.

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.