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.

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.

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.

Cleft palate associated with cervico-facial teratoma: report of two cases and review of the literature.

We report two original cases of association of cleft palate and lateral cervico-facial teratoma. We discuss the embryological explanation. The first child presented a cleft palate associated with two cervico-facial localisations of teratoma. The other had Pierre Robin sequence associated with lateropharyngeal teratoma with an extra sub maxillary localisation. Most reported cases were of midline teratomas, leading different authors to advance a mechanical origin to the cleft. Our cases are different: we could hardly find reported cases of associate lateral tumours and cleft palate, which would suggest two different embryologic mechanisms, or at least a combination of more complicated phenomenons.

Membrane molecule reorientation in an electric field recorded by attenuated total reflection Fourier-transform infrared spectroscopy.

Electric fields play an important role in the physiological function of macromolecules. Much is known about the role that electric fields play in biological systems, but membrane molecule structure and orientation induced by electric fields remain essentially unknown. In this paper, we present a polarized attenuated total reflection (ATR) experiment we designed to study the effect of electric fields on membrane molecule structure and orientation by Fourier-transform infrared (FTIR) spectroscopy. Two germanium crystals used as the internal reflection element for ATR-FTIR experiments were coated with a thin layer of polystyrene as insulator and used as electrodes to apply an electric field on an oriented stack of membranes made of dioleylphosphatidylcholine (DOPC) and melittin. This experimental set up allowed us for the first time to show fully reversible orientational changes in the lipid headgroups specifically induced by the electric potential difference.

Chromatin components as part of a putative transcriptional repressing complex.

The Drosophila HMG1-like protein DSP1 was identified by its ability to inhibit the transcriptional activating function of Dorsal in a promoter-specific fashion in yeast. We show here that DSP1 as well as its mammalian homolog hHMG2 bind to the mammalian protein SP100B and that SP100B in turn binds to human homologs of HP1. The latter is a Drosophila protein that is involved in transcriptional silencing. Each of these proteins represses transcription when tethered to DNA in mammalian cells. These results suggest how heterochromatin proteins might be recruited to specific sites on DNA with resultant specific effects on gene expression.

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.

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.

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.

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.

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.