Complete replacement of basic amino acid residues with cysteines in Rickettsia prowazekii ATP/ADP translocase

The ATP/ADP translocase (Tlc) of Rickettsia prowazekii is a basic protein with isoelectric point (pI)=9.84. It is conceivable, therefore, that basic residues in this protein are involved in electrostatic interactions with negatively charged substrates. We tested this hypothesis by individually mutating all basic residues in Tlc to Cys. Unexpectedly, mutations of only 20 out of 51 basic residues resulted in greater than 80% inhibition of transport activity. Moreover, 12 of 51Cys-substitution mutants exhibited higher than wild-type (WT) activity. At least in one case this up-effect was additive and the double mutant Lys422Cys Lys427Cys transported ATP five-fold better than WT protein. Since in these two single mutants and in the corresponding double mutant K(m)'s were similar to that of WT protein, we conclude that Tlc may have evolved a mechanism that limits the transporter's exchange rate and that at least these two basic residues play a key role in that mechanism. Based on the alignment of 16 Tlc homologs, the loss of activity in the mutants poorly correlates with charge conservation within the Tlc family. Also, despite the presence of three positively charged and one negatively charged intramembrane residues, we have failed to identify potential charge pairs (salt bridges) by either charge reversal or charge neutralization approaches.     

Deep Origin of Plastid/Parasite ATP/ADP Translocases

Abstract Membrane proteins that transport ATP and ADP have been identified in mitochondria, plastids, and obligate intracellular parasites. The mitochondrial ATP/ADP transporters are derived from a broad-specificity transport family of eukaryotic origin, whereas the origin of the plastid/parasite ATP/ADP translocase is more elusive. Here we present the sequences of five genes coding for ATP/ADP translocases from four species of Rickettsia. The results are consistent with an early duplication and divergence of the five ATP/ADP translocases within the rickettsial lineage. A comparison of the phylogenetic depths of the mitochondrial and the plastid/parasite ATP/ADP translocases indicates a deep origin for both transporters. The results provide no evidence for a recent acquisition of the ATP/ADP transporters in Rickettsia via horizontal gene transfer, as previously suggested. A possible function of the two types of ATP/ADP translocases was to allow switches between glycolysis and aerobic respiration in the early eukaryotic cell and its endosymbiont.     

Isolation and characterization of a rice cDNA clone encoding ATP/ADP translocator

We isolated a rice cDNA clone which encodes an open reading frame of 382 amino acids. Its deduced amino acid sequence corresponds to an ATP/ADP translocator protein. Its homology with a maize ATP/ADP translocator was 83.9% in nucleotide sequence, and 90.2% of the amino acid level. Expression of this gene is regulated by such external stresses as salinity and low temperature.     

Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate

The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use.By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate.The fluorescent transfer is inhibited by ADP, bongkrekate and carboxy-atractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix.The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment.The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.     

Relation between extra- and intramitochondrial ATP/ADP ratios in rat liver mitochondria

Changes of the extra- and intramitochondrial ATP/ADP ratios as a function of the respiratory state were measured in incubations with rat liver mitochondria. ATPase or creatine/creatine kinase was used to change the extramitochondrial ATP/ADP ratio; the separation of the mitochondrial pellet was performed by a Millipore filtration technique. Under all conditions tested, the intramitochondrial ratio changed in the same direction as the extramitochondrial one, except in the presence of atractylate where this correlation was not observed. Furthermore, it could be shown that the oxygen uptake and pyruvate carboxylase activity correlated with the intramitochondrial ATP/ADP ratio and not with the extramitochondrial one. These results do not support the proposal that the adenine nucleotide translocase is rate limiting for respiration.     

Control of the Mitochondrial Permeability Transition Pore by High-Affinity ADP Binding at the ADP/ATP Translocase in Permeabilized Mitochondria

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca²⁺-inducedpermeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was preventedby carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity forinhibition by ADP. This suggests that inhibition was related to the conformation of thetranslocase. Ca²⁺ addition was calculated to remove most of the free ADP. Ca²⁺ added after ADPinduced a slow decay of the inhibition, which probably reflected the dissociation of ADP fromthe translocator. We conclude that the probability of forming a permeability transition pore(PTP) is much greater when the translocase is in the CAT conformation than in the BKAconformation, and, in the absence of CAT and BKA, the translocator is shifted between theBKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Four mutations in transmembrane domains of the mitochondrial ADP/ATP carrier increase resistance to bongkrekic acid

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.     

Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters

ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H⁺-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters.     

Dialectics in carrier research: The ADP/ATP carrier and the uncoupling protein

A concise review is given of the research in our laboratory on the ADP/ATP carrier (AAC) and the uncoupling protein (UCP). Although homologous proteins, their widely different functions and contrasts are stressed. The pioneer role of research on the AAC, not only for the mitochondrial but also for other carriers, and the present state of their structure-function relationship is reviewed. The function of UCP as a highly regulated H⁺ carrier is described in contrast to the largely unregulated ADP/ATP exchange in AAC. General principles of carrier catalysis as derived from studies on the AAC and UCP are elucidated.     

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

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

Functional characterization of residues within the carnitine/acylcarnitine translocase RX2PANAAXF distinct motif

The mitochondrial carnitine/acylcarnitine carrier (CAC) is characterized by the presence of a distinct motif, RXXPANAAXF, within its sixth transmembrane α-helix. In this study, we analysed the role of the amino acids of this motif in the structure-function relationships of the human CAC by using two complementary approaches. First, we performed functional analysis in the model fungus Aspergillus nidulans of selected mutations with structural and functional relevance. Second, similar mutant human CACs were biochemically characterized after their reconstitution into liposomes. Both analyses have provided relevant information on the importance and role of the CAC motif residues in the activity and metabolic function of CAC. Only the two adjacent alanines, Ala281 and Ala282 in the human CAC, have been found not to be crucial for transport activity and in vivo function. Results obtained from amino acid substitutions of residues Arg275, Asn280 and Phe284 of human CAC together with structural analysis using molecular modelling of the carrier suggest that R275, N280 and F284 are involved in substrate binding during acylcarnitine/carnitine translocation. Furthermore, functional analysis of mutations of residues Pro278 and Ala279 in A. nidulans, together with kinetic data in reconstituted liposomes, suggest a predominant structural role for these amino acids.     

The delivery of ADP/ATP carrier protein to mitochondria probed by fusions with green fluorescent protein and beta-galactosidase

The import of proteins into mitochondria is an essential process, largely investigated in vitro with isolated mitochondria and radioactively labeled precursors. In this study, we used intact cells and fusions with genes encoding two reporter proteins, green fluorescent protein (GFP) and beta-galactosidase (lacZ), to probe the import of the ADP/ATP carrier (AAC). Typical mitochondrial fluorescence was observed with AAC-GFP fusions containing at least one complete transmembrane loop. This confirms the results of in vitro analysis demonstrating that an internal targeting signal was present in each one of the three transmembrane loops of the carrier. The fusions of AAC fragments to beta-galactosidase demonstrated that the targeting signal was capable of delivering the reporter molecule to the mitochondrial surface, but not to internalize it to a protease-inaccessible location. The delivery to a protease-inaccessible location required the presence of more distal sequences present within the third (C-terminal) transmembrane loop of the carrier molecule. The results of our study provide an alternative for investigation in a natural context of mitochondrial protein import in cells when the isolation of intact, functional mitochondria is not achievable.     

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.     

The mitochondrial ADP/ATP carrier: functional and structural studies in the route of elucidating pathophysiological aspects

The mitochondrial ADP/ATP carrier plays a central role in aerobic cell energetics by providing to the cytosol the ATP generated by oxidative phosphorylation. Though discovered around 40 years ago owing to the existence of unique inhibitors and in spite of numerous experimental approaches, this carrier, which stands as a model of the mitochondrial solute carriers keeps some long-standing mystery. There are still open challenging questions among them the precise ADP/ATP transport mechanism, the functional oligomeric state of the carrier and relationships between human ADP/ATP carrier dysfunctioning and pathologies. Deciphering the 3D structure of this carrier afforded a considerable progress of the knowledge but requires now additional data focused on molecular dynamics from this static picture. State of the art in this topic is reviewed and debated in this paper in view of better comprehending origin of the discrepancies in these questions and, finally, the multiple physiological roles of this carrier in eukaryotic cell economy.     

Cloning and Expression Pattern of a Gene Encoding a Putative Plastidic ATP/ADP Transporter from Helianthus tuberosus L.

Herein, we report the cloning and molecular characterization of a full cDNA encoding a putative plastidic ATP/ADP transporter, designated HtAATP, for Helianthus tuberosus L. The ATP/ADP translocator protein was isolated from the tuber-cDNA library of H. tuberosus for the first time. The predicted HtAATP protein was judged as a plastidic ATP/ADP translocator protein from its high homology at the amino acid sequence level to the two Arabidopsis thaliana plastidic ATP/ADP translocator proteins AATP1 and AATP2 （84.8% and 79.9% identity, respectively）. Amino acid sequence analysis of the primary structure of HtAATP revealed that it belonged to the plastidic ATP/ADP transporter family. Hydropathy prediction indicated that HtAATP gene product is a highly hydrophobic membrane protein that contains 10 transmembrane domains to form a spanning topology. Southern blotting analysis showed that the HtAATP gene is a single-copy gene in the H. tuberosus genome. Tissue distribution analysis showed that the HtAATP gene is prominently expressed in sink tissues. A stable expression pattern in tubers at different developmental stages implies an active involvement of HtAATP during carbohydrate formation.     

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 ADP^3– against mitochondrial ATP^4– 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 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 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.     

Conserved structural domains among species and tissues-specific differences in the mitochondrial phosphate-transport protein and the ADP/ATP carrier

Peptide maps were generated of the CNBr-digested mitochondrial phosphate-transport protein and ADP/ATP carrier from bovine and rat heart, rat liver and blowfly flight muscle. Total mitochondrial proteins from the same sources plus pig heart were separated by SDS-polyacrylamide gel electrophoresis. The peptide maps and the total mitochondrial proteins were electroblotted onto nitrocellulose membranes and reacted with rabbit antisera raised against the purified bovine heart phosphate-transport protein and the ADP/ATP carrier. On the basis of antibody specificity, mobility in SDS-polyacrylamide gel electrophoresis, and peptide maps the following was concluded. (1) Phosphate-transport protein and phosphate-transport protein β (pig and bovine heart) react equally with the first and also with the second of two independent phosphate-transport protein-antisera. (2) Tissue-specific structural domains exist for both the phosphate-transport protein and the ADP/ATP carrier, i.e., one phosphate-transport protein-antiserum reacts with the phosphate-transport protein from all assayed sources, the other only with the cardiac phosphate-transport protein. These differences may reflect tissue-specific regulation of phosphate and adenine nucleotide transport. (3) Homologies among the different species are found for the phosphate transport protein and the ADP/ATP carrier, except for the flight muscle ADP/ATP carrier. These conserved structural domains of the phosphate-transport protein may relate directly to catalytic activity. (4) Alkylation of the purified phosphate-transport proteins and the ADP/ATP carriers by the transport inhibitor N-ethylmaleimide affects electrophoretic mobilities but not the antibody binding. (5) Neither of the two phosphate-transport protein-antisera nor the ADP/ATP-carrier antiserum react with both phosphate transport protein and ADP/ATP carrier, even though these two proteins possess similarities in primary structure and function. Possible mechanisms for generating tissue-specific structural differences in the proteins are discussed.     

Structure, evolution and expression of the mitochondrial ADP/ATP translocator gene from Chlamydomonas reinhardtii

Summary The first AUG in the Chlamydomonas reinhardtii ADP/ATP translocator (CRANT) mRNA initiates an open reading frame (ORF) which is very similar (51–79% amino acid identity) to other ANT proteins. In contrast to higher plants, no evidence for a long amino-terminal extension was obtained. The 5 non-transcribed region of the single-copy CRANT gene contains sequence motifs present in other C. reinhardtii nuclear genes. Four introns, whose positions are not conserved in other ANT genes, interrupt the protein coding region. A short heat shock specifically reduces CRANT mRNA levels. CRANT mRNA levels were unaffected by a mutation in photosynthesis. In a dark/light regime CRANT mRNA levels are high in the dark phase and low in the early light phase. Data on translation initiation sites, splice junctions and the codon preferences of C. reinhardtii nuclear genes were compiled. With the exception of two rare codons, ACA and GGA, the CRANT gene exhibits the biased codon usage of C. reinhardtii nuclear genes that are highly expressed during normal vegetative growth.