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.     

Lawsonia intracellularis contains a gene encoding a functional rickettsia-like ATP/ADP translocase for host exploitation

ATP/ADP translocases are a hallmark of obligate intracellular pathogens related to chlamydiae and rickettsiae. These proteins catalyze the highly specific exchange of bacterial ADP against host ATP and thus allow bacteria to exploit their hosts' energy pool, a process also referred to as energy parasitism. The genome sequence of the obligate intracellular pathogen Lawsonia intracellularis (Deltaproteobacteria), responsible for one of the most economically important diseases in the swine industry worldwide, revealed the presence of a putative ATP/ADP translocase most similar to known ATP/ADP translocases of chlamydiae and rickettsiae (around 47% amino acid sequence identity). The gene coding for the putative ATP/ADP translocase of L. intracellularis (L. intracellularis nucleotide transporter 1 [NTT1(Li)]) was cloned and expressed in the heterologous host Escherichia coli. The transport properties of NTT1(Li) were determined by measuring the uptake of radioactively labeled substrates by E. coli. NTT1(Li) transported ATP in a counterexchange mode with ADP in a highly specific manner; the substrate affinities determined were 236.3 (+/- 36.5) microM for ATP and 275.2 (+/- 28.1) microM for ADP, identifying this protein as a functional ATP/ADP translocase. NTT1(Li) is the first ATP/ADP translocase from a bacterium not related to Chlamydiae or Rickettsiales, showing that energy parasitism by ATP/ADP translocases is more widespread than previously recognized. The occurrence of an ATP/ADP translocase in L. intracellularis is explained by a relatively recent horizontal gene transfer event with rickettsiae as donors.     

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.     

Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis

Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.     

Isolation and sequence analysis of a cDNA encoding the ATP/ADP translocator of Zea mays L.

A cDNA complementary to the mRNA for the ATP/ADP translocator of maize (Zea mays L.) has been identified by virtue of hybridisation with the homologous gene from yeast. The cloned cDNA has been shown by DNA sequence analysis to contain an open reading frame of 954bp., which encodes a polypeptide of molecular weight 40,519. This polypeptide exhibits a high degree of homology to the translocator polypeptides of beef heart and Neurospora crassa mitochondria.     

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.     

Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene

The Rickettsia prowazekii ATP/ADP translocase (Tlc) gene (tlc), previously cloned in Escherichia coli was localized to a 1.6-kb chromosomal fragment. Nucleotide sequence analysis of this fragment revealed an open reading frame of 1494 bp that could encode a hydrophobic protein of 497 amino acids (aa) with an M_r of 56 668. Analysis of the deduced aa sequence revealed that it contained twelve potential membrane-spanning regions. Comparisons between the deduced aa sequence of the R. prowazekii ATP/ADP Tlc and the sequences of mitochondrial (mt) Tic revealed no detectable homologies between the rickettsial and mt sequences. The major protein synthesized in E. coli minicells containing the rickettsial gene exhibited an M_r of approx. 34000.     

Nucleotide sequence of the cloned mRNA and gene of the ADP/ATP carrier from Neurospora crassa.

A cDNA complementary to the mRNA of the ADP/ATP carrier from Neurospora crassa was identified among ordered cDNA clones by hybridizing total polyadenylated RNA to pools of 96 cDNA recombinant plasmids and subsequent cell-free translation of hybridization-selected mRNA. Further carrier cDNAs were found by colony filter hybridization at a frequency of 0.2-0.3%. The gene of the carrier was cloned and isolated on a 4.6-kbp EcoRI fragment of total Neurospora DNA, and the start of the mRNA was determined by S1 nuclease mapping. From the nucleotide sequence of the cDNA and the genomic DNA, the primary structure of the gene, of the mRNA and of the ADP/ATP carrier protein could be deduced. The gene occurs in a single copy in the genome and related genes are absent. It contains two short introns, and a pyrimidine-rich promoter region. The mRNA has a 46-bp 5' end and a 219-bp 3' end. There is an open reading frame coding for the 313 amino acid residues of the Neurospora carrier protein. The amino acid sequence is homologous in 148 positions with the established primary structure of the beef heart carrier.     

Two bovine genes for mitochondrial ADP/ATP translocase expressed differences in various tissues

Two different bovine cDNAs have been characterized that encode closely related homologues of the mitochondrial membrane carrier protein ADP/ATP translocase. One of them codes for the protein that has been characterized previously from bovine heart mitochondria, and the other codes for a protein that differs from it in 33 amino acids out of 297. Including the base substitutions required to bring about these changes in amino acid sequence, the coding regions of the cDNAs differ at 184 positions. In addition, they are extensively diverged in their 3' noncoding sequences, which differ greatly in both length and sequence, and these segments of the cDNAs have been used as hybridization probes to demonstrate that the expression of the two genes giving rise to the two proteins is very different in various bovine tissues. Expression of one gene predominates in heart muscle and that of the other in intestine. Hybridization experiments with digests of genomic DNA have shown the presence of numerous sequences related to the two cDNAs in both the bovine and human genomes. Some of these probably arise from pseudogenes, but three expressed genes have been detected in the human genome. The study of the regulation of the expression of these genes may help to illuminate the basis of tissue-specific human mitochondrial diseases which arise because of defects in mitochondrial enzymes only in the affected tissue and not in other tissues of the same individual.     

Sequence of the bovine mitochondrial phosphate carrier protein: structural relationship to ADP/ATP translocase and the brown fat mitochondria uncoupling protein.

A cDNA encoding the precursor of the bovine mitochondrial phosphate carrier protein has been cloned from a bovine cDNA library using a mixture of 128 different 17-mer oligonucleotides as hybridisation probe. The protein has an N-terminal extension of 49 amino acids not present in the mature protein. This extension has a net positive charge and is presumed to direct the import of the protein from the cytoplasm to the mitochondrion. Comparison of the protein sequence of the mature phosphate carrier with itself, with ADP/ATP translocase and with the uncoupling protein from brown fat mitochondria shows that all three proteins contain a 3-fold repeated sequence approximately 100 amino acids in length, and that the repeats in the three proteins are related to each other. This implies that the three proteins have related three-dimensional structures and mechanisms and that they share a common evolutionary origin. The distribution of hydrophobic residues in the phosphate carrier protein suggests that each repeated 100 amino acid element is composed of two membrane-spanning alpha-helices linked by an extensive hydrophilic domain. This model is similar to that first proposed for the ADP/ATP translocase and later for the brown fat mitochondria uncoupling protein.     

Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.

The Rickettsia prowazekii ATP/ADP translocase was identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using antibodies raised against a synthetic peptide corresponding in sequence to the carboxyl-terminal 17 amino acids of the carrier. Both the translocase of R. prowazekii and that expressed by Escherichia coli transformants containing the rickettsial gene had an apparent molecular mass of 36,500 Da by SDS-PAGE analysis, a mass considerably less than that deduced from the sequence of the gene. The SDS-solubilized translocase aggregated upon heating at 100 degrees C in the presence of disulfide bond-reducing agents. Similar concentrations of disulfide bond-reducing agents inhibited the exchange transport of adenine nucleotides by both R. prowazekii and translocase-expressing E. coli. These data suggested that an intramolecular disulfide bond in the translocase was essential for transport activity. The antipeptide antibodies used for identification of the translocase bound preferentially to inside-out membrane vesicles of translocase-expressing E. coli relative to right-side-out spheroplasts, thus indicating that the carboxyl terminus of the carrier is located on the cytoplasmic side of the bacterial inner membrane. Protease studies were unable to localize the carboxyl terminus because of the resistance of this region of the native translocase to proteolytic cleavage. These data in conjunction with hydrophobicity analysis were used to construct an initial topological model of the translocase within the cell membrane.     

The substrate specificity of the human ADP/ATP carrier AAC1

Abstract

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [³²P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [¹⁴C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.     

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.     

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.     

Mitochondrial ATP synthasome. Cristae-enriched membranes and a multiwell detergent screening assay yield dispersed single complexes containing the ATP synthase and carriers for Pi and ADP/ATP

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.     

Substrate-induced fluorescence changes of the isolated ADP/ATP carrier protein in solution

Fluorescence studies were carried out on a purified preparation of the ADP/ATP carrier protein solubilized in 3-laurylamido-N-N-dimethylpropylaminoxide. The intrinsic fluorescence of the protein was modified upon addition of ADP and ATP and specific inhibitory ligands (carboxyatractyloside and bongkrekic acid). The fluorescence was transitorily enhanced by micromolar concentrations of ADP or ATP. The rise in fluorescence lasted for 10 sec at 25°C. It was suppressed by carboxyatractyloside and on the contrary enhanced by bongkrekic acid. These data were interpreted as reflecting conformational changes probably related to the functioning of the ADP/ATP carrier. Mg⁺⁺ inhibited the ADP- or ATP-induced rise in fluorescence, indicating that the free forms (and not the Mg⁺⁺ complexed forms) of ADP and ATP are the true substrates for the ADP/ATP carrier.     

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u⁻ revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is exclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed.     

Suggested mitochondrial ancestry of non-mitochondrial ATP/ADP

One of the major evolutionary events that transformed endosymbiotic bacterium into mitochondrion was an acquisition of ATP/ADP carrier in order to supply the host with respiration-derived ATP. Along with mitochondrial carrier, unrelated carrier is known which is characteristic of intracellular chlamydiae, plastids, parasitic intracellular eukaryote Encephalitozoon cuniculi, and the genus Rickettsia of obligate endosymbiotic alpha-Proteobacteria. This non-mitochondrial ATP/ADP carrier was recently described in rickettsia-like endosymbionts - a group of obligate intracellular bacteria, classified with the order Rickettsiales, which have diverged after free-living alpha-Proteobacteria but before sister groups of the Rickettsiaceae assemblage (true rickettsiae) and mitochondria. Published controversial phylogenetic data on the non-mitochondrial carrier were reanalysed in the present work using both DNA and protein sequences, and various methods including Bayesian analysis. The data presented are consistent with classic endosymbiont theory for the origin of mitochondria and also suggest that even last but one common ancestor of rickettsiae and organelles may have been an endosymbiotic bacterium in which ATP/ADP carrier has first originated.