Computational analysis of the transcriptional regulation of the adenine nucleotide translocator isoform 4 gene and its role in spermatozoid glycolytic metabolism.

Computational phylogenetic analysis coupled to promoter sequence alignment was used to understand mechanisms of transcriptional regulation and to identify potentially coregulated genes. Our strategy was validated on the human ANT4 gene which encodes the fourth isoform of the mitochondrial adenine nucleotide translocator specifically expressed during spermatogenesis. The movement of sperm flagella is driven mainly by ATP generated by glycolytic pathways, and the specific induction of the mitochondrial ANT4 protein presented an interesting puzzle. We analysed the sequences of the promoters, introns and exons of 30 mammalian ANT4 genes and constructed regulatory models. The whole human genome and promoter database were screened for genes that were potentially regulated by the generated models. 80% of the identified co-regulated genes encoded proteins with specific roles in spermatogenesis and with functions linked to male reproduction. Our in silico study enabled us to precise the specific role of the ANT4 isoform in spermatozoid bioenergetics.

Evolutionary genomics implies a specific function of Ant4 in mammalian and anole lizard male germ cells.

Most vertebrates have three paralogous genes with identical intron-exon structures and a high degree of sequence identity that encode mitochondrial adenine nucleotide translocase (Ant) proteins, Ant1 (Slc25a4), Ant2 (Slc25a5) and Ant3 (Slc25a6). Recently, we and others identified a fourth mammalian Ant paralog, Ant4 (Slc25a31), with a distinct intron-exon structure and a lower degree of sequence identity. Ant4 was expressed selectively in testis and sperm in adult mammals and was indeed essential for mouse spermatogenesis, but it was absent in birds, fish and frogs. Since Ant2 is X-linked in mammalian genomes, we hypothesized that the autosomal Ant4 gene may compensate for the loss of Ant2 gene expression during male meiosis in mammals. Here we report that the Ant4 ortholog is conserved in green anole lizard (Anolis carolinensis) and demonstrate that it is expressed in the anole testis. Further, a degenerate DNA fragment of putative Ant4 gene was identified in syntenic regions of avian genomes, indicating that Ant4 was present in the common amniote ancestor. Phylogenetic analyses suggest an even more ancient origin of the Ant4 gene. Although anole lizards are presumed male (XY) heterogametic, like mammals, copy numbers of the Ant2 as well as its neighboring gene were similar between male and female anole genomes, indicating that the anole Ant2 gene is either autosomal or located in the pseudoautosomal region of the sex chromosomes, in contrast to the case to mammals. These results imply the conservation of Ant4 is not likely simply driven by the sex chromosomal localization of the Ant2 gene and its subsequent inactivation during male meiosis. Taken together with the fact that Ant4 protein has a uniquely conserved structure when compared to other somatic Ant1, 2 and 3, there may be a specific advantage for mammals and lizards to express Ant4 in their male germ cells.

Adenine nucleotide transporters in organelles: novel genes and functions.

In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

Adenine nucleotide translocase family: four isoforms for apoptosis modulation in cancer.

Mitochondria have important functions in mammalian cells as the energy powerhouse and integrators of the mitochondrial pathway of apoptosis. The adenine nucleotide translocase (ANT) is a family of proteins involved in cell death pathways that perform distinctly opposite functions to regulate cell fate decisions. On the one hand, ANT catalyzes the adenosine triphosphate export from the mitochondrial matrix to the intermembrane space with the concomitant import of ADP from the intermembrane space to the matrix. On the other hand, during periods of stress, ANT could function as a lethal pore and trigger the process of mitochondrial membrane permeabilization, which leads irreversibly to cell death. In human, ANT is encoded by four homologous genes, whose expression is not only tissue specific, but also varies according to the pathophysiological state of the cell. Recent evidence revealed a differential role of the ANT isoforms in apoptosis and a deregulation of their expression in cancer. In this review, we introduce the current knowledge of ANT in apoptosis and cancer cells and propose a novel classification of ANT isoforms.

Adenine nucleotide translocase: a component of the phylogenetically conserved cell death machinery.

Lethal mitochondrial membrane permeabilization has been depicted as the result of two fundamentally distinct processes, namely primary mitochondrial outer membrane permeabilization (MOMP) versus permeability transition (PT) ignited at the level of the mitochondrial inner membrane. MOMP and PT have been connected to apoptosis and necrosis, respectively. Moreover, it has been thought that MOMP was mediated by pro-apoptotic multidomain proteins of the Bcl-2 family (Bax and Bak), which would operate near-to-independently from the permeability transition pore complex (PTPC) composed by voltage-dependent anion channel (VDAC), adenine nucleotide translocase (ANT) and cyclophilin D. A recent paper in Molecular and Cellular Biology now reveals the obligate contribution of one particular ANT isoform to the execution of developmental and homeostatic cell death in Caenorhabditis elegans. The physical and functional interaction between CED-9, the sole multidomain Bcl-2 protein of C. elegans, and ANT emphasizes the existence of an intricate, phylogenetically conserved crosstalk between Bcl-2 family proteins and constituents of the PTPC. In this issue of Cell Death and Differentiation, Malorni et al. further corroborate this notion by showing that type 2 transglutaminase (TG2) is essential for the correct assembly/function of ANT1, and that, at least in some experimental settings, TG2 might be required to enable and/or stabilize the pro-apoptotic association of Bax with ANT1.

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.

The conserved substrate binding site of mitochondrial carriers.

Mitochondrial carriers transport nucleotides, co-factors and metabolic intermediates across the inner mitochondrial membrane permeability barrier. They belong to a family of transporters unique to eukaryotes and they differ in structure and transport mechanism from other secondary transporters. The main structural fold consists of a barrel of six transmembrane alpha-helices closed at the matrix side by a salt-bridge network at the bottom of the cavity. The significant sequence conservation in the mitochondrial carrier family suggests that specific recognition of substrates is coupled to a common mechanism of transport. We have identified a common substrate binding site comprising residues that are highly conserved and, as demonstrated by mutagenesis, are essential for function. The binding site explains substrate selectivity, ion coupling and the effects of the membrane potential on transport. The main contact points in the site are related by threefold symmetry like the common structural fold. The substrate is bound at the midpoint of the membrane and may function as a pivot point for the movements of the transmembrane alpha-helices as the carrier changes conformation. The trigger for the translocation event is likely to be the substrate-induced perturbation of the salt bridge network at the bottom of the cavity.

A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.

Structural and functional implications of the instability of the ADP/ATP transporter purified from mitochondria as revealed by FTIR spectroscopy.

The ADP/ATP transporter shows a high instability when solubilized, making it difficult to obtain functional protein with sufficient purity for long-term spectroscopic studies. When solubilized in the detergent dodecyl maltoside the protein is in equilibrium between the so-called CATR and BA conformations and in a few hours it becomes nonfunctional, unable to bind either its inhibitors or its substrates. By Fourier transform infrared spectroscopy, we studied the structural changes involved in this denaturation process. To do so, the carboxyatractyloside-inhibited protein was used as a structural model for the protein in the CATR conformation and its spectrum was compared with that of the unliganded time-inactivated protein. From the difference spectra of the amide I, amide II, and amide A bands combined with dichroism spectra of the carboxyatractyloside-inhibited protein, we concluded that few structural differences exist between both states, affecting as few as 11 amino acids (3.5% of the protein); the structural changes consisted in the disappearance of large loop structure and the appearance of aggregated strands. We hypothesize that some mitochondrial loop (tentatively loop M1) shows a high tendency to aggregate, being responsible for the observed features. The functional consequences of this hypothesis are discussed.

Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp.

A mitochondrial-type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP--similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial-type AACs. Hydrogenosome-bearing anaerobic ciliates possess clearly distinct mitochondrial-type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial-type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.