Interspecific and sexual differences in riverine distribution of tropical eels Anguilla spp.

A total of 261 individuals of the four tropical eel species, Anguilla celebesensis, Anguilla marmorata, Anguilla bicolor pacifica and Anguilla interioris, were collected from 12 locations around Sulawesi Island, Indonesia, to gain knowledge about the riverine distribution of tropical eels. Anguilla marmorata was predominant in the lower reaches of Poso River (94·4% of total eel catch in the sampling area), Poso Lake (93·3%), three small inlet rivers of Tomini Bay (100%) and Laa River (92·3%). Anguilla celebesensis occurred frequently in the inlet rivers of Poso Lake (63·5%). Anguilla bicolor pacifica and Anguilla interioris were rare (1.5 and 0.4%, respectively). Otolith Sr:Ca ratio electron‐probe micro analysis (EPMA) for individual migratory histories revealed that 15 A. celebesensis caught in Poso Lake and its inlet rivers were categorized into 14 river eels (Sr:Ca < 2·5) showing upstream migration seemingly at their elver stage and only one sea eel (Sr:Ca ≥ 6·0) that stayed in the marine habitat for the majority of its life after recruiting to Sulawesi Island before its late upstream migration. In A. marmorata, 19 examined eels from Poso Lake and its inlet rivers were all river eels, while 17 eels from the lower reaches of Poso River were two river eels, six sea eels and nine estuarine eels (2·5 ≤ Sr:Ca < 6·0) that mostly lived in the brackish water. The sex ratio of A. celebesensis was highly skewed towards a dominance of females (99%). In A. marmorata, females were predominant in Poso Lake (95·2%), its inlet rivers (94·7%) and Laa River (100%), while males were more frequent in the lower reaches of Poso River (76·5%) and small inlet rivers of Tomini Bay (94·1%). These results indicate that the riverine distribution pattern of tropical eels differs among species and between sexes.     

中国3个不同地域藏族群体线粒体ATP6、ATP8和Cyt b基因的比较: 探查自然选择在基因组的印记

线粒体基因组(Mitochondrial DNA, mtDNA)具有区别于核基因组的诸多特性(例如缺乏重组等), 在人类进化历史的研究领域具有巨大的应用价值。文章通过对3个不同地域藏族群体线粒体基因组的ATP6、ATP8和Cyt b 基因约2 kb区域的测序研究发现: 随着海拔的增加, 3个基因的全序列、ATP6和ATP8基因逐渐偏离中性模式, 但未呈现显著性差异; 随着海拔的降低, Cyt b 基因受到纯化选择的作用逐渐增大; ATP6基因可能存在适应性选择, 并且随着海拔的增高呈现出适应性选择增强的趋势; 选择的主要因素应该是该群体所处的特殊的地理环境, 即不同的地理环境具有直接的选择作用。     

Human-specific amino acid changes found in 103 protein-coding genes

We humans have many characteristics that are different from those of the great apes. These human-specific characters must have arisen through mutations accumulated in the genome of our direct ancestor after the divergence of the last common ancestor with chimpanzee. Gene trees of human and great apes are necessary for extracting these human-specific genetic changes. We conducted a systematic analysis of 103 protein-coding genes for human, chimpanzee, gorilla, and orangutan. Nucleotide sequences for 18 genes were newly determined for this study, and those for the remaining genes were retrieved from the DDBJ/EMBL/GenBank database. The total number of amino acid changes in the human lineage was 147 for 26,199 codons (0.56%). The total number of amino acid changes in the human genome was, thus, estimated to be about 80,000. We applied the acceleration index test and Fisher's synonymous/nonsynonymous exact test for each gene tree to detect any human-specific enhancement of amino acid changes compared with ape branches. Six and two genes were shown to have significantly higher nonsynonymous changes at the human lineage from the acceleration index and exact tests, respectively. We also compared the distribution of the differences of the nonsynonymous substitutions on the human lineage and those on the great ape lineage. Two genes were more conserved in the ape lineage, whereas one gene was more conserved in the human lineage. These results suggest that a small proportion of protein-coding genes started to evolve differently in the human lineage after it diverged from the ape lineage.     

Positive selection on the mitochondrial ATP synthase 6 and the NADH dehydrogenase 2 genes across 22 hare species (genus Lepus)

Amino acid changes in mitochondrial (mt) oxidative phosphorylation (OXPHOS) genes have been suggested as a key adaptation to environmental variation. Here, we analyzed 416 sequences of ATPase synthase 6 (MT‐ATP6) and NADH dehydrogenase 2 (MT‐ND2) in 22 different hare (Lepus) species from across a wide range of habitats and climates. We used site‐ and branch‐based methods to test for positive selection on specific codons and lineages. We found four codons in MT‐ATP6 and five in MT‐ND2 under positive selection, affecting several species lineages. We investigated the association of protein variants at each locus with climate zone, using multinomial generalized linear models (glm), including species, regions, historical introgression events, and the co‐occurring protein variant at the other locus as additional explanatory variables. A significant climate effect as based on the “Köppen climate classification” was observed for MT‐ND2 protein variants as translated from our nucleotide sequences. Moreover, MT‐ND2 protein variants were significantly affected by the co‐occurring MT‐ATP6 protein variant in the same mtDNA molecule. Contrary to the expectation for non‐recombining mitochondrial DNA molecules, the presence of an evolutionarily relatively ancestral protein variant at one locus was associated with a relatively derived protein at the other locus in the same mitochondrial molecule, respectively. The relative evolutionary status of a protein variant was evaluated according to its positions relative to the respective out‐group protein variant in a network analysis of nucleotide sequences. All our results suggest a complex effect of various climatic parameters acting on multiple mtOXPHOS genes in a co‐adaptive way, favoring combinations of ancestral and derived variants.     

Identification of camelid specific residues in mitochondrial ATP synthase subunits

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.     

Identification and biochemical characterization of the novel mutation m.8839G>C in the mitochondrial ATP6 gene associated with NARP syndrome

Mutations in the ATP6 gene are reported to be associated with Leber hereditary optic neuropathy, bilateral striatal necrosis, coronary atherosclerosis risk and neuropathy, ataxia and retinitis pigmentosa (NARP)/maternally inherited Leigh syndromes. Here, we present a patient with NARP syndrome, in whom a previously undescribed mutation was detected in the ATP6 gene: m.8839G>C. Several observations support the concept that m.8839G>C is pathogenically involved in the clinical phenotype of this patient: (1) the mutation was heteroplasmic in muscle; (2) mutation load was higher in the symptomatic patient than in the asymptomatic carriers; (3) cybrids carrying this mutation presented lower cell proliferation, increased mitochondrial DNA (mtDNA) copy number, increased steady‐state OxPhos protein levels and decreased mitochondrial membrane potential with respect to isogenic wild‐type cybrids; (4) this change was not observed in 2959 human mtDNAs from different mitochondrial haplogroups; (5) the affected amino acid was conserved in all the ATP6 sequences analyzed; and (6) using in silico prediction, the mutation was classified as ‘probably damaging’. However, measurement of ATP synthesis showed no differences between wild‐type and mutated cybrids. Thus, we suggest that m.8839G>C may lower the efficiency between proton translocation within F₀ and F₁ rotation, required for ATP synthesis. Further experiments are needed to fully characterize the molecular mechanisms involved in m.8839G>C pathogenicity .     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

Excess of amino acid substitutions relative to polymorphism between X-linked duplications in Drosophila melanogaster

We have obtained sequence polymorphism data from 13 genes belonging to 5 gene families in Drosophila melanogaster where the K(a)/K(s) between copies is greater than 1. Twelve of these 13 loci are X-linked. In general, there is evidence of purifying selection in all families, as inferred both from levels of silent and replacement variation and insertion/deletion variation, suggesting that the loci are likely functional. Shared polymorphisms indicative of gene conversion between paralogs are rare among the X-linked families, in contrast to available data from autosomal duplicates. McDonald-Kreitman tests between duplicates reveal an excess of amino-acid fixations between copies in the X-linked families, suggesting that the divergence between these loci was driven by positive selection. In contrast, available data from autosomal duplicates show a deficit of fixations, consistent with gene conversion being a strong homogenizing force.     

Distribution of ATP and reducing equivalents in Corynebacterium glutamicum during amino acid resolution

The distributions of ATP and reducing equivalents were estimated with the aid of a pre-constructed bionetwork describing the growth characteristics of Corynebacterium glutamicum cultivated in a pH-auxostat. Results showed that the specific growth rate (μ) imposed different degrees of influences on both ATP and reducing equivalents. The ATP generated from the TCA cycle increased about 33% as μ was raised from 0.23 to 0.45 h⁻¹, while the ATP obtained from the EMP pathway decreased from 32.90 to 2.12% over the same growth span. In contrast, the reducing equivalents contributed from both the TCA cycle and the EMP pathway varied slightly. The ATP obtained through glucose utilization was insufficient to balance the microbial demands. As a result, the transformation among amino acids to compensate the ATP requirement was noticed. To enhance product resolution from an amino acid mix, metabolic nodes at AKG, PYR, PEP, and RU5P have been identified and could be considered concurrently as the potential nodes for manipulating metabolic flows in C. glutamicum.     

GUG is an efficient initiation codon to translate the human mitochondrial ATP6 gene

A maternally inherited and practically homoplasmic mitochondrial (mtDNA) mutation, 8527A>G, changing the initiation codon AUG into GUG, normally coding for a valine, was observed in the ATP6 gene encoding the ATPase subunit a. No alternate Met codon could replace the normal translational initiator. The patient harboring this mutation exhibited clinical symptoms suggesting a mitochondrial disease but his mother who carried the same mtDNA mutation was healthy. The mutation was absent from 100 controls and occurred once amongst 44 patients suspected of Leber Hereditary Optic Neuropathy (LHON) but devoid of typical LHON mutations. In patient fibroblasts, no effect of 8527A>G mutation could be demonstrated on the biosynthesis of mtDNA-encoded proteins, on size and the content of ATPase subunit a, on ATP hydrolysis and on mitochondrial membrane potential. In addition, ATP synthesis was barely decreased. Therefore, GUG is a functional initiation codon for the human ATP6 gene.     

Expression and purification of a novel rice (Oryza sativa L.) mitochondrial ATP synthase small subunit in Escherichia coli

To clarify the function of the rice mitochondrial ATP synthase 6 kDa subunit (RMtATP6), a method of producing large quantities of this protein is needed. Here, we describe an Escherichia coli expression system for the rapid and economic expression of RMtATP6. The RMtATP6 gene (GenBank Accession No. ) was cloned into the pGEX-6p-3 vector to allow expression of RMtATP6 as a glutathione S-transferase (GST) fusion protein. The RMtATP6-GST fusion protein was purified by affinity chromatography using a glutathione-Sepharose 4B column. A Western blot analysis using anti-GST antibody showed that the fusion protein was not degraded. After enzymatic cleavage of the GST tail, the RMtATP6 protein showed a molecular weight of around 6 kDa. The predicted pI of this protein is 10.01. After improving the conditions of expression and the purification procedures, the final yield of the entire expression and purification process was about 4.6 mg of pure RMtATP6 protein per liter of bacterial culture.     

ATP5B regulates mitochondrial fission and fusion in mammalian cells

Mitochondria are essential organelles that produce ATP and regulate cell growth, proliferation, and cell death. To maintain homeostasis, fusion and fission of mitochondria must be strictly regulated. Even though oligomerization of ATP synthase could affect the mitochondrial morphology, the exact mechanism is not clear. We confirmed that structure and function of ATP5B, which is a major component of the catalytic center of ATP synthase complexes, are closely connected to the mitochondrial morphology. ATP5B itself can enhance elongation of mitochondria. Moreover, mutations of the threonine residue at β-barrel domain, and the serine residue at nucleotide-binding domain of ATP5B, produce the opposite effect on the fission and fusion of mitochondrial networks. Here, we demonstrate that ATP5B is clearly involved in the mechanism of regulation for mitochondrial fusion and fission in mammalian cells.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10

In an earlier study, the ATP10 gene of Saccharomyces cerevisiae was shown to code for an inner membrane protein required for assembly of the F(0) sector of the mitochondrial ATPase complex (Ackerman, S., and Tzagoloff, A. (1990) J. Biol. Chem. 265, 9952-9959). To gain additional insights into the function of Atp10p, we have analyzed a revertant of an atp10 null mutant that displays partial recovery of oligomycin-sensitive ATPase and of respiratory competence. The suppressor mutation in the revertant has been mapped to the OLI2 locus in mitochondrial DNA and shown to be a single base change in the C-terminal coding region of the gene. The mutation results in the substitution of a valine for an alanine at residue 249 of subunit 6 of the ATPase. The ability of the subunit 6 mutation to compensate for the absence of Atp10p implies a functional interaction between the two proteins. Such an interaction is consistent with evidence indicating that the C-terminal region with the site of the mutation and the extramembrane domain of Atp10p are both on the matrix side of the inner membrane. Subunit 6 has been purified from the parental wild type strain, from the atp10 null mutant, and from the revertant. The N-terminal sequences of the three proteins indicated that they all start at Ser(11), the normal processing site of the subunit 6 precursor. Mass spectral analysis of the wild type and mutants subunit 6 failed to reveal any substantive difference of the wild type and mutant proteins when the mass of the latter was corrected for Ala --> Val mutation. These data argue against a role of Atp10p in post-translational modification of subunit 6. Although post-translational modification of another ATPase subunit interacting with subunit 6 cannot be excluded, a more likely function for Atp10p is that it acts as a subunit 6 chaperone during F(0) assembly.     

Influence of two cell harvesting methods on intracellular ATP and amino acid concentrations in human fibroblast cultures

The intracellular ATP and amino acid concentrations were determined in human fibroblast cultures reaching confluence. The values obtained were very different, depending on the cell harvesting method: trypsinization or scraping. Trypsinization appeared to be the better method for measuring the ATP concentrations (21.25 +/- 0.96 nmol per mg cell protein), this level being much lower with scraping. On the contrary, scraping was the most appropriate method for amino acid measurement. This work underlines the importance of harvesting methods for metabolic studies in human cell cultures.     

骨髓瘤细胞中阿霉素诱导的ATP变化与自噬的关系

目的：检测用阿霉素（doxorubicin DOXO）处理的骨髓瘤细胞株NCI-H929中ATP与自噬表达水平的变化,探讨两者之间的关联。方法：分别以DOXO 2umol/l 24h、DOXO 2umol/l联用自噬抑制剂3MA 10mmol/l 24h处理H-929细胞后,采用MTT法检测细胞存活率;ATP生物发光法检测ATP表达量;Western Blot检测靶细胞自噬标志分子LC3蛋白的表达。结果：各组相对未处理组存活率分别为54%、35%;相对未处理组%ATP分别为400%、150%;DOXO 24h LC3表达显著上调。结论：经DOXO处理H-929细胞系自噬形成,进而ATP上升以保护细胞。     

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.     

Two mutations in mitochondrial ATP6 gene of ATP synthase,related to human cancer,affect ROS,calcium homeostasis and mitochondrial permeability transition in yeast

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.     

Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes

Mitochondrial ATP synthase is mostly isolated in monomeric form, but in the inner mitochondrial membrane it seems to dimerize and to form higher oligomeric structures from dimeric building blocks. Following a period of electron microscopic single particle analyses that revealed an angular orientation of the membrane parts of monomeric ATP synthases in the dimeric structures, and after extensive studies of the monomer-monomer interface, the focus now shifts to the potentially dynamic state of the oligomeric structures, their potential involvement in metabolic regulation of mitochondria and cells, and to newly identified interactions like physical associations of complexes IV and V. Similarly, larger structures like respiratory strings that have been postulated to form from individual respiratory complexes and their supercomplexes, the respirasomes, come into the focus. Progress by structural investigations is paralleled by insights into the functional roles of respirasomes including substrate channelling and stabilization of individual complexes. Cardiolipin was found to be important for the structural stability of respirasomes which in turn is required to maintain cells and tissues in a healthy state. Defects in cardiolipin remodeling cause devastating diseases like Barth syndrome. Novel species-specific roles of respirasomes for the stability of respiratory complexes have been identified, and potential additional roles may be deduced from newly observed interactions of respirasomes with components of the protein import machinery and with the ADP/ATP translocator.