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The efficiency of the cellular oxidative phosphorylation system was recently shown to be modulated by common mitochondrial tRNA(A) (rg) haplotypes. The molecular mechanism by which some mt-Tr haplotypes induce these functional differences remains undetermined. Common polymorphisms in mouse mt-Tr genes affect the size of the dihydrouridine loop in the mature tRNA, producing loops of between five and seven nucleotides, the largest being a rare variant among mammals. Here, we analyzed a new mt-Tr variant identified in C3H mice, and found that it is mitochondrial tRNA loop size, but not the specific sequence, that is responsible for the observed differences in cellular respiration. We further found that the sensitivity of mitochondrial protein synthesis to specific inhibitors is dependent on the mt-Tr gene haplotype, and confirmed that the differences in oxidative phosphorylation performance are masked by a reactive oxygen species-induced compensatory increase in mitochondrial biogenesis.

Mitochondrial-DNA diseases have no effective treatments. Allotopic expression—synthesis of a wild-type version of the mutated protein in the nuclear-cytosolic compartment and its importation into mitochondria—has been proposed as a gene-therapy approach. Allotopic expression has been successfully demonstrated in yeast, but in mammalian mitochondria results are contradictory. The evidence available is based on partial phenotype rescue, not on the incorporation of a functional protein into mitochondria. Here, we show that reliance on partial rescue alone can lead to a false conclusion of successful allotopic expression. We recoded mitochondrial mt-Nd6 to the universal genetic code, and added the N-terminal mitochondrial-targeting sequence of cytochrome c oxidase VIII (C8) and the HA epitope (C8Nd6HA). The protein apparently co-localized with mitochondria, but a significant part of it seemed to be located outside mitochondria. Complex I activity and assembly was restored, suggesting successful allotopic expression. However, careful examination of transfected cells showed that the allotopically-expressed protein was not internalized in mitochondria and that the selected clones were in fact revertants for the mt-Nd6 mutation. These findings demonstrate the need for extreme caution in the interpretation of functional rescue experiments and for clear-cut controls to demonstrate true rescue of mitochondrial function by allotopic expression.

Human mitochondrial DNA (mtDNA) shows extensive within-population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic, metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation, insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

A puzzling observation in patients with oxidative phosphorylation (OXPHOS) deficiencies is the presence of combined enzyme complex defects associated with a genetic alteration in only one protein-coding gene. In particular, mutations in the mtDNA encoded cytochrome b gene are associated either with combined complex I+III deficiency or with only complex III deficiency. We have reproduced the combined complex I+III defect in mouse and human cultured cell models harboring cytochrome b mutations. In both, complex III assembly is impeded and causes a severe reduction in the amount of complex I, not observed when complex III activity was pharmacologically inhibited. Metabolic labeling in mouse cells revealed that complex I was assembled, although its stability was severely hampered. Conversely, complex III stability was not influenced by the absence of complex I. This structural dependence among complexes I and III was confirmed in a muscle biopsy of a patient harboring a nonsense cytochrome b mutation.

Sperm motility is dependent on mitochondrial ATP production that relies on the coordinated expression of the mitochondrial and nuclear genomes. It is generally accepted that mammalian ejaculated spermatozoa retain the ability to synthesize mtDNA-encoded proteins but not most of the nuclear ones. This implies an asynchronous regulation of the oxidative phosphorylation-related genes encoded by each genome. Trying to investigate this issue, we unexpectedly found that ejaculated human spermatozoa do not synthesize mtDNA-encoded proteins. Moreover, we estimated that the discrepancy between our observations and those published elsewhere was due to a chloramphenicol-sensitive protein synthesis attributed to mitochondria that instead corresponds to contaminating bacteria.

The existence of reliable mtDNA reference sequences for each species is of great relevance in a variety of fields, from phylogenetic and population genetics studies to pathogenetic determination of mtDNA variants in humans or in animal models of mtDNA-linked diseases. We present compelling evidence for the existence of sequencing errors on the current mouse mtDNA reference sequence. This includes the deletion of a full codon in two genes, the substitution of one amino acid on five occasions and also the involvement of tRNA and rRNA genes. The conclusions are supported by: (i) the re-sequencing of the original cell line used by Bibb and Clayton, the LA9 cell line, (ii) the sequencing of a second L-derivative clone (L929), and (iii) the comparison with 12 other mtDNA sequences from live mice, 10 of them maternally related with the mouse from which the L cells were generated. Two of the latest sequences are reported for the first time in this study (Balb/cJ and C57BL/6J). In addition, we found that both the LA9 and L929 mtDNAs also contain private clone polymorphic variants that, at least in the case of L929, promote functional impairment of the oxidative phosphorylation system. Conse quently, the mtDNA of the strain used for the mouse genome project (C57BL/6J) is proposed as the new standard for the mouse mtDNA sequence.

Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

SummaryElectron flux in the mitochondrial electron transport chain is determined by the superassembly of mitochondrial respiratory complexes. Different superassemblies are dedicated to receive electrons derived from NADH or FADH2, allowing cells to adapt to the particular NADH/FADH2 ratio generated from available fuel sources. When several fuels are available, cells adapt to the fuel best suited to their type or functional status (e.g., quiescent versus proliferative). We show that an appropriate proportion of superassemblies can be achieved by increasing CII activity through phosphorylation of the complex II catalytic subunit FpSDH. This phosphorylation is mediated by the tyrosine-kinase Fgr, which is activated by hydrogen peroxide. Ablation of Fgr or mutation of the FpSDH target tyrosine abolishes the capacity of mitochondria to adjust metabolism upon nutrient restriction, hypoxia/reoxygenation, and T cell activation, demonstrating the physiological relevance of this adaptive response.

The light strand promoter of mammalian mitochondrial DNA gives rise to a primary transcript, but also to the RNA primer necessary for initiation of replication and 7S DNA synthesis as well as 7S RNA. Here we have studied the turnover of 7S DNA in isolated rat liver mitochondria and whether import of mitochondrial transcription factor A (mtTFA), which is necessary for transcription initiation, increases its rate of synthesis. 7S DNA was present as two species, probably due to two different sites of RNA–DNA transition. Time course and pulse–chase experiments showed that the half-life of this DNA is ∼45 min. Import of mtTFA, produced in vitro, into the mitochondrial matrix in stoichiometric amounts significantly increased the rate of 7S DNA formation. We conclude that isolated rat liver mitochondria faithfully synthesize and degrade 7S DNA and that increased matrix levels of mtTFA are sufficient to increase its rate of synthesis, strongly supporting the hypothesis that this process is transcription primed.

Report.-- et al., The textbook description of mitochondrial respiratory complexes (RCs) views them as free-moving entities linked by the mobile carriers coenzyme Q (CoQ) and cytochrome c (cyt c). This model (known as the fluid model) is challenged by the proposal that all RCs except complex II can associate in supercomplexes (SCs). The proposed SCs are the respirasome (complexes I, III, and IV), complexes I and III, and complexes III and IV. The role of SCs is unclear, and their existence is debated. By genetic modulation of interactions between complexes I and III and III and IV, we show that these associations define dedicated CoQ and cyt c pools and that SC assembly is dynamic and organizes electron flux to optimize the use of available substrates.

Nature 535, 561–565 (2016); doi:10.1038/nature18618 In this Letter, the number of mice monitored in Fig. 1a should have stated: ‘n = 31 BL/6C57 and n = 29 BL/6NZB’, rather than: ‘n = 31 per genotype (19 males, 12 females)’. In addition, in Extended Data Table 1, the non-synonymous mutation at nucleotide position 9985 (mt-Nd4l) was inadvertently omitted.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., About half of the mitochondrial DNA (mtDNA) mutations causing diseases in humans occur in tRNA genes. Particularly intriguing are those pathogenic tRNA mutations than can reach homoplasmy and yet show very different penetrance among patients. These mutations are scarce and, in addition to their obvious interest for understanding human pathology, they can be excellent experimental examples to model evolution and fixation of mitochondrial tRNA mutations. To date, the only source of this type of mutations is human patients. We report here the generation and characterization of the first mitochondrial tRNA pathological mutation in mouse cells, an m. 3739G > A transition in the mitochondrial mt-Ti gene. This mutation recapitulates the molecular hallmarks of a disease-causing mutation described in humans, an m. 4290T > C transition affecting also the human mt-Ti gene. We could determine that the pathogenic molecular mechanism, induced by both the mouse and the human mutations, is a high frequency of abnormal folding of the tRNA(Ile) that cannot be charged with isoleucine. We demonstrate that the cells harboring the mouse or human mutant tRNA have exacerbated mitochondrial biogenesis triggered by an increase in mitochondrial ROS production as a compensatory response. We propose that both the nature of the pathogenic mechanism combined with the existence of a compensatory mechanism can explain the penetrance pattern of this mutation. This particular behavior can allow a scenario for the evolution of mitochondrial tRNAs in which the fixation of two alleles that are individually deleterious can proceed in two steps and not require the simultaneous mutation of both., This work was supported by the Spanish Ministry of Education (SAF2009-08007 and CSD2007-00020), the EU (EUMITOCOMBAT-LSHM-CT-2004-503116), and Group of Excellence grant DGA (B55). The CNIC is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation.

Complex I (CI) is the largest enzyme of the mammalian mitochondrial respiratory chain. The biogenesis of the complex is a very complex process due to its large size and number of subunits (45 subunits). The situation is further complicated due to the fact that its subunits have a double genomic origin, as seven of them are encoded by the mitochondrial DNA. Understanding of the assembly process and characterization of the involved factors has advanced very much in the last years. However, until now, a key part of the process, that is, how and at which step the mitochondrially encoded CI subunits (ND subunits) are incorporated in the CI assembly process, was not known. Analyses of several mouse cell lines mutated for three ND subunits allowed us to determine the importance of each one for complex assembly/stability and that there are five different steps within the assembly pathway in which some mitochondrially encoded CI subunit is incorporated.

Cisplatin accumulates in mitochondria, which are a major target for this drug in cancer cells. Thus alterations in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents. Moreover, cisplatin toxic side effects seem to be associated with mitochondrial injury in vivo and in vitro. In order to clarify the potential effect of cisplatin in mtDNA (mitochondrial DNA) maintenance and expression, we have analysed rat liver mtDNA and mtRNA (mitochondrial RNA) synthesis as well as their stability under the influence of in vivo treatment or in vitro exposure to cisplatin. We show that cisplatin causes a direct and significant impairment of mtDNA and mtRNA synthesis and decreases steady-state levels of mtRNAs in isolated mitochondria. Furthermore, in vivo treatment of the animals with cisplatin exerts a protective effect from the impairment of mtRNA metabolism caused by in vitro exposure to the drug, by means of increased mitochondrial GSH levels after in vivo cisplatin treatment.

The structural organization of the mitochondrial respiratory complexes as four big independently moving entities connected by the mobile carriers CoQ and cytochrome c has been challenged recently. Blue native gel electrophoresis reveals the presence of high-molecular-weight bands containing several respiratory complexes and suggesting an in vivo assembly status of these structures (respirasomes). However, no functional evidence of the activity of supercomplexes as true respirasomes has been provided yet. We have observed that (1) supercomplexes are not formed when one of their component complexes is absent; (2) there is a temporal gap between the formation of the individual complexes and that of the supercomplexes; (3) some putative respirasomes contain CoQ and cytochrome c; (4) isolated respirasomes can transfer electrons from NADH to O(2), that is, they respire. Therefore, we have demonstrated the existence of a functional respirasome and propose a structural organization model that accommodates these findings.

2 páginas -- PAGS nros. 571-572, Reactive oxygen species and the segregation of mtDNA sequence variants

Common mitochondrial DNA (mtDNA) haplotypes in humans and mice have been associated with various phenotypes, including learning performance and disease penetrance. Notably, no influence of mtDNA haplotype in cell respiration has been demonstrated. Here, using cell lines carrying four different common mouse mtDNA haplotypes in an identical nuclear background, we show that the similar level of respiration among the cell lines is only apparent and is a consequence of compensatory mechanisms triggered by different production of reactive oxygen species. We observe that the respiration capacity per molecule of mtDNA in cells with the NIH3T3 or NZB mtDNA is lower than in those with the C57BL/6J, CBA/J or BALB/cJ mtDNA. In addition, we have determined the genetic element underlying these differences. Our data provide insight into the molecular basis of the complex phenotypes associated with common mtDNA variants and anticipate a relevant contribution of mtDNA single nucleotide polymorphisms to phenotypic variability in humans. © 2006 Nature Publishing Group.

Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

We report here the identification of a cell line containing single and double missense mutations in cytochrome c oxidase (COX) subunit I gene of mouse mitochondrial DNA. When present in homoplasmy, the single mutant displays a normal complex IV assembly but a significantly reduced COX activity, while the double mutant almost completely compensates the functional defect of the first mutation. We discuss the potential structural consequences of those mutations based on the modeled structure of mouse complex IV. Based on genetic, biochemical and molecular analyses of cultured mouse cells we infer that: (i) deleterious mutations can arise and become predominant; (ii) cultured cells can maintain several mtDNA haplotypes at stable frequencies; (iii) the respiratory chain has little spare COX capacity; and (iv) the size of a cavity in the vicinity of Val421 in CO I of animal COX may affect the function of the enzyme.

Sperm mitochondria play an important role in spermatozoa because of the high ATP demand of these cells. Different mitochondrial DNA (mtDNA) mutations and haplogroups influence sperm function. The mtDNA dose also contributes to genetic variability and pathology in different tissues and organs, but nothing is known about its relevance in the performance of spermatozoa. We estimated the variability in mtDNA content within a population of men. Different mtDNA:nuclear DNA ratios were characteristic of progressive and nonprogressive spermatozoa, confirming the influence of mtDNA content on sperm functionality. We also estimated that the absolute content of mtDNA was 700 and 1200 mtDNA copies per cell in progressive and nonprogressive human spermatozoa, respectively. These results suggest that a marked increase of mtDNA copy number per cell volume takes place during spermatogenesis.

Living isolated mitochondria incubated with iron(II) show a major alteration in mitochondrial DNA (mtDNA) conformational forms as assessed by Southern blot analysis of undigested mtDNA. In the presence of iron(II), form I is transformed into form III in a dose-dependent manner. This alteration in mtDNA conformation shows a strong correlation with a decrease in the mtDNA transcription rate (r=0.965, P

A variety of mtDNA mutations responsible for human diseases have been associated with molecular defects in the OXPHOS system. It has been proposed that mtDNA genetic alterations can also be responsible for sperm dysfunction. In addition, it was suggested that if sperm dysfunction is the main phenotypic consequence, these mutations could be fixed as stable mtDNA variants, because mtDNA is maternally inherited. To test this possibility, we have performed an extensive analysis of the distribution of mtDNA haplogroups in white men having fertility problems. We have found that asthenozoospermia, but not oligozoospermia, is associated with mtDNA haplogroups in whites. Thus, haplogroups H and T are significantly more abundant in nonasthenozoospermic and asthenozoospermic populations, respectively, and show significant differences in their OXPHOS performance.

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria.

Until now, little attention has been paid to the contribution of mitochondrial dysfunction to germinal tissue disorders. The target of this study was to investigate the relationship between sperm motility and mitochondrial respiratory chain enzyme activities. The results obtained showed that semen samples of control individuals (n = 33) have substantially higher activities of complexes I, II, and IV compared with those of asthenozoospermic subjects (n = 86). Moreover, a direct and positive correlation was found in the whole population studied between spermatozoa motility and all the mitochondrial respiratory complex activities assayed (I, II, I+III, II+III, and IV). The ratio of these enzymes to citrate synthase (a reliable enzymatic marker of mitochondrial volume) activities did not correlate with sperm motility. This suggests that motility depends largely on the mitochondrial volume within the sperm midpiece. These observations could be of physiopathological relevance because they suggest that factors affecting the mitochondrial energy production could be then responsible for particular cases of idiopathic asthenozoospermia.

The dependence for the maintenance of the synthesis and maturation of mitochondrial RNA on the supply of nucleo-cytoplasmic factors has been investigated by a novel in organello RNA synthesis system. We found that mitochondrial DNA transcription can be maintained for several hours in isolated mitochondria. Analysis of the individual mitochondrial RNA species revealed that: the processing of the rRNA precursors and the stability of the mature rRNAs, but not the transcription itself, is severely impaired after short periods of incubation, indicating that these processes are strongly dependent on the mitochondrial interaction with the nucleo-cytoplasmic compartment; the events that lead to the synthesis, processing and turnover of the mitochondrial mRNAs do not require the continuous supply of nucleo-cytoplasmic factors, that are accumulated in excess by mitochondria. Furthermore, we present evidence indicating an inhibition of high ATP levels on the mitochondrial RNA polymerase activity, both in organello and in vitro. Consequently, it is proposed that mitochondrial mRNA synthesis can be regulated in response to changes in intramitochondrial ATP levels. This regulation of mitochondrial mRNA synthesis together with their very rapid turnover described here and elsewhere [Gelfand, R. & Attardi, G. (1981) Mol. Cell Biol. 1, 497-511], could represent a mechanism that would allow each individual mitochondrion to adjust its optimal levels of mRNA, and hence its translation capacity, in response to local energetic demands.

An in vitro transcription system utilizing isolated mitochondria has been used to study the effect of chronic ethanol consumption on liver mitochondrial DNA transcription. The results obtained showed an overall increase of RNA synthesis and a dramatic accumulation of a discrete polyadenylated RNA species. This effect is a consequence of the chronic ethanol consumption since these changes do not occur when isolated control mitochondria are incubated in the presence of ethanol.