The correction here implies the transfection of mammalian cells with either mitochondrial or nuclear genes from other organisms encoding the protein of interest DiMauro et al. This has also been applied to human cybrids harboring the m. Also this approach could correct the biochemical defect in these cells Ojaimi et al. For example, the m. The gene for Sma I was fused to a mitochondrial targeting sequence and expressed in heteroplasmic mutant cybrid cells, which lost mutant mtDNA and recovered biochemically Tanaka et al.
It has been shown that culturing heteroplasmic m. It has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease Sato et al. Briefly, this means that the pronucleus of an oocyte, or zygote, of a woman carrying mutated mtDNA could be transferred to a donor enucleated oocyte, or zygote, carrying wild type mtDNA DiMauro et al.
In that way, the offspring will carry all the nuclear — and physiognomonic - traits of the parents, but not the mutated mtDNA of the mother DiMauro et al. Two successful approaches have recently been described. It has been demonstrated in mature non-human primate oocytes Macaca mulatta that the mitochondrial genome can be efficiently replaced by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg Tachibana et al.
Subsequently, it was possible to have normal fertilization and embryo development. The offspring was healthy Tachibana et al. This is essentially the same procedure, except that the nuclear material, both the male and female pronucleus, is removed after fertilization Tachibana et al. It has been shown that transfer of pronuclei between abnormally fertilized human zygotes resulted in minimal carry-over of donor zygote mtDNA and is compatible with onward development of the blastocyst stage in vitro Craven et al.
There have been few randomized controlled trials for the treatment of mitochondrial disease Chinnery et al. To date, there is no clear evidence supporting the use of pharmacological agents, non-pharmacological treatments vitamins and food supplements , and physical training in patients with mitochondrial disorders Chinnery et al.
Although very promising, all genetic techniques are still in an experimental phase and different technical, ethical and safety issues still have to be solved DiMauro et al.
Nevertheless, they do allow cautious optimism for the future. Since current therapeutic options for mitochondrial diseases are insufficient, the possibility of prenatal diagnosis for fetuses at risk is a valuable alternative. If it concerns a known nuclear genetic defect, the mutation can directly be searched for in fetal tissue. Second, the heteroplasmy level differs between tissues and in one tissue through time Poulton and Marchington In this context, the m.
Finally, it is suggested that the heteroplasmy level remains stable after 10 weeks of gestation Steffann et al. Hitherto termination of pregnancy has been preferred in case of intermediate mutant loads Steffann et al. Remarkably, the intermediate mutant loads question the observation that the m.
Post-zygotic drift might explain this discrepancy Steffann et al. The interpretation of PGD results nevertheless demands a known correlation between mutation load and clinical phenotype. In addition, caution is warranted since some pathogenic mutations could exhibit different segregation behavior Dean et al. In case the genetic examination of an index case has revealed no mutations in both mtDNA and nDNA, prenatal diagnosis could still be possible.
In Nijmegen, complex V activity can be measured spectrophotometrically in native chorionic villi, cultured chorionic cells or cultured amniotic cells if there is a clear isolated complex V deficiency in fibroblasts and muscle tissue or other tissue of the index patient Niers et al. As mentioned briefly, most of the structure of the bovine mitochondrial enzyme has been resolved.
The structure of the membrane extrinsic part of bovine ATP synthase is complete Rees et al. The structure of the c-ring has been resolved recently Watt et al. The structures of the membrane domain of subunit b, subunit a, and the accessory subunits e, f, g, and A6L remain to be determined Rees et al. Still, understanding the enzyme fully at a molecular level will require further efforts, both experimental and theoretical for a review, see Junge et al.
Next to structure and function of the monocomplex, also the role of di- and oligomerization of complex V, shaping the inner mitochondrial membrane, has been addressed in many studies both in yeast and in mammalian mitochondria Paumard et al. The role of IF 1 in this process has been shown to be important Campanella et al. Despite this huge progress, lots of questions remain to be answered.
As mentioned, the assembly of the different subunits into the holocomplex continues to be puzzling. Most of the research has been done in yeast. However, the yeast assembly process probably differs from the one in mammalian mitochondria, since there are substantial differences between higher and lower eukaryotes such as the number of F o subunit c-genes, ATP synthase-specific assembly factors, and factors regulating transcription of ATP synthase genes Houstek et al.
To gain further insight into the assembly of complex V, techniques like blue native and clear native PAGE, combined with incorporation and knock-down experiments of different subunits as described in Wagner et al. They both have a role in F 1 assembly. TMEM70 maintains normal expression levels of complex V, and has been suggested to have a role in complex V biogenesis Cizkova et al.
The exact mechanism however still remains to be elucidated. Moreover, the existence of specific factors involved in mammalian F o formation is probable Houstek et al.
A possible approach could be to study the evolution of complex V subunits and complex V chaperones by comparative genomics. For example, the yeast F o assembly factor Atp23p has a human homolog for which, however, no involvement in ATP synthase assembly could be demonstrated Kucharczyk et al.
Also a homology of complex V chaperones with other human proteins could be of interest in the search of specific assembly factors. Another intriguing fact is that to date, only one mutation has been found in a nuclear structural complex V gene Mayr et al.
It could be possible that mutations in some of the structural subunits are incompatible with life. On the other hand, given the lower frequency of complex V deficiency compared to the other OXPHOS deficiencies, routine screening of all nuclear structural genes is rarely implemented in a diagnostic setting.
Whole genome or whole exome screening could counter this problem and possibly solve some of the hitherto unknown genetic defects causing complex V deficiency.
Finally, the biggest challenge will be to find a tailored curative therapy for this patient group. Large-scale and high-throughput compound screening is needed to find a possible pharmacological approach. For mtDNA defects, gene-shifting and germline techniques are promising, but much more and thorough experimental research is needed before this can be implemented in the patient setting. In conclusion, mitochondrial ATP synthase has been and still is a popular research topic.
Thanks to sustained effort, many aspects of this intriguing protein have been elucidated. This knowledge will guide further physio patho logical studies, paving the way for future therapeutic interventions. The authors confirm independence from the sponsors; the content of the article has not been influenced by the sponsors.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited. Competing interest: None declared.
National Center for Biotechnology Information , U. Journal of Inherited Metabolic Disease. J Inherit Metab Dis. Published online Aug Jonckheere , Jan A.
Smeitink , and Richard J. Jan A. Richard J. Author information Article notes Copyright and License information Disclaimer. Corresponding author. This article has been cited by other articles in PMC. Abstract Human mitochondrial mt ATP synthase, or complex V consists of two functional domains: F 1 , situated in the mitochondrial matrix, and F o , located in the inner mitochondrial membrane.
ATP synthase: architecture Fig. Open in a separate window. Table 1 Subunit composition of human, yeast and E. Stoichiometry Bacteria Mitochondria E.
Complex V assembly Current knowledge about the assembly of ATP synthase is mainly based on research performed on assembly-deficient yeast mutants Kucharczyk et al. Complex V di- and oligomerization An important role of subunits a and A6L is the stabilization of holocomplex V Wittig et al. Complex V and mitochondrial morphology The association of ATP synthase dimers as generating the tubular cristae has been hypothesized by Allen Allen Biochemical diagnosis Measurement of the mitochondrial energy-generating system MEGS capacity in fresh muscle tissue is a powerful tool to assess mitochondrial function and to detect deficiencies of complex V and other OXPHOS complexes.
Modifiers Phenotypical variations between patients harboring the same mtDNA mutation have classically been attributed to mtDNA heteroplasmy. Therapy Current available treatment options for patients with mitochondrial diseases are mainly supportive.
Antioxidants As mentioned above, complex V mutations can increase ROS production which is deleterious for the cell. Affecting heteroplasmy of the mtDNA gene-shifting This genetic approach aims to force a shift in heteroplasmy, reducing the ratio of mutant to wild-type genomes also called gene-shifting DiMauro et al.
Allotopic expression Here, a normal version of a mutant mtDNA-encoded protein is imported into the nucleus. Xenotopic expression The correction here implies the transfection of mammalian cells with either mitochondrial or nuclear genes from other organisms encoding the protein of interest DiMauro et al. Oligomycin It has been shown that culturing heteroplasmic m. Germline therapy It has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease Sato et al.
Metaphase II spindle transfer between unfertilized metaphase II oocytes It has been demonstrated in mature non-human primate oocytes Macaca mulatta that the mitochondrial genome can be efficiently replaced by spindle-chromosomal complex transfer from one egg to an enucleated, mitochondrial-replete egg Tachibana et al. Pronuclear transfer between zygotes This is essentially the same procedure, except that the nuclear material, both the male and female pronucleus, is removed after fertilization Tachibana et al.
Prenatal and preimplantation diagnosis Since current therapeutic options for mitochondrial diseases are insufficient, the possibility of prenatal diagnosis for fetuses at risk is a valuable alternative. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author s and source are credited.
Footnotes Competing interest: None declared. Mitochondrial abnormalities in patients with LHON-like optic neuropathies. Invest Ophthalmol Vis Sci. Biochim Biophys Acta. Coupling of rotation and catalysis in F 1 -ATPase revealed by single-molecule imaging and manipulation. Gene Ther. Membrane tubulation and proton pumps. Sequence and organization of the human mitochondrial genome. Self-assembly of ATP synthase subunit c rings.
FEBS Lett. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J. Mitochondrial DNA mutation stimulates prostate cancer growth in bone stromal environment. Eur J Biochem. J Biol Chem. Recent advances in structure-functional studies of mitochondrial factor B. J Bioenerg Biomembr. Factor B and the mitochondrial ATP synthase complex. Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F 0 F 1 ATP synthase.
Am J Hum Genet. A model for conformational coupling of membrane potential and proton translocation to ATP synthesis and to active transport. The ATP synthase—a splendid molecular machine. Annu Rev Biochem. The present status of the binding-change mechanism and its relation to ATP formation by chloroplasts. Energy coupling in photosynthesis. Amsterdam: Elsevier; Transmission of mitochondrial DNA disorders: possibilities for the future.
Nat Struct Biol. Systematic identification of human mitochondrial disease genes through integrative genomics. Nat Genet. Cell Metab. Trends Biochem Sci. Biochemical-clinical correlation in patients with different loads of the mitochondrial DNA TG mutation. Arch Neurol. A microspectrophotometric method for the determination of cytochrome oxidase. The mechanism of ATP synthase.
Conformational change by rotation of the beta-subunit. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Towards reliable prenatal diagnosis of mtDNA point mutations: studies of nt mutations in oocytes, fetal tissues, children and adults. Hum Reprod. Hum Mol Genet. Bilateral striatal necrosis with a novel point mutation in the mitochondrial ATPase 6 gene. Pediatr Neurol. Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP J Med Genet.
A second missense mutation in the mitochondrial ATPase 6 gene in Leigh's syndrome. Ann Neurol. Prospect of preimplantation genetic diagnosis for heritable mitochondrial DNA diseases. Three conformations corresponding to different rotational states of the enzyme were identified by ab-initio 3D classification and refined to high resolution.
Estimation of local resolution suggests that the F 1 regions of the maps, which are larger than the F O regions and appear to dominate the image alignment process, are mostly at between 2. Focused refinement Bai et al. An improved map of the F O region was obtained by focused refinement of the membrane-embedded region only, excluding the soluble portion of subunit b with particle images from all three classes Figure 1—figure supplement 2.
The soluble region of the two b -subunits was modeled as poly-alanine Supplementary file 1. The general architecture of the enzyme resembles E. However, there are more ionic interactions in the F 1 -ATPase structures from thermophiles than from mesophiles, suggesting that these interactions may play a role in stabilizing the complexes. In the F O region, one copy of subunit b is positioned at a location equivalent to that of the mitochondrial subunit b , while the second copy occupies the position of yeast subunit 8 mammalian A6L on the other side of subunit a Figure 1B.
Rather than reflecting true differences between E. By comparing the positions of equivalent c -subunits in different rotational states, the observed rotational step sizes in the three rotational states of the ATP synthase appear to be almost exactly 3, 4 and 3 c -subunits Figure 2B.
At the present resolution, the structures of subunit a and the c- ring do not appear to differ between rotary states. Similar integer step sizes were found in yeast ATP synthase Vinothkumar et al. However, non-integer steps were seen in the chloroplast 14 c -subunits Hahn et al. B Top view of the c -ring and subunit a of the three rotational states from the cytoplasm when the F 1 regions of the three states are aligned.
The b subunits appear to be the most flexible part of the enzyme. This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing.
Alternatively, flexibility in the enzyme could maintain a constant rotational velocity. In Bacillus PS3 ATP synthase, the peripheral stalk is structurally simpler and more flexible than in yeast mitochondria Srivastava et al. Given that these structures represent resting states of the bacterial ATP synthase, additional subunits, such as those in the central stalk, may show flexibility while under strain during rotation. Subunit a and the first copy of subunit b occupy the same positions as their yeast counterparts, while the second copy of subunit b is found at a position equivalent to subunit 8 in the yeast enzyme, which is known as A6L in mammals.
Atomic models for ATP synthase from mitochondria Guo et al. Subunit a from Bacillus PS3 shares The sequence for this loop varies significantly among species, suggesting that it is unlikely to be involved in the core function of proton translocation, despite being proximal to the periplasmic proton half-channel. The loop forms an additional interface with subunit b near the periplasmic side of the membrane region and may interact with the N terminus of subunit b in the periplasm as well.
The structure suggests that two interfaces are necessary for subunits a and b to maintain a stable interaction. B Cross sections through a surface representation of the F O region simulated with rolling of a 1. The proton then rotates with the c -ring until it reaches the cytoplasmic half-channel formed between subunit a and the c -ring.
In the cytoplasmic half-channel, the proton is released from the Glu residue due to its interaction with the positively charged Arg of subunit a. A Glu 56 residue from each protomer of the c -ring is shown. Arg is in purple, important residues for proton translocation identified by mutagenesis in E.
The Bacillus PS3 ATP synthase structure implies a path for proton translocation through the bacterial complex involving two half-channels similar to the paths described for the mitochondrial and chloroplast enzymes. The cytoplasmic half-channel consists of an aqueous cavity at the interface of subunit a and the c -ring Figure 4B , left.
In the atomic model, both channels are visible when modeling the surface with a 1. The channels are wide and hydrophilic, suggesting that water molecules could pass freely through each of the channels before accessing the conserved Glu 56 of the c -subunits. During ATP synthesis, protons travel to the middle of the c -ring via the periplasmic half-channel and bind to the Glu 56 residue of a subunit c Figure 4C.
Protonation of the glutamate allows rotation of the ring counter-clockwise, when viewed from F 1 toward F O , delivering the subunit c into the hydrophobic lipid bilayer.
Protonation of the remaining nine subunits in the c -ring returns the first glutamate to subunit a , now into the cytoplasmic half-channel, where it releases its proton to the cytoplasm due to interaction with the positively charged Arg of subunit a.
The proposed channels are consistent with a series of experiments probing water accessibility of residues in the E. Together, these results suggest that ion selectivity in ATP synthases is probably determined by the c -ring, not subunit a.
In eukaryotes, subunit a is encoded by the mitochondrial genome, limiting genetic interrogation of the roles of different residues. In contrast, numerous mutagenesis studies have been performed on bacterial subunits a and b , with E. A single G9D mutation in the E. Therefore, the G9D mutation in E.
Cross-linking experiments suggested that the N terminus of the two copies of subunit b are in close proximity to each other Dmitriev et al. Recent structures of rotary ATPases suggest that the importance of this residue derives from its role in releasing protons bound to the Glu residues of the c -subunits as they enter the cytoplasmic half-channel, as well as preventing short-circuiting of the proton path by protons flowing between half-channels without rotation of the c -ring Morales-Rios et al.
Other residues in the E. Extensive mutations of E. Therefore, the negative surface charge from Glu Glu near the cytoplasmic half-channel facilitates proton transport across the lipid bilayer. The atomic model of subunit a also suggests that other residues such as Bacillus PS3 Thr , Asn , Glu , Tyr , and His , which are close to the cytoplasmic half-channel, may contribute to channel formation.
Many functional residues identified by mutagenesis are clustered around the periplasmic half-channel. In the atomic model of the Bacillus PS3 subunit a , Asp 19 and Glu are close to the periplasm, while Ser , Asn , and Gln are deeper inside the membrane.
Among these residues, Glu and Ser are considered to be more important to enzyme function than Asn and Gln , as mutations of corresponding residues in E. These residues do not appear to be close enough to form a hydrogen bond in the S. In Bacillus PS3 subunit a , the His residue is replaced by a serine Ser that similarly does not appear to close enough to Glu to form a hydrogen bond.
Interestingly, although many of these functional residues appear important, their mutation to amino acids that cannot be protonated or deprotonated often does not completely abolish proton translocation Vik et al.
The atomic model of Bacillus PS3 subunit a shows that the proton half-channels are wide enough for water molecules to pass through freely. This observation suggests that the function of these conserved polar and charged residues is not the direct transfer of protons during translocation.
Rather, their presence may help maintain a hydrophilic environment for water-filled proton channels. This role allows different species to use unique sets of polar and charged residues to form their proton half-channels. This variability suggests a remarkably flexible proton translocation mechanism for this highly efficient macromolecular machine. Transformed E. Download citation. Issue Date : 18 November Anyone you share the following link with will be able to read this content:.
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