Inefficient coupling between proton transport and ATP synthesis may be the pathogenic mechanism for NARP and Leigh syndrome resulting from the T8993G mutation in mtDNA

Mutations in the ATP6 gene of mtDNA (mitochondrial DNA) have been shown to cause several different neurological disorders. The product of this gene is ATPase 6, an essential component of the F1F0-ATPase. In the present study we show that the function of the F1F0-ATPase is impaired in lymphocytes from ten individuals harbouring the mtDNA T8993G point mutation associated with NARP (neuropathy, ataxia and retinitis pigmentosa) and Leigh syndrome. We show that the impaired function of both the ATP synthase and the proton transport activity of the enzyme correlates with the amount of the mtDNA that is mutated, ranging from 13-94%. The fluorescent dye RH-123 (Rhodamine-123) was used as a probe to determine whether or not passive proton flux (i.e. from the intermembrane space to the matrix) is affected by the mutation. Under state 3 respiratory conditions, a slight difference in RH-123 fluorescence quenching kinetics was observed between mutant and control mitochondria that suggests a marginally lower F0 proton flux capacity in cells from patients. Moreover, independent of the cellular mutant load the specific inhibitor oligomycin induced a marked enhancement of the RH-123 quenching rate, which is associated with a block in proton conductivity through F0 [Linnett and Beechey (1979) Inhibitors of the ATP synthethase system. Methods Enzymol. 55, 472-518]. Overall, the results rule out the previously proposed proton block as the basis of the pathogenicity of the mtDNA T8993G mutation. Since the ATP synthesis rate was decreased by 70% in NARP patients compared with controls, we suggest that the T8993G mutation affects the coupling between proton translocation through F0 and ATP synthesis on F1. We discuss our findings in view of the current knowledge regarding the rotary mechanism of catalysis of the enzyme.     

A yeast model of the neurogenic ataxia retinitis pigmentosa (NARP) T8993G mutation in the mitochondrial ATP synthase-6 gene

NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F(1) coupled to F(0)-mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.     

Biochemical phenotypes associated with the mitochondrial ATP6 gene mutations at nt8993

Two point mutations (T>G and T>C) at the same 8993 nucleotide of mitochondrial DNA (at comparable mutant load), affecting the ATPase 6 subunit of the F1F0-ATPase, result in neurological phenotypes of variable severity in humans. We have investigated mitochondrial function in lymphocytes from individuals carrying the 8993T>C mutation: the results were compared with data from five 8993T>G NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) patients. Both 8993T>G and 8993T>C mutations led to energy deprivation and ROS overproduction. However, the relative contribution of the two pathogenic components is different depending on the mutation considered. The 8993T>G change mainly induces an energy deficiency, whereas the 8993T>C favours an increased ROS production. These results possibly highlight the different pathogenic mechanism generated by the two mutations at position 8993 and provide useful information to better characterize the biochemical role of the highly conserved Leu-156 in ATPase 6 subunit of the mitochondrial ATP synthase complex.     

The study of the pathogenic mechanism of mitochondrial diseases provides information on basic bioenergetics

Mitochondrial F(1)F(0)-ATPase was studied in lymphocytes from patients with neuropathy, ataxia, and retinitis pigmentosa (NARP), caused by a mutation at leu-156 in the ATPase 6 subunit. The mutation giving the milder phenotype (Leu156Pro) suffered a 30% reduction in proton flux, and a similar loss in ATP synthetic activity. The more severe mutation (Leu156Arg) also suffered a 30% reduction in proton flux, but ATP synthesis was virtually abolished. Oligomycin sensitivity of the proton translocation through F(0) was enhanced by both mutations. We conclude that in the Leu156Pro mutation, rotation of the c-ring is slowed but coupling of ATP synthesis to proton flux is maintained, whereas in the Leu156Arg mutation, proton flux appears to be uncoupled. Modelling indicated that, in the Leu156Arg mutation, transmembrane helix III of ATPase 6 is unable to span the membrane, terminating in an intramembrane helix II-helix III loop. We propose that the integrity of transmembrane helix III is essential for the mechanical function of ATPase 6 as a stator element in the ATP synthase, but that it is not relevant for oligomycin inhibition.     

Antioxidant defence systems and generation of reactive oxygen species in osteosarcoma cells with defective mitochondria: Effect of selenium

Mitochondrial diseases originate from mutations in mitochondrial or nuclear genes encoding for mitochondrial proteome. Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) syndrome is associated with the T8993G transversion in ATP6 gene which results in substitution at the very conservative site in the subunit 6 of mitochondrial ATP synthase. Defects in the mitochondrial respiratory chain and the ATPase are considered to be accompanied by changes in the generation of reactive oxygen species (ROS). This study aimed to elucidate effects of selenium on ROS and antioxidant system of NARP cybrid cells with 98% of T8993G mutation load. We found that selenium decreased ROS generation and increased the level and activity of antioxidant enzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR). Therefore, we propose selenium to be a promising therapeutic agent not only in the case of NARP syndrome but also other diseases associated with mitochondrial dysfunctions and oxidative stress.     

Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene

A T --> G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.     

Biochemical phenotypes associated with the mitochondrial ATP6 gene mutations at nt8993

Two point mutations (T > G and T > C) at the same 8993 nucleotide of mitochondrial DNA (at comparable mutant load), affecting the ATPase 6 subunit of the F₁F₀-ATPase, result in neurological phenotypes of variable severity in humans. We have investigated mitochondrial function in lymphocytes from individuals carrying the 8993T > C mutation: the results were compared with data from five 8993T > G NARP (Neuropathy, Ataxia and Retinitis Pigmentosa) patients. Both 8993T > G and 8993T > C mutations led to energy deprivation and ROS overproduction. However, the relative contribution of the two pathogenic components is different depending on the mutation considered. The 8993T > G change mainly induces an energy deficiency, whereas the 8993T > C favours an increased ROS production. These results possibly highlight the different pathogenic mechanism generated by the two mutations at position 8993 and provide useful information to better characterize the biochemical role of the highly conserved Leu-156 in ATPase 6 subunit of the mitochondrial ATP synthase complex.     

Structure, functioning, and assembly of the ATP synthase in cells from patients with the T8993G mitochondrial DNA mutation. Comparison with the enzyme in Rho(0) cells completely lacking mtdna

The structure and functioning of the ATP synthase of human fibroblast cell lines with 91 and 100%, respectively, of the T8993G mutation have been studied, with MRC5 human fibroblasts and Rho(0) cells derived from this cell line as controls. ATP hydrolysis was normal but ATP synthesis was reduced by 60% in the 100% mutants. Both activities were highly oligomycin-sensitive. The levels of F(1)F(0) were close to normal, and the enzyme was stable. It is concluded that the loss of ATP synthesis is because of disruption of the proton translocation step within the F(0) part. This is supported by membrane potential measurements using the dye JC-1. Cells with a 91% mutation load grew well and showed only a 25% loss in ATP synthesis. This much reduced effect for only a 9% difference in mutation load mirrors the reduced pathogenicity in patients. F(1)F(0) has been purified for the first time from human cell lines. A partial complex was obtained from Rho(0) cells containing the F(1) subunits associated with several stalk, as well as F(0) subunits, including oligomycin sensitivity conferring protein, b, and c subunits. This partial complex no longer binds inhibitor protein.     

Bioenergetics of mitochondrial diseases associated with mtDNA mutations

This mini-review summarizes our present view of the biochemical alterations associated with mitochondrial DNA (mtDNA) point mutations. Mitochondrial cytopathies caused by mutations of mtDNA are well-known genetic and clinical entities, but the biochemical pathogenic mechanisms are often obscure. Leber's hereditary optic neuropathy (LHON) is due to three main mutations in genes for complex I subunits. Even if the catalytic activity of complex I is maintained except in cells carrying the 3460/ND1 mutation, in all cases there is a change in sensitivity to complex I inhibitors and an impairment of mitochondrial respiration, eliciting the possibility of generation of reactive oxygen species (ROS) by the complex. Neurogenic muscle weakness, Ataxia and Retinitis Pigmentosa (NARP), is due to a mutation in the ATPase-6 gene. In NARP patients ATP synthesis is strongly depressed to an extent proportional to the mutation load; nevertheless, ATP hydrolysis and ATP-driven proton translocation are not affected. It is suggested that the NARP mutation affects the ability of the enzyme to couple proton transport to ATP synthesis. A point mutation in subunit III of cytochrome c oxidase is accompanied by a syndrome resembling MELAS: however, no major biochemical defect is found, if we except an enhanced production of ROS. The mechanism of such enhancement is at present unknown. In this review, we draw attention to a few examples in which the overproduction of ROS might represent a common step in the induction of clinical phenotypes and/or in the progression of several human pathologies associated with mtDNA point mutations.     

A novel mitochondrial mutation m.8989G>C associated with neuropathy,ataxia, retinitis pigmentosa — The NARP syndrome

The archetypal NARP syndrome is almost exclusively associated with the m.8993T>C/G mutation in the sixth subunit of the mitochondrial ATP synthase, whereas other mutations in the MT-ATP6 gene primarily associate with Leigh syndrome or Leber's hereditary optic neuropathy (LHON). We report a novel mitochondrial point mutation, m.8989G>C, in a patient presenting with neuropathy, ataxia and retinitis pigmentosa constituting the classical NARP phenotype. This mutation alters the amino acid right next to canonical NARP mutation. We suggest that classic NARP syndrome relates to a defined dysfunction of p.MT-ATP6.     

Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H(+)-F(O)F(1)-ATP synthase couples proton flow through its membrane portion, F(O), to the synthesis of ATP in its headpiece, F(1). Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the epsilon subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the gamma subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced gammaLys23 with the DELSEED region of subunit beta stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit gamma rotation which is necessary for the activation.     

Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene

Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.     

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.     

Pathogenesis of primary defects in mitochondrial ATP synthesis

Maternally inherited mutations in the mtDNA-encoded ATPase 6 subunit of complex V (ATP synthase) of the respiratory chain/oxidative phosphorylation system are responsible for a subgroup of severe and often-fatal disorders characterized predominantly by lesions in the brain, particularly in the striatum. These include NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh syndrome), and FBSN (familial bilateral striatal necrosis). Of the five known pathogenic mutations causing these disorders, four are located at two codons (156 and 217), each of which can suffer mutations converting a conserved leucine to either an arginine or a proline. Based on the accumulating data on both the structure of ATP synthase and the mechanism by which rotary catalysis couples proton flow to ATP synthesis, we propose a model that may help explain why mutations at codons 156 and 217 are pathogenic.     

ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization

The molecular pathogenic mechanism of the human mitochondrial diseases neurogenic ataxia and retinitis pigmentosa and maternally inherited Leigh syndrome was determined in cultured human cells harboring homoplasmic T8993G/T8993C point mutations in the mitochondrial ATP6 gene, which encodes subunit 6 of the F1F0-ATP synthase. Immunoprecipitation and blue native electrophoresis showed that F1F0-ATP synthase assembles correctly in homoplasmic mutant mitochondria. The mutants exhibited a tendency to have an increased sensitivity to subsaturating amounts of oligomycin; this provided further evidence for complete assembly and tight coupling between the F1 and F0 sectors. Furthermore, human ATP synthase dimers and higher homo-oligomers were observed for the first time, and it was demonstrated that the mutant enzymes retain enough structural integrity to oligomerize. A reproducible increase in the proportion of oligomeric-to-monomeric enzyme was found for the T8993G mutant suggesting that F1F0 oligomerization is regulated in vivo and that it can be modified in pathological conditions. Despite correct assembly, the T8993G mutation produced a 60% inhibition in ATP synthesis turnover. In vitro denaturing conditions showed F1F0 instability conferred by the mutations, although this instability did not produce enzyme disassembly in the conditions used for determination of ATP synthesis. Taken together, the data show that the primary molecular pathogenic mechanism of these deleterious human mitochondrial mutations is functional inhibition in a correctly assembled ATP synthase. Structural instability may play a role in the progression of the disease under potentially denaturing conditions, as discussed.     

The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli

A new mutation in human F(1)F(0) ATPase6, T9176G, which changes Leu 217 to an Arg, has been described in two siblings with Leigh syndrome [Carrozzo et al. (2000) Neurology, in press]. This mutation was modeled in Escherichia coli by changing Leu 259 (the equivalent residue) to Arg and the properties of the altered ECF(1)F(0) were compared to those of previously characterized ATPase6 mutants also modeled in the E. coli enzyme. The L259R change produced a fully assembled ECF(1)F(0) which had no significant ATP hydrolysis, ATP synthesis or proton pumping functions. This is very different from previously described human ATPase6 mutations. The presence of Arg at position 259 in subunit a did not make membranes permeable to protons. We conclude that the mutation inhibits functioning by blocking the rotary motor action of the enzyme.     

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.     

De novo mutation in the mitochondrial ATP synthase subunit 6 gene (T8993G) with rapid segregation resulting in Leigh syndrome in the offspring

The mutation in the mitochondrial ATP synthase subunit 6 gene (ATP6 T8993G) was identified in a male infant who died at age 15 months of Leigh syndrome. He had 94% mutated mitochondrial DNA (mtDNA) in muscle and 92% in lymphocytes. His mother was healthy but had 37% mutated mtDNA in muscle and 38% in lymphocytes. The proband's brother, who was also healthy, had 44% mutated mtDNA in lymphocytes. No mutated mtDNA was detected in muscle and lymphocytes from the maternal grandmother of the proband or in lymphocytes from 15 other maternal relatives, showing that the first carrier of the ATP6 T8993G mutation in this family was the mother of the proband. This study shows that this point mutation may occur at substantial levels in a carrier of a de novo mutation and rapid segregation with high levels of mutated mtDNA causing neurodegenerative disease may occur in the second generation.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.     

Mutation of the mitochrondrially encoded ATPase 6 gene modeled in the ATP synthase of Escherichia coli.

Defects of respiratory chain protein complexes and the ATP synthase are becoming increasingly implicated in human disease. Recently, mutations in the ATPase 6 gene have been shown to cause several different neurological disorders. The product of this gene is homologous to the a subunit of the ATP synthase of Escherichia coli. Here, mutations equivalent to those described in humans have been introduced into the a subunit of E. coli by site-directed mutagenesis, and the effects of these mutations on the ATPase activity, ATP synthesis and ability of the enzyme to pump protons studied in detail. The effects of the mutations varied considerably. The mutation L262P (9185 T-C equivalent) caused a 70% loss of ATP synthesis activity, reduced DCCD sensitivity, and lowered proton pumping activity. The L207P (8993 T-C equivalent) reduced ATP synthesis by 50%, affected DCCD sensitivity, while proton pumping was only marginally affected when measured by the standard AMCA quenching assay. The other mutations studied affected the functioning of the ATP synthase much less. The results confirm that modeling of these point mutations in the E. coli enzyme is a useful approach to determining how alterations in the ATPase 6 gene affect enzyme function and, therefore, how a pathogenic effect can be exerted.