Abstract

The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane α-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.

INTRODUCTION

Most of the ATP in human cells is produced by an F1FO-type ATP synthase or complex V located in the mitochondrial inner membrane (1). This enzyme utilizes the energy of the electrochemical proton gradient established by the mitochondrial electron transport chain to drive the synthesis of ATP from ADP and inorganic phosphate. It is therefore not surprising that pronounced defects in the ATP synthase can be responsible for very deleterious disorders in humans, especially in the highly energy-demanding neuromuscular system (2–4). Such disorders like NARP (neuropathy ataxia retinitis pigmentosa) and MILS (maternally-inherited Leigh's syndrome) have been associated to several mutations in the mitochondrial ATP6 gene encoding the subunit a of the ATP synthase (referred to as Atp6p in yeast). This subunit is a trans-membrane protein containing most of the residues involved in the proton translocating activity of the FO domain of the ATP synthase (5). Proton movements mediated by subunit a lead to the rotation of a ring of c subunits (referred to as Atp9p in yeast) that contacts subunit a in the membrane. The resulting mechanical energy is then used to induce conformational changes at the level of the catalytic sites in the F1 extra-membrane domain of the enzyme that favor the synthesis of ATP and its subsequent release into the mitochondrial matrix.

One of the known pathogenic ATP6 mutations, T9176G, leads to replacement of a highly conserved leucine by arginine within a presumed trans-membrane α-helical segment, at amino acid position 217 of subunit a in proximity of the c-ring (6,7). The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation (6). However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created a yeast strain bearing an equivalent of this mutation and analyzed its properties. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on a previous partial biochemical characterization of human T9176G cells (6), it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been previously suggested (8). Interestingly, the T9176G yeast exhibited important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176 may not be limited to a bioenergetic deficiency.

RESULTS

Genetic stability and respiratory growth of a yeast mutant atp6-L247R

The leucine residue 217 of human subunit a that is changed into arginine in the T9176G mutation corresponds to the leucine residue 247 of the yeast homologous protein Atp6p (4). We converted the TTA triplet encoding this residue into the AGA codon for arginine (see Materials and Methods). Independent yeast clones with the atp6-L247R mutation (called RKY25, see Table 1 for complete genotype) all failed to grow on medium containing a non-fermentable carbon source like glycerol (Fig. 1). In view of earlier findings (reviewed in 9) that mutations of the ATP synthase can promote instability of the mitochondrial genome in yeast in the form of cytoplasmic petites issued from large deletions (ρ) or complete loss (ρ0) of that genome, two independent atp6-L247R clones were tested for the percentage of ρ/ρ0 cells in cultures grown under non-selective conditions, in rich glucose or galactose media. The glucose cultures contained only 20% ρ/ρ0 cells versus 2% for the corresponding wild-type strain (MR6) showing that the respiratory growth deficiency of the atp6-L247R mutant could not be attributed to defects in mtDNA maintenance alone. The cultures made with galactose, a fermentable carbon source not eliciting repression of the mitochondrial functions, contained somewhat more petites (30–40%, versus 3–4% for the wild-type). Those atp6-L247R cells that contained a complete (ρ+) mitochondrial genome fully recovered respiratory competence in crosses with SDC30, a synthetic ρ strain whose mitochondrial genome encodes only for wild-type ATP6 gene (10). This proved that the respiratory growth defect of these cells was truly and solely caused by the atp6-L247R mutation.

Figure 1.

The atp6-L247R mutation prevents the growth of yeast on respiratory substrates. Freshly grown cells of wild-type yeast (MR6) and the atp6-L247R mutant (RKY25) were serially diluted and 5 µl of each dilution were spotted onto glucose (YPGA) and glycerol (N3) plates. The plates were incubated at 28°C and photographed after three (YPGA) or seven (N3) days.

Figure 1.

The atp6-L247R mutation prevents the growth of yeast on respiratory substrates. Freshly grown cells of wild-type yeast (MR6) and the atp6-L247R mutant (RKY25) were serially diluted and 5 µl of each dilution were spotted onto glucose (YPGA) and glycerol (N3) plates. The plates were incubated at 28°C and photographed after three (YPGA) or seven (N3) days.

Table 1.

Genotypes and sources of yeast strains

Strain Nuclear genotype mtDNA Source 
DFS160 MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1 ρ0 (63
NB40-3C MATa lys2 leu2-3,112 ura3-52 his3ΔHinDIII arg8::hisG ρ+cox2-62 (63
MR6 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+ wt (10
MR10 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6::ARG8m (10
MR14 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6-L183R(18
RKY14 MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1 ρatp6-L247R This study 
RKY25 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6-L247R This study 
Strain Nuclear genotype mtDNA Source 
DFS160 MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1 ρ0 (63
NB40-3C MATa lys2 leu2-3,112 ura3-52 his3ΔHinDIII arg8::hisG ρ+cox2-62 (63
MR6 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+ wt (10
MR10 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6::ARG8m (10
MR14 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6-L183R(18
RKY14 MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1 ρatp6-L247R This study 
RKY25 MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 ρ+atp6-L247R This study 

Consequences of the atp6-L247R mutation on several activities related to respiration and oxidative phosphorylation

Oxygen consumption.

Mitochondria isolated from the atp6-L247R mutant (RKY25) and wild-type strain (MR6) were assayed for oxygen consumption. Using NADH as an electron donor, the oxygen consumption rate in the mutant was <10% compared with the wild-type, both in presence of an excess of ADP (state 3, phosphorylating conditions) or CCCP (uncoupled respiration) (Table 2). The atp6-L247R mutant respired very slowly also when electrons were delivered from ascorbate/TMPD at the level of complex IV, the last complex of the respiratory chain (Table 2).

Table 2.

Influence of the atp6-L247R mutation on yeast mitochondrial respiration, ATP hydrolysis, and ATP synthesis activities

Strain +NADH +ADP +NADH +CCCP Asc/TMPD +CCCP ATP synthesis (nmol/min/mg) ATPase (μmol/min/mg)
 
−oligo +oligo 
wt 613 ± 12 1081 ± 107 2013 ± 290 637 ± 18 4.474 ± 0.222 0.665 ± 0.108 
atp6-L247R 44 ± 3 70 ± 1 89 ± 19 26 ± 0.3 3.108 ± 0.020 2.797 ± 0.100 
Strain +NADH +ADP +NADH +CCCP Asc/TMPD +CCCP ATP synthesis (nmol/min/mg) ATPase (μmol/min/mg)
 
−oligo +oligo 
wt 613 ± 12 1081 ± 107 2013 ± 290 637 ± 18 4.474 ± 0.222 0.665 ± 0.108 
atp6-L247R 44 ± 3 70 ± 1 89 ± 19 26 ± 0.3 3.108 ± 0.020 2.797 ± 0.100 

Mitochondria were isolated from wild-type strain MR6 and the atp6-L247R mutant (RKY25) grown for five to six generations in YPGALA medium (rich galactose) at 28°C. Additions were 0.15 mg/ml proteins, 4 mm NADH, 150 µm ADP, 12.5 mm ascorbate (Asc), 1.4 mmN,N,N,N,-tetramethyl-p-phenylenediamine (TMPD), 4 µm CCCP, 3 µg/ml oligomycin (oligo). The cultures of the wild-type and atp6-L247R mutant contained <5 and 34% ρ/ρ0 cells, respectively. The values reported are averages of triplicate assays ± standard deviation. Respiration and ATP synthesis activities were measured on freshly isolated osmotically protected mitochondria buffered at pH 6.8. For the ATPase assays, mitochondria kept at –80°C were thawed and the reaction performed in the absence of osmotic protection and at pH 8.4.

To determine the reason of this severe respiratory deficiency, spectral analyses were performed from whole atp6-L247R cells chemically reduced with dithionite. The Cytochromes aa3 of complex IV were almost undetectable in these cells (Fig. 2). Even though the Cytochromes c1 and b of complex III were less abundant, they were much less affected. Cytochrome c, a soluble protein that electronically connects the complexes III and IV in the intermembrane space, was present in normal amounts in the mutant. These data were corroborated by SDS–PAGE analyses of mitochondrial proteins showing that the level of the Cox2p subunit of complex IV was barely detectable in the mutant, while the amount of the Cytochrome b subunit of complex III was much less reduced, by <50% (Fig. 3A). Both Cox2p and Cytochrome b are encoded by the mitochondrial DNA. The partial decrease in Cytochrome b content may be due in part to the presence of 30–40% ρ/ρ0 cells in the cultures of the mutant. It is evident that the almost complete absence of Cox2p cannot only be attributed to the increased propensity of the atp6-L247R mutant to produce ρ/ρ0 cells.

Figure 2.

Cytochrome spectral analysis. The tracings are optical spectra of mitochondrial cytochromes recorded from whole cells (grown in rich galactose) in liquid nitrogen after chemical reduction with dithionite. The positions of the α band absorption maxima of Cytochrome c (c), Cytochrome c1 (c1), Cytochrome b (b) and Cytochromes aa3 (aa3) are indicated.

Figure 2.

Cytochrome spectral analysis. The tracings are optical spectra of mitochondrial cytochromes recorded from whole cells (grown in rich galactose) in liquid nitrogen after chemical reduction with dithionite. The positions of the α band absorption maxima of Cytochrome c (c), Cytochrome c1 (c1), Cytochrome b (b) and Cytochromes aa3 (aa3) are indicated.

Figure 3.

SDS- and BN–PAGE analyses of mitochondrial proteins. (A) SDS–PAGE analysis of total mitochondrial proteins of wild-type strain MR6 and the atp6-L247R mutant. After their electrophoretic separation the proteins (10 µg) were transferred onto a nitrocellulose membrane and probed with the indicated antibodies. The immunological responses were vizualized and quantified using a PhosphoImager. The values are the levels of each protein in the mutant expressed as percentages of the wild-type levels after normalization to porin. (B) BN–PAGE analyses. For the analysis of the ATP synthase (C), the mitochondria were solubilized with 2 g of digitonin per gram of proteins while 10 g of digitonin per gram of proteins were used to analyze the complexes III and IV (B). After their electrophoretic separation the digitonin-extracted proteins (50 µg) were transferred to a nitrocellulose membrane and hybrized with the indicated antibodies.

Figure 3.

SDS- and BN–PAGE analyses of mitochondrial proteins. (A) SDS–PAGE analysis of total mitochondrial proteins of wild-type strain MR6 and the atp6-L247R mutant. After their electrophoretic separation the proteins (10 µg) were transferred onto a nitrocellulose membrane and probed with the indicated antibodies. The immunological responses were vizualized and quantified using a PhosphoImager. The values are the levels of each protein in the mutant expressed as percentages of the wild-type levels after normalization to porin. (B) BN–PAGE analyses. For the analysis of the ATP synthase (C), the mitochondria were solubilized with 2 g of digitonin per gram of proteins while 10 g of digitonin per gram of proteins were used to analyze the complexes III and IV (B). After their electrophoretic separation the digitonin-extracted proteins (50 µg) were transferred to a nitrocellulose membrane and hybrized with the indicated antibodies.

We further analyzed the impact of the atp6-L247R mutation on complexes III and IV by western blotting of BN–PAGE gels using antibodies against Cytochrome b or Cox2p. In the conditions used, these complexes are mainly resolved as two supercomplexes consisting of one complex III dimer combined with either one (III2IV1) or two (III2IV2) complexes IV, and a small amount of free complex III (11). The amount of assembled complex IV was extremely low in the mutant, while complex III content was less affected (Fig. 3B). From the blots made with antibodies against Cytochrome b, it is clear that the mutant contained more free complex III compared with the wild-type. This is due to the pronounced effect of atp6-L247R on complex IV, which reduces the amount of complex III that can be incorporated into supercomplexes.

Mitochondrial ATP synthesis/hydrolysis and membrane potential.

The consequences of the atp6-L247R mutation were analyzed further by measuring the rate of mitochondrial ATP synthesis, at state 3 with NADH as a respiratory substrate. Not surprisingly, because of their extremely poor oxygen consumption activity, the atp6-L247R mitochondria showed almost no ATP synthesis (Table 2). Other measurements not dependent on the electron transport chain were needed to know whether the atp6-L247R mutation was detrimental to the ATP synthase. First evidence for defects in the mutated ATP synthase was provided by mitochondrial ATPase activity assays. These were performed using non-osmotically protected mitochondria buffered at pH 8.4, i.e. in conditions where the hydrolysis of ATP by the F1 cannot sustain a mitochondrial membrane potential. The mitochondrial ATPase activity of the atp6-L247R mutant was only modestly decreased, by ∼30%, compared with the wild-type (Table 2). However, it was very poorly inhibited by oligomycin by only 10 versus 80% for the wild-type.

We next examined the influence of the atp6-L247R mutation on the proton pumping capacity of the F1FO complex using Rhodamine 123. This is a fluorescent cationic dye that can be used to monitor changes in the mitochondrial membrane potential (ΔΨ) on intact (osmotically protected) mitochondria (12). Increasing ΔΨ is followed by the uptake of the dye inside the matrix space and concomitant fluorescence quenching. Prior to testing for ATP-driven proton translocation, the mitochondria were first energized with ethanol to remove the natural inhibitory peptide (IF1) of the F1-ATPase (Fig. 4). The mitochondrial membrane potential was then collapsed with KCN, and less than one minute later, thus well before rebinding of IF1 (13), ATP was added. The external ATP is counter-exchanged against ADP from the matrix by the ADP/ATP translocase. No ΔΨ is required, the presence of ADP in the matrix suffices to trigger the import of ATP into the mitochondria. The ATP can then be hydrolyzed by the F1 coupled to the transport of protons out of the matrix through the FO. As expected, the ATP addition promoted a large and stable fluorescence quenching of the dye in wild-type mitochondria, which was reversed upon inhibition of the FO with oligomycin (Fig. 4). In the mutant, the addition of ATP was followed by only a small and transient variation in fluorescence. This variation is due to the adenine nucleotide exchange which is electrogenic by nature (one negative charge is translocated across the membrane for each exchange). It has to be noted that the addition of ethanol on the mutant mitochondria resulted in a weak but significant ΔΨ (Fig. 4). This observation is consistent with the results of the respiration assays showing a strong but not complete loss of respiratory activity in the mutant. It can be inferred that the almost complete absence of ATP-driven proton translocation in the atp6-L247R mutant was not due to a loss of physical integrity of the mitochondria used in these experiments but rather to defects in the F1FO complex itself.

Figure 4.

Energization of mitochondria. Energization of the mitochondrial inner membrane was monitored by rhodamine 123 fluorescence quenching with intact mitochondria from wild-type yeast strain MR6 and the atp6-L247R mutant. The additions were 0.5 µg/ml rhodamine 123, 0.15 mg/ml mitochondrial proteins (Mito), 10 µl of ethanol (EtOH), 6 µg/ml oligomycin (oligo), 0.2 mm potassium cyanide (KCN), 1 mm ATP and 3 µm CCCP. Data are representative of at least three experiments.

Figure 4.

Energization of mitochondria. Energization of the mitochondrial inner membrane was monitored by rhodamine 123 fluorescence quenching with intact mitochondria from wild-type yeast strain MR6 and the atp6-L247R mutant. The additions were 0.5 µg/ml rhodamine 123, 0.15 mg/ml mitochondrial proteins (Mito), 10 µl of ethanol (EtOH), 6 µg/ml oligomycin (oligo), 0.2 mm potassium cyanide (KCN), 1 mm ATP and 3 µm CCCP. Data are representative of at least three experiments.

Assembly/stability of the ATP synthase in the atp6-L247R mutant.

We next examined the influence of the atp6-L247R mutation on the ATP synthase assembly/stability. We first determined the steady-state levels of a number of subunits (Atp6p, Atp9p, α-F1 and β-F1) by western blotting of total mitochondrial proteins separated by SDS–PAGE. The Atp9p, α-F1 and β-F1 proteins showed a good accumulation in the mutant while Atp6p was present in trace amounts only compared with the wild-type (Fig. 3A). The nearly complete absence of Atp6p in the mutant was apparently due to some post-translational defect. Indeed, pulse chase experiments showed that the Atp6p-L247R protein was efficiently synthesized and the newly synthesized polypeptides did not have an increased turnover compared with wild-type Atp6p (Fig. 5).

Figure 5.

Influence of the atp6-L247R mutation on the rate of production and stability of newly synthesized Atp6p. Proteins encoded by the mtDNA were in vivo labeled with [35S]-(methionine + cysteine) for 20 min at 28°C in the presence of cycloheximide to inhibit cytosolic protein synthesis. Turnover of newly synthesized proteins was evaluated by the addition of excess cold (methionine + cysteine) and incubation for the indicated periods of time. After the labeling reactions, total protein extracts were prepared from the cells (0.2 OD at 650 nm) and loaded on a 12.5% polyacrylamide–4 m urea gel containing 25% glycerol. After electrophoresis the gel was dried and radioactive proteins visualized with a phosphoImager.

Figure 5.

Influence of the atp6-L247R mutation on the rate of production and stability of newly synthesized Atp6p. Proteins encoded by the mtDNA were in vivo labeled with [35S]-(methionine + cysteine) for 20 min at 28°C in the presence of cycloheximide to inhibit cytosolic protein synthesis. Turnover of newly synthesized proteins was evaluated by the addition of excess cold (methionine + cysteine) and incubation for the indicated periods of time. After the labeling reactions, total protein extracts were prepared from the cells (0.2 OD at 650 nm) and loaded on a 12.5% polyacrylamide–4 m urea gel containing 25% glycerol. After electrophoresis the gel was dried and radioactive proteins visualized with a phosphoImager.

We then performed BN–PAGE analyses using conditions where the ATP synthase is mainly resolved as dimeric and monomeric F1FO complexes. After their electrophoretic separation, the digitonin-extracted mitochondrial proteins were transferred onto a nitrocellulose membrane and successively hybridized with antibodies against Atp6p, Atp9p and α-F1. Between two hybridizations, the membrane was stripped to completely remove the previously hybridized antibodies. As expected from their very low content in Atp6p, the mitochondrial extracts prepared from the mutant reacted very poorly with the antibodies against this subunit (Fig. 3C). However, trace amounts of apparently complete F1FO complexes were clearly visible. The antibodies against Atp9p and α-F1 revealed for the mutant a number of additional signals that were virtually absent in the wild-type: the Atp9p ring, the F1 and unassembled α subunits. Interestingly, these subcomplexes are observed also in a strain lacking the entire ATP6 gene (Δatp6) (10) and in a strain where has been deleted the ATP6 leader peptide sequence (14), a stretch of 10 amino acids on the N-terminal side that is cleaved during the assembly of Atp6p (15,16). These data indicate that the atp6-L247R protein cannot be properly folded or inserted into the ATP synthase which ultimately causes its elimination from the cells (see Discussion).

A distinct situation has been described when another mutation of the ATP6 gene found in NARP/MILS patients, T8993G (17), was modeled in Saccharomyces cerevisiae (18). In this case, although this mutation also dramatically affected the ATP synthesis activity (by >90%), the assembly/stability of the yeast ATP synthase was mostly unaffected (18) (Fig. 3C).

Mitochondrial morphology is profoundly altered in the mutant atp6-L247R

Mitochondrial structure was found to be altered in several yeast ATP synthase mutants (10,19–26) giving support to the notion that in addition to its energetic function, the ATP synthase has some role in the biogenesis of the organelle (20,27,28) (see Discussion). We therefore decided to determine whether the atp6-L247R mutation influences the mitochondrial morphology. For this, the mutated cells were embedded in araldite resin and their ultrastructure examined by transmission electron microscopy. In electron micrographs of wild-type cells, the mitochondrial sections showed a rather circular or oval contour and were well separated from each other (Fig. 6A). In the atp6-L247R cells, most of the mitochondrial sections that were examined exhibited a variety of aberrant structures (Fig. 6C and D). A striking feature was the presence of unusual membranous structures within the atp6-L247R mitochondria. Normally, the inner membrane system of mitochondria consists of two contiguous but distinct membranes, the inner boundary membrane which opposes the outer membrane, and the cristal membrane which forms tubules or lamellae in the interior (29). Cristae organization allows greater amounts of membrane-bound energy transducing enzymes to be packed in the interior of the organelle (30). In wild-type yeast, the cristae are small and poorly defined structures (Fig. 6A). Cristae-like structures were discernable in atp6-L247R mitochondria (denoted c* in Fig. 6B and C), although they appeared quite longer and larger than wild-type cristae. Another typical feature in the atp6-L247R mutant was the presence of membranous partitions going from one point to another of the mitochondrial peripheral contour (Fig. 6C and D), something never observed in the wild-type. These anomalies in mitochondrial structure were not found in a yeast model (atp6-L183R) of another mutation (T8993G) of the ATP6 gene associated with the NARP/MILS diseases (18) (Fig. 6E). This is an intriguing observation since both the T9176G and T8993G mutations drastically decrease the rate of ATP synthesis in yeast mitochondria (see Discussion).

Figure 6.

Ultrastructure of yeast cells. The figure shows ultrastructure micrographs of araldite 80-nm-thick sections of the wild-type strain MR6 (A), the atp6-L247R (BD) and atp6-L183R (E) mutants. m, mitochondria; V, vacuole; c, cristae; c*, cristae-like structures; IM, inner mitochondrial membrane; OM, outer mitochondrial membrane.

Figure 6.

Ultrastructure of yeast cells. The figure shows ultrastructure micrographs of araldite 80-nm-thick sections of the wild-type strain MR6 (A), the atp6-L247R (BD) and atp6-L183R (E) mutants. m, mitochondria; V, vacuole; c, cristae; c*, cristae-like structures; IM, inner mitochondrial membrane; OM, outer mitochondrial membrane.

DISCUSSION

The present study describes the construction and properties of a yeast strain bearing an equivalent of the T9176G mutation of human mtDNA found in NARP/MILS patients. This mutation leads to the replacement of a highly conserved leucine residue by arginine in the Atp6p (or a) subunit (217 in humans, 247 in yeast) of the ATP synthase (7,31). This subunit mediates the transport of protons across the FO which is coupled to the rotation of a trans-membrane ring of Atp9p (or c) subunits that contacts Atp6p (5). The leucine residue modified by T9176G would be located within an α-helical trans-membrane segment of Atp6p in proximity of residues (c-D61 and a-R159) known to be critical for the activity of the FO (32). From such an arrangement it can be inferred that converting a highly hydrophobic residue into a positively charged one in this region of the ATP synthase might be extremely detrimental to the Atp9p/Atp6p system. Indeed, human cells containing high loads of T9176G showed a major deficit in ATP production (6). However, how the leucine to arginine pathogenic change affects the ATP synthase remains unknown. This prompted us to determine the consequences of this mutation in yeast, an organism easily tractable to mitochondrial genetic transformation (33) and biochemical analysis of the ATP synthase (34). The yeast ATP synthase is highly similar to the human enzyme (35,36), especially in the region of Atp6p modified by T9176G which shows strong sequence similarities among species (4). We thus reasoned that the yeast and human ATP synthases may be similarly affected by the leucine to arginine pathogenic change.

We found that, as in humans, the T9176G mutation resulted in a drastic reduction (>95%) in the rate of mitochondrial ATP synthesis in yeast. The ATP production deficit correlates with severe alterations of the ATP synthase. First, the mutant mitochondrial ATPase activity, although modestly decreased, was very poorly inhibited by oligomycin, by <10 versus 85% for the wild-type, and this activity was not coupled to any proton translocation across the mitochondrial inner membrane. Second, although efficiently synthesized Atp6p was present in very low amounts in T9176G yeast. Finally, BN analysis of atp6-L247R mitochondrial extracts revealed the presence of several ATP synthase subcomplexes (the Atp9p ring, the F1 and unassembled α-F1 subunits) and only trace amounts of fully assembled F1FO complexes.

These results indicate that the atp6-L247R mutation impairs the incorporation of Atp6p within the yeast ATP synthase, according to the following rationale. Numerous studies of yeast ATP synthase-deficient strains, issued from mutations in its individual subunits or factors required for its assembly, led to the proposition that Atp6p might be incorporated in a late step of the ATP synthase assembly pathway (36–40). Consistent with this, we have provided direct biochemical evidence that large assemblies lacking Atp6p can form in Δatp6 yeast (10). These assemblies are however very fragile and easily dissociate into several subcomplexes upon detergent-extraction and/or during electrophoresis (10) (Fig. 3C). Exactly the same ATP synthase subcomplexes than those seen in Δatp6 yeast are found in a yeast strain lacking the leader peptide of Atp6p (14) (Fig. 3C), a stretch of 10 amino acid residues on the N-terminal side that is cleaved during ATP synthase assembly (15,16). This peptide is not absolutely required for, but significantly enhances, the association of Atp6p with the Atp9p ring (14). Thus, when missing, incomplete ATP synthase complexes lacking Atp6p form, which gives rise to the presence in BN–PAGE gels of those partial assemblies found in Δatp6 yeast, in addition to complete F1FO complexes (Fig. 3C). The strong detection of the same ATP synthase subcomplexes in atp6-L247R yeast with only trace amounts of fully assembled (but possibly inactive) F1FO complexes (Fig. 3C) indicates that most of the ATP synthase in atp6-L247R cells consists of large assemblies lacking Atp6p. As Atp6p was efficiently synthesized in the atp6-L247R mutant, one can ascribe the presence of incomplete ATP synthase complexes in this mutant to a decreased stability or a less efficient assembly of Atp6p rather than to a lack of production of this subunit. One possibility is that the atp6-L247R mutation may delay the folding/insertion of Atp6p by weakening the interactions with chaperone(s) specifically acting on Atp6p [Atp10p (39), Atp23p (16)]. In this regard, it is worth noting that a null mutation of the gene encoding Atp10p can be compensated for by a single amino acid change in Atp6p (V249A), only two residues away from position 247 (41), which indicates that the region modified by T9176G may specifically interact with Atp10p during the assembly of the ATP synthase. Alternatively, the T9176G mutation may result in a less efficient interaction of Atp6p with the Atp9p ring. The lack of Atp6p in the atp6-L247R mutant may reasonably be accounted for by elimination of the unassembled Atp6p proteins from the cells, as was previously observed in most yeast mutants where the assembly of Atp6p is compromised (16,36,39,42).

When modeling the T9176G mutation in Escherichia coli (43) by changing the leucine 259 of the homologous ATP synthase subunit a by arginine, the rate of ATP synthesis was found to be strongly reduced, like in yeast (this study) and in humans (6). It was reasoned that the mutated bacterial enzyme was correctly assembled due the normal accumulation of the a subunit in the mutant (43). However, the authors did not provide any direct biochemical evidence for the presence of the mutated a subunit within the ATP synthase. Based on the properties of the T9176G E. coli model, it has been initially suggested that the severe lack of ATP synthesis in human T9176G cells might be due to a block in the rotation of the subunit c-ring (8). However, Carrozzo et al. reported latter 2d-gel analyses of T9176C patients tissues revealing that all OXPHOS complexes were found to be normally assembled except complex V (6,44). The presence of large amounts of F1-part in these tissues points to an assembly or stability deficiency rather than to a mechanistic defect like in T9176G yeast. The authors of these studies were surprised by the observation that two subunits of the ATP synthase, α and d, showed a normal content in the T9176G tissues. Now that a yeast model of this mutation is available, these 2d-gel and SDS–PAGE data can be reconciled. A lack of Atp6p has indeed little influence on the steady-state levels and assembly of the other ATP synthase subunits in yeast (10). However, the resulting assemblies are very fragile and easily dissociate when analyzed by BN–PAGE. It is possible that human Atp6p is inserted in a late step of the ATP synthase assembly pathway like in yeast, concerning the conservation of specific chaperones in humans, among those Atp23p, a protein which assists the folding/insertion of Atp6p in yeast (16).

The impact of the T9176G mutation on the ATP synthase is clearly distinct from that of T8993G, another ATP6 mutation found in NARP/MILS patients (17). This mutation leads like T9176G to the replacement of a conserved leucine residue by arginine within a region of Atp6p presumed to be located in the membrane, at position 156 in humans (183 in yeast). Like the T9176G mutation, the T8993G mutation severely reduces the rate of mitochondrial ATP synthesis, both in humans (45–48) and in yeast (18). However, contrary to T9176G, the T8993G mutation has little effect on the assembly or stability of the ATP synthase, and this was observed both in humans (49) and in yeast (18). Thus, if T9176G prevents in some way the association of Atp6p to the remainder of the ATP synthase complex, the T8993G mutation is responsible for a more local structural perturbation that impairs the functioning of the ATP synthase proton channel. The similar sensitivities of yeast and human ATP synthases to the T8993G and T9176G mutations validate the use of yeast as an experimental model to decipher the primary molecular mechanisms involved in the diseases induced by these mutations.

The yeast atp6-L247R mutant showed strongly reduced mitochondrial respiration apparently because of a very low content in complex IV. Similar observations were made with other yeast atp6 mutants (10,18,50). These findings indicate, as we had previously discussed (10,18,50), that complex IV expression is in some way under control of the ATP synthase, perhaps a means to adjust the rate of electron transfer to the ATP needs of the cell or to modulate the mitochondrial membrane potential when glycolytic ATP has to be imported into the organelle, like in anaerobic conditions (51). There is no evidence for the existence of a control of complex IV abundance by the ATP synthase in human cells (52). However, short regulatory responses involving the Cytochrome c oxidase have been described both in yeast and mammals (for a review see 53). For instance, it is well established that mitochondrial respiration rate is modulated by the binding of adenine nucleotides to the Cytochrome c oxidase, indicating that the ADP/ATP ratio is an important effector in the co-regulation of Cytochrome c oxidase and ATP synthase.

Considering mitochondria beyond their bioenergetic function might be crucial for a complete understanding of pathogenic pathways at a molecular level. In recent years, mitochondria have come to be seen as highly dynamic organelles that continuously move, fuse and divide (54). Furthermore, it has been established that mitochondrial membrane dynamics can modulate mitochondrial and cellular functions (55). In humans, mutations of genes encoding components of the mitochondrial fusion/fission machineries are associated with various neuropathies (56–58). In addition, mitochondrial dysfunction generates changes in mitochondrial morphology as exemplified in several models of mitochondrial pathologies (59), indicating a close relationship between membrane dynamics and energy metabolism. Furthermore, oligomerization of the ATP synthase apparently plays an important role in the structural organization of the inner mitochondrial membrane in cristae (20,24,25,60,61). Therefore, changes in the morphology and ultrastructure of mitochondria may also contribute to the underlying pathogenic mechanisms of ATPase-based diseases. The present study points to the possible involvement of such defects in the disease caused by the T9176G mutation. Indeed, a striking feature in most yeast atp6-L247R cell electron micrographs was the presence of tightly associated and reciprocally deformed mitochondria reminiscent of a defect in the fusion of inner mitochondrial membranes (62). In addition, the atp6-L247R mitochondria contained unusually large and long cristae-like structures compared with the wild-type control (Fig. 6). Very similar anomalies were found in yeast cells lacking the entire ATP6 gene (Δatp6) (10), but not in yeast atp6 mutants showing a major deficit in mitochondrial ATP synthesis but where Atp6p is correctly assembled, like in yeast models of the NARP/MILS T8993G (18) (Fig. 6E) and T8851C (Kucharczyk and di Rago, unpublished data) mutations. These findings indicate that it is the lack of Atp6p rather than an ATP production deficit that modifies the overall structure of mitochondria. These observations point to the possibility that in addition to a bioenergetic deficiency changes in the mitochondrial structure may also contribute to the NARP/MILS diseases caused by ATP6 mutations where incorporation of Atp6p within the ATP synthase is compromised.

MATERIALS AND METHODS

Yeast strains and media

The S. cerevisiae strains and their genotypes are listed in Table 1. The media used for growth of yeast were: YPGA [1% (w/v) yeast extract, 1% (w/v) peptone, 2% (w/v) glucose and 40 mg l−1 adenine]; N3 [1% (w/v) yeast extract, 1% (w/v) peptone, 2% (w/v) glycerol and 50 mm potassium phosphate buffer pH 6.2]; YPGALA [1% (w/v) yeast extract, 1% (w/v) peptone, 2% (w/v) galactose and 40 mg l−1 adenine]; WO [0.17% (w/v) yeast nitrogen base without amino acids or ammonium sulfate, 0.5% (w/v) ammonium sulfate, 2% (w/v) glucose, and other supplements depending on the strain's auxotrophic markers]. Solid media contained 2% (w/v) agar.

Construction of a yeast atp6-L247R mutant.

Using the QuikChange XL Site-directed Mutagenesis Kit of Stratagene, the TTA codon 247 of the yeast ATP6 gene was changed into the arginine AGA codon with the primers 5′ GGGATATGTCTGGGCTATTAGAACAGCATCATATTTAAAAGATGC-3′, 5′-GCATCTTTTAAATATGATGCTGTTCTAATAGCCCAGACATATCCC-3′ (the mutagenic bases are in bold). The mutagenesis was performed on an EcoRI–BamHI fragment containing the last 38 codons of ATP6 cloned in pUC19 (plasmid pSDC9) (18). The mutated fragment was liberated by restriction with EcoRI and SapI and ligated with pSDC14 (18) cut with the same enzymes to reconstruct a whole ATP6 gene with the L247R mutation. The resulting plasmid (pRK3) also contains the yeast mitochondrial COX2 gene as a marker for mitochondrial transformation. The pRK3 plasmid was introduced by co-transformation with the nuclear selectable LEU2 plasmid pFL46 into the ρ0 strain DFS160 by microprojectile bombardment using a biolistic PDS-1000/He particle delivery system (Bio-Rad) as described (33). Mitochondrial transformants (synthetic ρ RKY14) were identified among the Leu+ nuclear transformants by their ability to produce respiring clones when mated to the non-respiring NB40-3C strain bearing a deletion in the mitochondrial COX2 gene. One RKY14 clone was crossed to the atp6::ARG8m deletion strain MR10 (10) for the production of clones (called RKY25) harboring the MR10 nucleus and where the ARG8m ORF (63) had been replaced by recombination with the mutated atp6-L247R gene. These clones were identified by their inability to grow in the absence of an external source of arginine. Sequencing of the mutated atp6 locus in RKY25 revealed no other changes than L247R.

Miscellaneous procedures.

Determination of ρ/ρ0 cells in yeast cultures, mitochondrial respiratory and ATP synthesis/hydrolysis activities, mitochondrial membrane potential and cytochrome spectral analyses, SDS–PAGE and BN–PAGE, western blottings, pulse labeling of mtDNA encoded proteins and EM experiments were performed as described in (10).

FUNDING

R.K. was supported by post-doctoral fellowships from the French Ministry of Research and the Agence Nationale de la Recherche (ANR). This work was supported by grants from the Association Française contre les Myopathies (AFM), the GIS-Maladies rares, the ANR and the Conseil de la Région Aquitaine.

ACKNOWLEDGEMENTS

We are grateful to J. Velours for the generous gift of antibodies and M. Ding for English revisions.

Conflict of Interest statement. None declared.

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