Abstract

Adenine nucleotide translocase (Ant) is the most abundant protein on the mitochondrial inner membrane (MIM) primarily involved in ADP/ATP exchange. Ant also possesses a discrete membrane uncoupling activity. Specific mis-sense mutations in the human Ant1 cause autosomal dominant Progressive External Ophthalmoplegia (adPEO), mitochondrial myopathy and cardiomyopathy, which are commonly manifested by fractional mitochondrial DNA (mtDNA) deletions. It is currently thought that the pathogenic mutations alter substrate preference (e.g. ATP versus ADP) thereby dominantly disturbing adenine nucleotide homeostasis in mitochondria. This may interfere with mtDNA replication, consequently affecting mtDNA stability and oxidative phosphorylation. Here, we showed that the adPEO-type A128P, A106D and M114P mutations in the yeast Aac2p share the following common dominant phenotypes: electron transport chain damage, intolerance to moderate over-expression, synthetic lethality with low Δψm conditions, hypersensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) and mtDNA instability. More interestingly, the aac2A137D allele mimicking ant1A123D in mitochondrial myopathy and cardiomyopathy exhibits similar dominant phenotypes. Because Aac2A137D is known to completely lack transport activity, it is strongly argued that the dominant mitochondrial damages are not caused by aberrant nucleotide transport. The four pathogenic mutations occur in a structurally dynamic gating region on the cytosolic side. We provided direct evidence that the mutant alleles uncouple mitochondrial respiration. The pathogenic mutations likely enhance the intrinsic proton-conducting activity of Ant, which excessively uncouples the MIM thereby affecting energy transduction and mitochondrial biogenesis. mtDNA disintegration is a phenotype co-lateral to mitochondrial damages. These findings provide mechanistic insights into the pathogenesis of the Ant1-induced diseases.

INTRODUCTION

Adenine nucleotide translocase (Ant) catalyzes ADP/ATP exchange across the mitochondrial inner membrane (MIM) (1). It contributes 1–10% of total mitochondrial proteins, depending on different tissues and species. This nuclear-encoded protein of 300–320 residues forms six tilted transmembrane helices and a central pore of ∼20 Å in diameter that is proposed to translocate the bulky adenine nucleotides (2). In addition to its primary function in ADP/ATP exchange, it has been extensively documented that Ant has also an intrinsic uncoupling activity (1,3). This activity contributes half to two-thirds of the basal proton conductance in muscle mitochondria (4). How Ant mechanistically uncouples the membrane remains unclear. Current view posits that the uncoupling activity results from a passive proton leakage either through the central substrate translocation channel or on the protein-phospholipid interface, likely as a side effect of drastic conformational changes during the transport process.

Mis-sense mutations in ANT1, encoding the muscle/heart/brain isoform of Ant, cause autosomal dominant Progressive External Ophthalmoplegia (adPEO) (5–9), an adult/later-onset degenerative disorder manifested by ptosis, progressive ophthalmoplegia, muscle weakness, exercise intolerance and dementia (10). As the yeast Aac2 variants equivalent to the pathogenic ant1 alleles retain some basic kinetic properties for nucleotide exchanges, and cells co-expressing the wild-type AAC2 and the mutant aac2 alleles are largely respiratory competent ( (11,12), also see below), the mechanism for the dominant penetrance of the disease remains enigmatic. Given that fractional mtDNA deletions are commonly detected in skeletal muscle, it is currently thought that the mutant Ant may cause an adenine nucleotide imbalance in mitochondria, which sequentially affects dATP biosynthesis, mtDNA replication/stability and ultimately, oxidative phosphorylation. The nucleotide imbalance model is supported by the observation that the yeast Aac2 mutants have a noticeable preference towards the transport of ATP versus ADP in in vitro reconstituted proteoliposomes (12). A potential implication of this finding is that excessive ATP import may be causative for nucleotide imbalance and mtDNA instability, although it remains undetermined whether the altered transport specificity actually affects adenine nucleotide homeostasis in the mitochondrial matrix especially in the context of heterozygous diploid cells. Alternatively, because expression of the adPEO-type mutations in yeast causes electron transport chain damages (12), and more importantly, induces cell death even on glucose medium where respiration is dispensable, it is argued that the pathogenic mutations may directly interfere with a vital function in mitochondrial biogenesis (11,13).

In addition to adPEO, a specific mis-sense allele of ANT1 has been found to be associated with mitochondrial myopathy and cardiomyopathy in a sporadic homozygous patient (14). In this case, multiple mtDNA deletions are also manifested. Interestingly, the mutant allele completely lacks nucleotide transport activity. This is reminiscent of the multiple mtDNA deletions in skeletal and cardiac muscles of Ant1-knockout mice. In the latter case, the loss of the ADPcytosol/ATPmatrix exchange activity depletes ADP in the mitochondrial matrix, which causes ATP synthase stagnation, membrane hyperpolarization, increased ROS production and mtDNA damages (15).

In the present report, we show in the yeast model that the mutations responsible for adPEO, mitochondrial myopathy and cardiomyopathy share common properties which include dominant damages to mitochondria and mtDNA, and loss of cell viability. We provide direct evidence that the pathogenic mutations uncouple the MIM, which directly affects energy transduction and mitochondrial biogenesis. mtDNA instability is secondary to defects in mitochondrial biogenesis.

RESULTS

Common dominant phenotypes associated with the adPEO-type mutations

A salient feature of Ant1-induced adPEO is the dominant penetrance of the disease trait in heterozygous individuals. We thus searched for common dominant phenotypes associated with the yeast aac2A128P, aac2A106D and aac2M114P alleles, equivalent to the pathogenic ant1A114P, ant1A90D and ant1L98P alleles in humans (Fig. 1A). Yeast cells co-expressing the mutant aac2 alleles and the wild-type AAC2 did not exhibit noticeable growth defect on glucose (YPD) or the non-fermentable glycerol (YPGly) medium when incubated at 30°C (Fig. 1B), suggesting that the mutant alleles do not significantly affect ADP/ATP exchange and oxidative phosphorylation. However, cell growth was strongly inhibited at 25°C even on YPD, with an effect in the order of aac2A128P>aac2M114P>aac2A106D. Under these conditions, although mtDNA levels remain unchanged (data not shown), cellular respiration was reduced even after the addition of the uncoupler, CCCP (Fig. 1C). These observations suggest severe damages to the electron transport chain independent of mtDNA stability. The mutant aac2 alleles are hypersensitive to moderate over-expression. For instance, in a cross involving parental strains that express a chromosomally integrated copy of aac2A128P at either the lys2 or trp1 locus, meiotic spores receiving both lys2Δ::aac2A128P and trp1Δ::aac2A128P are segregated into poorly growing and highly sectoring white colonies (Fig. 1D). These cells are mostly non-viable when transferred onto a fresh YPD plate, indicating that the mutant protein directly interferes with a vital function in mitochondrial biogenesis.

Figure 1.

Dominant phenotypes associated with aac2 alleles equivalent to the adPEO-type pathogenic mutations in human Ant1. (A) Projected localization of Ala106, Met114, Ala128 and Ala137 (red) in the yeast Aac2p on the crystal structure of the bovine Ant1 in the cytosolic conformation bound by carboxyatractyloside (yellow) (2). M, matrix; IMS, inter-membrane space; H2, α-helix 2; H3, α-helix 3. (B) The aac2A128P, aac2A106D and aac2M114P alleles dominant-negatively inhibit cell growth on YPD medium at 25°C and on YPD supplemented with EB. The strains 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D) and 6A/UP2U (+aac2M114P) were grown in YPD at 30°C for overnight, serially diluted with water and spotted onto YPD, YPGly and YPD supplemented with EB. The plates were incubated at the temperatures as indicated for 5–7 days before being photographed. (C) Relative oxygen consumption rate of 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D) and 6A/UP2U (+aac2M114P) cells incubated at 30°C, or at 25°C for approximately 5, 10 and 15 generations. The relative oxygen consumption rates are normalized against 6A/UAU. (D) Representative tetrads showing the growth phenotype of the meiotic segregants that express two extra copies of AAC2 and aac2A128P (indicated by the arrows). The asci were dissected on YPD plates which were incubated at 30°C for 4 days.

Figure 1.

Dominant phenotypes associated with aac2 alleles equivalent to the adPEO-type pathogenic mutations in human Ant1. (A) Projected localization of Ala106, Met114, Ala128 and Ala137 (red) in the yeast Aac2p on the crystal structure of the bovine Ant1 in the cytosolic conformation bound by carboxyatractyloside (yellow) (2). M, matrix; IMS, inter-membrane space; H2, α-helix 2; H3, α-helix 3. (B) The aac2A128P, aac2A106D and aac2M114P alleles dominant-negatively inhibit cell growth on YPD medium at 25°C and on YPD supplemented with EB. The strains 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D) and 6A/UP2U (+aac2M114P) were grown in YPD at 30°C for overnight, serially diluted with water and spotted onto YPD, YPGly and YPD supplemented with EB. The plates were incubated at the temperatures as indicated for 5–7 days before being photographed. (C) Relative oxygen consumption rate of 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D) and 6A/UP2U (+aac2M114P) cells incubated at 30°C, or at 25°C for approximately 5, 10 and 15 generations. The relative oxygen consumption rates are normalized against 6A/UAU. (D) Representative tetrads showing the growth phenotype of the meiotic segregants that express two extra copies of AAC2 and aac2A128P (indicated by the arrows). The asci were dissected on YPD plates which were incubated at 30°C for 4 days.

We then tested whether, as in adPEO, a dominant and massive induction of mtDNA mutations can be recapitulated in cells co-expressing the mutant aac2 alleles and the wild-type AAC2. A direct measurement for the frequency of respiratory-deficient petite colonies using the above strains was precluded, because the aac2A128P, aac2M114P and aac2A106D mutants in the M2915-6A background are unable to tolerate the loss of mtDNA (or ρ°-lethal) on YPD (Fig. 1B, see below). To overcome this, we integrated the mutant alleles into the chromosomes of the W303-1B strain which is known to have a relatively high tolerance to ρ°-conditions (16). It was found that at both 30 and 25°C, strains expressing one copy of the mutant alleles form petite colonies at a frequency of <5% (Fig. 2), comparable with spontaneous petite frequency observed in wild-type cells. However, the petite frequencies of cells expressing two copies of aac2A128P, aac2M114P and aac2A106D are drastically increased to 36.7, 15.3 and 47.3%, respectively, at 30°C and to 63.8, 100 and 57.8%, respectively, at 25°C. On medium containing raffinose, a fermentable carbon source that derepresses mitochondrial respiration, the viability of the cells expressing two copies of aac2A128P and aac2A106D is reduced by 3–5-fold, suggesting that mitochondrial biogenesis is severely affected which compromises cell viability. The survivors are almost completely converted into petites at 25°C. mtDNA instability appears to manifest as a phenotype secondary to defects in mitochondrial biogenesis. Remarkably, the growth of cells expressing two copies of aac2M114P is completely inhibited on raffonose medium at 25°C. Respiratory growth on raffonose medium up-regulates the expression of the AAC2 locus (17). This would be expected to further increase the toxicity of the mutant alleles beyond the threshold compatible with cell viability.

Figure 2.

Petite formation and cell death in strains expressing one or two copies of chromosomally integrated aac2A128P, aac2A106D and aac2M114P. Cells were grown at 30°C for 2–3 days, diluted in water and plated onto YPD (Glu) and YPGal+Raf (Gal+Raf) to an estimated cell density of 200 cells per plate. The plates were incubated at 30 or 25°C for 5–7 days.

Figure 2.

Petite formation and cell death in strains expressing one or two copies of chromosomally integrated aac2A128P, aac2A106D and aac2M114P. Cells were grown at 30°C for 2–3 days, diluted in water and plated onto YPD (Glu) and YPGal+Raf (Gal+Raf) to an estimated cell density of 200 cells per plate. The plates were incubated at 30 or 25°C for 5–7 days.

The dominant aac2A137D allele

The close proximity of Ala128, M114P and Ala106 (Fig. 1A) suggests that the three aac2 mutations may damage mitochondria through a similar mechanism, by inducing structural changes to the cytosolic gating domain which encompasses the helix 2–loop–helix 3 region. To provide further support for the cell-killing property of this domain, we performed a random mutagenesis to screen similar mutations that induce cell death when expressed from the GAL10 promoter. One such mutation was identified (Fig. 1A), which converts the codon 137 from alanine to threonine at a position only nine amino acids downstream of Ala128. More importantly, an Ala→Asp mutation in the equivalent Ala123 in human Ant1 has been described in a sporadic homozygous individual suffering from mitochondrial myopathy and cardiomyopathy (14). This prompted us to investigate whether the yeast aac2A137D allele shares common properties with the adPEO-type mutations.

Like the adPEO-type alleles, the chromosomally integrated aac2A137D mutation does not cause significant respiratory growth defect in cells co-expressing the wild-type AAC2 (Fig. 3A). In W303 background, expression of one single copy of aac2A137D is sufficient to dominantly increase petite production to 29% on YPD plates at 25°C (Fig. 3B). The aac2A137D allele is also sensitive to moderate over-expression and to cold temperature. When two copies of aac2A137D are expressed, ∼50 and 95% of cells form abortive micro-colonies at 30 and 25°C, respectively, on YPD. Cells expressing one copy of aac2A137D (Fig. 3C, lanes 2 and 5) have mtDNA levels comparable to that in a wild-type strain (Fig. 3C, lanes 1 and 4). However, expression of Cox2p, an mtDNA-encoded subunit of the cytochrome c oxidase, is reduced by approximately 2-fold (Fig. 3D, lanes 2 and 5). In cells with two copies of aac2A137D, the relative mtDNA levels were reduced by 2.5- and 2.0-fold at 25 and 30°C, respectively (Fig. 3C, lanes 3 and 6), whereas the Cox2p levels were decreased by ∼5-fold (Fig. 3D, lanes 3 and 6). Loss of the electron transport chain is therefore part of the co-lateral damages that precede mtDNA instability, and might be caused by defects in mitochondrial gene expression and in the biogenesis of the respiratory complexes. On raffinose plus galactose medium, expression of two copies of aac2A137D reduces cell viability by 5-fold at 30°C (Fig. 3B), while cell growth was completely inhibited at 25°C. The relative mtDNA levels were reduced by only 22 and 40%, respectively (Fig. 3E and F). Thus, cell death is also independent of mtDNA loss.

Figure 3.

Dominant mitochondrial damages induced by the aac2A137D allele. (A) Growth phenotype of cells of M2915-6A background co-expressing aac2A137D and AAC2 on YPD, YPGly and YPD supplemented with EB at 30°C. (B) Petite formation and loss of cell viability in strains of W303 background expressing one or two copies of the chromosomally integrated aac2A137D allele. Cells were grown at 30°C for 2–3 days, diluted in water and plated onto YPD (Glu) and YPGal+Raf (Gal+Raf) to an estimated cell density of 200 cells per plate. The plates were incubated at 30 or 25°C for 5–7 days. (C) A representative southern blot showing mtDNA content in M2915-6A (wild-type; lanes 1 and 4), CS1481 (lys2Δ::aac2A137D-kan; lanes 2 and 5) and CS1487-1C (lys2Δ::aac2A137D-kan, trp12Δ::aac2A137D-URA3; lanes 3 and 6). Cells were grown in YPD at 25 and 30°C, respectively. (D) Western-blot analysis of isolated mitochondria showing the levels of the mtDNA-encoded Cox2p in the same cultures as in (C). Ilv5p is used as a marker for mitochondrial proteins. (E) A representative southern blot showing mtDNA content in M2915-6A (wild-type; lanes 1 and 3) and CS1487-1C (lys2Δ::aac2A137D-kan, trp12Δ::aac2A137D-URA3; lanes 2 and 4), grown in YPGal+Raf at 25 and 30°C, respectively. (F) Relative mtDNA copy number in CS1487-1C grown in YPGal+Raf at 25 and 30°C, respectively. Errors bars are standard deviations of three independent experiments.

Figure 3.

Dominant mitochondrial damages induced by the aac2A137D allele. (A) Growth phenotype of cells of M2915-6A background co-expressing aac2A137D and AAC2 on YPD, YPGly and YPD supplemented with EB at 30°C. (B) Petite formation and loss of cell viability in strains of W303 background expressing one or two copies of the chromosomally integrated aac2A137D allele. Cells were grown at 30°C for 2–3 days, diluted in water and plated onto YPD (Glu) and YPGal+Raf (Gal+Raf) to an estimated cell density of 200 cells per plate. The plates were incubated at 30 or 25°C for 5–7 days. (C) A representative southern blot showing mtDNA content in M2915-6A (wild-type; lanes 1 and 4), CS1481 (lys2Δ::aac2A137D-kan; lanes 2 and 5) and CS1487-1C (lys2Δ::aac2A137D-kan, trp12Δ::aac2A137D-URA3; lanes 3 and 6). Cells were grown in YPD at 25 and 30°C, respectively. (D) Western-blot analysis of isolated mitochondria showing the levels of the mtDNA-encoded Cox2p in the same cultures as in (C). Ilv5p is used as a marker for mitochondrial proteins. (E) A representative southern blot showing mtDNA content in M2915-6A (wild-type; lanes 1 and 3) and CS1487-1C (lys2Δ::aac2A137D-kan, trp12Δ::aac2A137D-URA3; lanes 2 and 4), grown in YPGal+Raf at 25 and 30°C, respectively. (F) Relative mtDNA copy number in CS1487-1C grown in YPGal+Raf at 25 and 30°C, respectively. Errors bars are standard deviations of three independent experiments.

Hypersensitivity to low Δψm conditions

Another common dominant phenotype shared by the four aac2 alleles is the intolerance to the elimination of mtDNA by ethidium bromide (EB) on glucose medium (Figs 1B and 3A). The ρ°-lethal phenotype suggests that the mutant cells have reduced MIM potential (Δψm), such that they are unable to support mitochondrial biogenesis upon a further loss of the Δψm-generating electron transport chain. To support this, we first tested whether the mutant aac2 alleles can create synthetic defects with genetic conditions that have compromised membrane integrity and decreased Δψm. The aac2A128P, aac2M114P, aac2A106D and aac2A137D alleles are all synthetically lethal with the disruption of YME1 (Fig. 4A). Yme1p is an AAA protease on MIM involved in protein turnover (18,19). Disruption of YME1 does not affect the steady-state level of Aac2 (our unpublished observation), but leads to over accumulation of various proteins on the membrane (20,21) which is manifested by low Δψm and ρ°-lethality (18,21,22). Our data suggest that Δψm may be additively reduced by the mutant Aac2 proteins and yme1Δ to a level below the threshold compatible with cell survival.

Figure 4.

The mutant aac2 alleles are hypersensitive to low Δψm conditions in a dominant manner. (A) Hypersensitivity to yme1Δ. Diploid strains with the indicated phenotypes were sporulated and dissected on YPD. The plates were incubated at 30°C for 4–5 days. Circled are spores deduced to have the combination of yme1Δ::LEU2 with the chromosomally integrated copy of AAC2-URA3, aac2A128P-URA3, aac2A106D-URA3, aac2M114P-URA3 or aac2A137D-URA3. (B) Hypersensitivity to CCCP. The strains 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D), 6A/UP2U (+aac2M114P) and CS1426/1 (+aac2A137D) were grown on YPD at 30°C for 4 days in the presence or absence of CCCP (120 µm).

Figure 4.

The mutant aac2 alleles are hypersensitive to low Δψm conditions in a dominant manner. (A) Hypersensitivity to yme1Δ. Diploid strains with the indicated phenotypes were sporulated and dissected on YPD. The plates were incubated at 30°C for 4–5 days. Circled are spores deduced to have the combination of yme1Δ::LEU2 with the chromosomally integrated copy of AAC2-URA3, aac2A128P-URA3, aac2A106D-URA3, aac2M114P-URA3 or aac2A137D-URA3. (B) Hypersensitivity to CCCP. The strains 6A/UAU (+AAC2), 6A/UPU (+aac2A128P), 6A/UDU (+aac2A106D), 6A/UP2U (+aac2M114P) and CS1426/1 (+aac2A137D) were grown on YPD at 30°C for 4 days in the presence or absence of CCCP (120 µm).

Further support for the low Δψm model has come from the experiment showing that cells co-expressing the mutant aac2 alleles with the wild-type AAC2 are hypersensitive to the chemical uncoupler, CCCP (Fig. 4B). In response to CCCP, the control cells expressing only the wild-type AAC2 form white colonies, suggesting altered mitochondrial function. Under these conditions, the growth of the cells expressing the mutant aac2 alleles is severely inhibited. The aac2A106D mutant exhibits the least sensitivity to CCCP, consistent with its relatively mild effect on mitochondrial function (Fig. 1C).

Respiratory uncoupling

To directly test the hypothesis that the mutant Aac2 proteins dominantly uncouple the MIM, we determined the respiratory coupling efficiency of isolated mitochondria by measuring the respiratory control ratio (RCR). To minimize the damage to the electron transport chain, which would interfere with an accurate measurement of respiratory coupling efficiency, we introduced the mutant aac2 alleles into the diploid cells of W303 background expressing the endogenous wild-type AAC2. At 30°C, the RCR was significantly reduced in mitochondria from the aac2A106D and aac2A137D, but not from the aac2M114P and aac2A128P mutants compared with the wild-type control (Fig. 5). This correlates with the petite frequencies of the mutants under active respiring conditions at 30°C (Figs 2 and 3B), with the aac2A106D and aac2A137D mutants having the strongest phenotype compared with aac2M114P and aac2A128P. At 25°C, mitochondria from the aac2A137D mutant remain uncoupled. Those from the aac2M128P and aac2M114P mutants become significantly uncoupled, consistent with the cold-sensitive nature of these alleles. Respiratory coupling in the relatively cold-insensitive aac2A106D mutant (Fig. 2) is only marginally reduced at 25°C. Taken together, these data demonstrate that mitochondrial uncoupling is a common property associated with the four aac2 alleles.

Figure 5.

The aac2A128P, aac2M114P, aac2A106D and aac2A137D alleles induce membrane uncoupling. Yeast cells were grown in YPGal at 30°C (A) or 25°C (B) and harvested at later exponential phase. Isolated mitochondria were immediately used for respiratory assays using ethanol as substrate. RCR was deduced by dividing the State 3 respiratory rate (induced by ADP) by the State 4 rate. A low RCR is an indication of uncoupled respiration and an RCR of 1 would indicate that the mitochondria are completely uncoupled. For all experiments, the values are averages of 4–6 measurements and are compared with the wild-type control. The standard errors and P-values (Unpaired Student's t-test) are indicated.

Figure 5.

The aac2A128P, aac2M114P, aac2A106D and aac2A137D alleles induce membrane uncoupling. Yeast cells were grown in YPGal at 30°C (A) or 25°C (B) and harvested at later exponential phase. Isolated mitochondria were immediately used for respiratory assays using ethanol as substrate. RCR was deduced by dividing the State 3 respiratory rate (induced by ADP) by the State 4 rate. A low RCR is an indication of uncoupled respiration and an RCR of 1 would indicate that the mitochondria are completely uncoupled. For all experiments, the values are averages of 4–6 measurements and are compared with the wild-type control. The standard errors and P-values (Unpaired Student's t-test) are indicated.

DISCUSSION

In this report, we captured an array of dominant phenotypes commonly associated with the aac2A128P, aac2M114P, aac2A106D and aac2A137D alleles in strains co-expressing the wild-type AAC2. These include cold-induced respiratory deficiency, ρ°-lethality, intolerance to chemical uncoupler and to compromised MIM integrity, hypersensitivity to moderately increased expression, gene dosage-dependent cell death, mtDNA instability and uncoupled respiration. All the phenotypes can be best explained by the proposal that the mutant proteins primarily uncouple the inner membrane, which subsequently affects the mitochondrial gene expression, electron transport chain assembly, mitochondrial biogenesis, mtDNA integrity and cell viability.

We have recapitulated the dominant mtDNA instability phenotype of Ant1-induced human diseases in the yeast model. In response to moderately increased gene dosage and to respiratory conditions, mtDNA is drastically destabilized in cells co-expressing aac2A128P, aac2M114P, aac2A106D or aac2A137D with the wild-type AAC2. This is in sharp contrast to an early observation that aac2A128P can only induce a marginal increase in petite production when expressed from a centromeric vector (12). Our data suggest that mtDNA instability arises independent of adenine nucleotide homeostasis. Aac2A137D is known to completely lack nucleotide transport activity (14). Likewise, haploid cells expressing only Aac2A106D are also respiratory deficient (data not shown), indicating a severe defect in ADP/ATP exchange. Thus, it can be excluded that the gain of a novel substrate selectivity or transport kinetics, which can potentially cause ATP/ADP imbalance in the matrix, is a prerequisite for the dominant mitochondrial damages. On the other hand, neither the heterozygous AAC2/aac2Δ diploid cells nor an aac2Δ null mutant exhibit cell death and mtDNA instability (our unpublished data). Clearly, the dominant mitochondrial damages are likely induced by the gain of some non-catalytic properties by the mutant proteins, rather than being caused by simply the loss of the nucleotide transport activity. Furthermore, Aac2A128P and Aac2M114P are known to retain some basic kinetic properties for nucleotide exchange (12). We also found that consistent with the respiratory competent phenotype, mitochondria isolated from diploid cells heterozygous for aac2A128P have a Km of 8.97 ± 0.24 µm and a Vmax of 0.385 ± 0.005 for ADP/ATP exchange, which are only marginally different from a Km of 7.5 ± 0.22 µm and a Vmax of 0.398 ± 0.01 for wild-type mitochondria (data not shown). These observations further argue against the idea that mitochondrial damages by the pathogenic alleles are caused by catalytic deficiency.

Our data strongly support the proposal that the pathogenic mutations uncouple the MIM and induce severe defects in mitochondrial biogenesis, which secondarily causes mtDNA instability. mtDNA stability is known to be affected by mutations in genes ostensibly unrelated to mtDNA replication (23). Although adPEO is most frequently caused by mutations in genes involved in mtDNA replication, including the DNA polymerase γ and the Twinkle helicase (24–26), mtDNA deletions are also associated with diseases such as autosomal dominant optic atrophy with mutations in OPA1. OPA1 plays a role in mitochondrial fusion and cristae organization rather than directly in mtDNA metabolism (27–29).

The human ant1A123D allele has been identified in a sporadic homozygous patient of mitochondrial myopathy and cardiomyopathy (14). The detailed inheritance pattern of this allele is unknown. The equivalent aac2A137D mutation in yeast is reportedly recessive, based on the observation that yeast cells co-expressing the mutant allele with the wild-type AAC2 are ρ°-viable and do not induce any detectable respiratory deficiency and mtDNA instability in the W303 background (14). By using strains of M2915-6A background, which have relatively higher sensitivity to low Δψm conditions compared with W303 (16), we clearly demonstrated that aac2A137D shares common dominant phenotypes with the adPEO-type alleles, which include ρ°-lethality, cold-induced cell death, sensitivity to CCCP and synthetic lethality to the yme1 mutation. Even in W303 strains, we found that mtDNA can be dramatically destabilized by cold shock, respiratory growth and moderately increased expression in a dominant manner. Thus, under these conditions, aac2A137D is unambiguously a dominant mutation like the adPEO alleles.

All the four pathogenic mutations analyzed in this study occur in a specific region that connects α-helices 2 and 3 on the cytosolic side (the cytosolic H2–L–H3 domain). This domain is likely very vulnerable to structural alterations, which results in mitochondrial damage and cell death. The cytosolic H2–L–H3 domain of Aac2p is known to undergo dynamic structural changes during nucleotide transport (30). We speculate that the introduction of the helix-breaking proline and the negatively charged aspartic acid residues may induce drastic conformational changes that impede its gating function. Improper gating consequently magnifies the proton-conducting activity intrinsically associated with Ant (1,3,4,31), although induction of other forms of membrane-permeabilizaing events cannot be completely excluded (1,32). The proton-conducting activity of Ant has been shown to be cold-inducible (33). Remarkably, the cytosolic H2–L–H3 domain also coincides with the binding site of the HIV-1 derived Vpr peptide in human Ant1. The binding of Vpr specifically increases proton conductance of the MIM and uncouples mitochondrial respiration (34).

Our findings provide a potential mechanistic explanation for Ant1-induced adPEO. The hypersensitivity of cells to the dosage of the mutant Ant is reminiscent of the clinical pattern of the disease, which selectively affects skeletal muscle where Ant1 is most abundantly expressed. With the overwhelming abundance of Ant1, excessive membrane uncoupling could have far-reaching implications for mitochondrial bioenergetics. In state 3 respiration, the maximum ATP/ADP ratio maintained by mitochondria can decrease by 10-fold for every 14 mV decrease in Δp (35). Therefore, ant1-induced membrane uncoupling could directly affect phosphorylation potential and ATP output. Excessive membrane depolarization in a subpopulation of mitochondria of individual cells impairs the biogenesis of these organelles leading to fractional destabilization of mtDNA. Thus, from the therapeutic perspectives, future efforts may be focused on reducing the toxicity of the mutant proteins and to alleviate the MIM from excessive uncoupling. In ant1A123D -induced mitochondrial myopathy and cardiomyopathy, respiratory uncoupling and mitochondrial damages may also contribute to the pathogenesis in addition to deficiency in ADP/ATP exchange in homozygous patients. In light of the present study, it would also be predicted that a dominant penetrance of this allele may occur in heterozygous individuals.

MATERIALS AND METHODS

Strains and media

Yeast strains used in this study are listed in Table 1. To construct the aac2 mutants, AAC2 was first placed adjacent to URA3 or kan. The aac2A106D, aac2M114P, aac2A128P and aac2A137D alleles were generated by in vitro mutagenesis using the QuickChange kit (Stratagene). The aac2-URA3 and aac2-kan cassettes were amplified by PCR and integrated into the trp1, lys2 or ura3 loci through homologous recombination by selecting for Ura+ or G418R transformants. Complete medium for growth of yeast (YPD) contains 1% Bacto yeast extract, 2% Bacto peptone and 2% glucose. Galactose plus raffinose medium (Gal+Raf) contain 2% galactose and 2% raffinose instead of glucose. Minimal medium (YNBD) contains 0.17% Difco yeast nitrogen base without amino acids, 0.5% ammonium sulfate and 2% glucose. EB plates were YPD supplemented with EB at 16 µg/ml.

Table 1.

Genotypes and source of yeast strains used in this study

Strain name Genotype Source 
M2915-6A background 
M2915-6A MATa,ade2, leu2, ura3 This laboratory 
UPU5-6B as M2915-6A, but aac2::kan, ura3Δ::aac2A128P-URA3 This study 
6A/UAU as M2915-6A, but ura3Δ::AAC2-URA3 This study 
6A/UPU as M2915-6A, but ura3Δ::aac2A128P-URA3 This study 
6A/UP2U as M2915-6A, but ura3Δ::aac2M114P-URA3 This study 
6A/UDU as M2915-6A, but ura3Δ::aac2A106D-URA3 This study 
CS1426/1 as M2915-6A, but ura3Δ::aac2A137D-URA3 This study 
CS1482/1 as M2915-6A, but lys2Δ::aac2A137D-kan This study 
CS1481/1 α, but trp1Δ::aac2A137D-URA3 This study 
CS1487-1C as M2915-6A, but trp1Δ::aac2A137D-URA3, lys2Δ::aac2A137D-kan This study 
CS1380 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A128P-URA3/+, +/lys2Δ:: aac2A128P-kan This study 
CS1381 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::AAC2-URA3/+, +/lys2Δ::AAC2-kan This study 
CS1483 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A106D-URA3/+, +/lys2Δ:: aac2A106D-kan This study 
CS1486 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2M114P-URA3/+, +/lys2Δ:: aac2M114P-kan This study 
CS1487 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A137D-URA3/+, +/lys2Δ:: aac2A137D-kan This study 
CS1039 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A128P-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1476 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A137D-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1406 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A106D-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1407 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2M114P-URA3/ura3, +/yme1Δ::LEU2 This study 
W303 background 
W303-1B MATα, ade2, trp1, his3, leu2, ura3 R. Rothstein 
W303-1B/A MATa,ade2, trp1, his3, leu2, ura3 R. Rothstein 
W303/a/α MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2 R. Rothstein 
CS1382-4A as W303-1B, but trp1Δ::aac2A128P-URA3 This study 
CS1382-1C as W303-1B, but trp1Δ::aac2A128P-URA3, lys2Δ::AAC2A128P-kan This study 
CS1442/2 as W303-1B, but trp1Δ::aac2M114P-URA3 This study 
CS1460-2D as W303-1B, but trp1Δ::aac2M114P-URA3, lys2Δ::AAC2M114P-kan This study 
CS1444/1 as W303-1B, but trp1Δ::aac2A106D-URA3 This study 
CS1462-1C as W303-1B, but trp1Δ::aac2A106D-URA3, lys2Δ::AAC2A106D-kan This study 
CS1458/1 as W303-1B, but trp1Δ::aac2A137D-URA3 This study 
CS1466-1D as W303-1B, but trp1Δ::aac2A137D-URA3, lys2Δ::AAC2A137D-kan This study 
KLS204 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A128P-kan This study 
KLS203 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2M114P-kan This study 
KLS202 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A106D-kan This study 
KLS205 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A137D-kan This study 
Strain name Genotype Source 
M2915-6A background 
M2915-6A MATa,ade2, leu2, ura3 This laboratory 
UPU5-6B as M2915-6A, but aac2::kan, ura3Δ::aac2A128P-URA3 This study 
6A/UAU as M2915-6A, but ura3Δ::AAC2-URA3 This study 
6A/UPU as M2915-6A, but ura3Δ::aac2A128P-URA3 This study 
6A/UP2U as M2915-6A, but ura3Δ::aac2M114P-URA3 This study 
6A/UDU as M2915-6A, but ura3Δ::aac2A106D-URA3 This study 
CS1426/1 as M2915-6A, but ura3Δ::aac2A137D-URA3 This study 
CS1482/1 as M2915-6A, but lys2Δ::aac2A137D-kan This study 
CS1481/1 α, but trp1Δ::aac2A137D-URA3 This study 
CS1487-1C as M2915-6A, but trp1Δ::aac2A137D-URA3, lys2Δ::aac2A137D-kan This study 
CS1380 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A128P-URA3/+, +/lys2Δ:: aac2A128P-kan This study 
CS1381 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::AAC2-URA3/+, +/lys2Δ::AAC2-kan This study 
CS1483 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A106D-URA3/+, +/lys2Δ:: aac2A106D-kan This study 
CS1486 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2M114P-URA3/+, +/lys2Δ:: aac2M114P-kan This study 
CS1487 MATa/α, ade2/ade2, leu2/leu2, ura3/ura3, trp1Δ::aac2A137D-URA3/+, +/lys2Δ:: aac2A137D-kan This study 
CS1039 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A128P-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1476 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A137D-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1406 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2A106D-URA3/ura3, +/yme1Δ::LEU2 This study 
CS1407 MATa/α, ade2/ade2, leu2/leu2, ura3Δ::aac2M114P-URA3/ura3, +/yme1Δ::LEU2 This study 
W303 background 
W303-1B MATα, ade2, trp1, his3, leu2, ura3 R. Rothstein 
W303-1B/A MATa,ade2, trp1, his3, leu2, ura3 R. Rothstein 
W303/a/α MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2 R. Rothstein 
CS1382-4A as W303-1B, but trp1Δ::aac2A128P-URA3 This study 
CS1382-1C as W303-1B, but trp1Δ::aac2A128P-URA3, lys2Δ::AAC2A128P-kan This study 
CS1442/2 as W303-1B, but trp1Δ::aac2M114P-URA3 This study 
CS1460-2D as W303-1B, but trp1Δ::aac2M114P-URA3, lys2Δ::AAC2M114P-kan This study 
CS1444/1 as W303-1B, but trp1Δ::aac2A106D-URA3 This study 
CS1462-1C as W303-1B, but trp1Δ::aac2A106D-URA3, lys2Δ::AAC2A106D-kan This study 
CS1458/1 as W303-1B, but trp1Δ::aac2A137D-URA3 This study 
CS1466-1D as W303-1B, but trp1Δ::aac2A137D-URA3, lys2Δ::AAC2A137D-kan This study 
KLS204 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A128P-kan This study 
KLS203 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2M114P-kan This study 
KLS202 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A106D-kan This study 
KLS205 MATa/α, ade2/ade2, trp1/trp1, his3/his3, ura3/ura3, leu2/leu2, trp1/trp1Δ:: aac2A137D-kan This study 

Preparation of mitochondria and measurement of respiration rates

Cells were grown at 30 or 25°C in YPGal medium to late exponential phase. Mitochondria were isolated by differential centrifugation after the homogenization of spheroplasts. The isolated mitochondria were resuspended in 0.6 M mannitol, 2 mm EGTA, 10 mm Tris-maleate, 10 mm phosphate (pH 6.8) and 0.1% fatty acid-free BSA. Respiration was measured with a Clark-type electrode (Yellow Springs Instrument Co.) in the same buffer using a mitochondrial concentration of 0.4 mg/ml. Ethanol (1 mm) was used as a substrate. State 3 respiration was stimulated by the addition of ADP at 50 µm. For measuring O2 consumption by intact cells, strains were grown in YPD at 30°C, diluted 1:1000 to fresh medium and incubated at 25°C, repeatedly diluted to fresh medium every 24 h. Cells were collected by centrifugation for 5 min at 600×g, washed with H2O and resuspended in H2O to OD600=60. Fifty microliter of cell suspension was placed into the electrode chamber containing 2.95 ml of 100 mm glucose and 10 mm Tris-maleate (pH 6.8) to obtain a final OD600 of 1.0. After stabilization of the oxygen consumption rate, CCCP (carbonyl cyanide m-chlorophenylhydrazone, Sigma) was added to a final concentration of 10 µM.

Other procedures

All the strains used carry the ade2 mutation, which turn red under respiring conditions in the presence of a functional mitochondrial genome. The petite frequency was measured by determining the percentage of white colonies. For determining mtDNA profile and content, total yeast DNA was isolated and analysed by Southern blotting. CsCl-purified entire yeast genomic DNA was labeled with [32P] and used as a probe. Relative mtDNA content was estimated by phosphoimaging-based quantitation. Western blot was used for determining Cox2p levels by using antibodies specific for Cox2p and Ilv5p which are provided by Molecular Probes and the Butow laboratory (University of Texas Southwestern Medical Center), respectively. Hydroxylamine mutagenesis was performed to introduce random mutations into AAC2. Briefly, 10 µg of the plasmid pCXJ28-GAL10-AAC2, in which the AAC2 coding sequence was placed under the control of the galactose-inducible GAL10 promoter, was mixed with 500 µl of freshly made hydroxylamine solution (1 M hydroxylamine–HCl, 0.45 N NaOH). After incubation for 20 h at 56°C, the DNA was purified on a Quick-Spin column (QIAGEN). The mutagenized plasmids were transformed into M2915-6A on YNBD medium. Ura+ transformants were screened for sensitivity to galactose induction on YNBGal. The plasmids in the galactose-sensitive colonies were rescued and sequenced for identifying the mutations.

FUNDING

This work was supported by National Institute of Health (AG023731) and the American Heart Association (0435047N).

Conflict of Interest statement. None declared.

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