Role of Adenine Nucleotide Translocator 1 in mtDNA Maintenance

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Science  04 Aug 2000:
Vol. 289, Issue 5480, pp. 782-785
DOI: 10.1126/science.289.5480.782


Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

Mitochondrial dysfunction caused by instability of mtDNA (depletion or multiple deletions) has been associated with a variety of inherited diseases, as well as with aging and exposure to the antiviral drug zidovudine (AZT) (1). Studies of an interesting subgroup of human mitochondrial disorders have revealed that nuclear mutations can affect the mtDNA copy number or its integrity. These diseases include mtDNA depletion syndrome (2), mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) (3), and recessive and dominant forms of progressive external ophthalmoplegia with multiple deletions of mtDNA (4, 5).

Autosomal dominant progressive external ophthalmoplegia (adPEO) with multiple mtDNA deletions is an adult-onset mitochondrial disorder with an incidence of ∼1:100,000 in Finland and in Italy. The typical clinical features are progressive external ophthalmoplegia, ptosis, and exercise intolerance. Ataxia, depression, hypogonadism, hearing deficit, peripheral neuropathy, and cataract are found in some families (5–8). The skeletal muscle shows ragged-red fibers and mildly reduced activities of the respiratory-chain enzymes, as well as multiple mtDNA deletions (Fig. 1). There are three distinct autosomal loci for this disorder on chromosomes 10q24 (MIM 157640) (9), 3p14-21 (MIM 601226) (10), and 4q34-35 (MIM 601227) (11).

Figure 1

Multiple mtDNA deletions in adPEO patient muscle. Total muscle DNA from a control subject (C) and an adPEO patient (P, patient from family C, Fig. 2) was digested with Pvu II, which linearizes the circular mtDNA. Southern blot analysis was done as described (5), with total human mtDNA as the hybridization probe. The 16.6-kb band indicates the full-size mtDNA, and the additional bands on the patient's sample represent the mutant mtDNA molecules with multiple deletions of different sizes.The amount of mutant mtDNA is about 50% of total mtDNA. MtDNA hybridization signal on each lane was compared with that of a nuclear 18S rDNA sequence by densitometry, and no sign of reduced total mtDNA was detected (39).

The critical region of the 4q-adPEO locus includes the gene encoding the heart- and skeletal muscle–specific isoform of the adenine nucleotide translocator (ANT1). ANT, or the ADP/ATP translocator, is the most abundant protein in the inner mitochondrial membrane (12). It forms as a homodimer, a gated channel by which ADP is brought into and ATP brought out of the mitochondrial matrix. ANT regulates the adenine nucleotide concentrations in the cytoplasm and within the mitochondria and mediates signals of nucleo-cytoplasmic energy consumption to the mitochondrial respiratory chain. In addition to the translocase activity, ANT is a core structural element of the mitochondrial permeability transition pore (MPTP) (13) and has an important role in mitochondrial-mediated apoptosis (14). Human ANT exists as three isoforms: ANT1 is expressed predominantly in postmitotic cell types in skeletal muscle, heart, and brain; ANT2 is expressed mainly in proliferating tissue types; and ANT3 is expressed ubiquitously (15, 16).

We analyzed the genomic sequence of ANT1 in the 4q-linked Italian family A (Fig. 2) (17) and identified a heterozygous G→C transversion in exon 2, codon 114, which produces an Ala→Pro substitution (Fig. 3A). The nucleotide change was present in all the affected family members, but not in 860 Finnish or 150 Italian control individuals. A114 and its flanking sequence are strictly conserved between species, and thus likely to be functionally significant (Fig. 3, B and C). Next we analyzed ANT1in patients and healthy subjects of all the PEO families of our study material, including those with previous linkage to other chromosomal loci or with unknown gene loci (Table 1). The heterozygous A114P change was present in four additional families (B to E), two of which, surprisingly, had been previously linked to the chromosome 3 locus (10). We constructed 13.5-cM-long haplotypes with DNA markers flanking the ANT1 locus and identified a common disease haplotype with identical marker alleles, shared by patients in the three Italian families A, B, and C (Fig. 2). This suggests that there is one founder mutation and common ancestry (Fig. 2), although this could not be genealogically confirmed. The A114P mutation segregated with the disease in all these families, with the exception of subject A/402 (Fig. 2). In one sporadic patient with PEO and multiple mtDNA deletions, we identified another missense mutation, a G→A transition in exon 4, codon 289, which produces a Val→Met substitution (Fig. 3, A and C, and Table 1). This sequence change was not present in the patient's clinically healthy parents [paternity tested (18)] or in 156 Italian or 921 Finnish control subjects and was therefore considered a new mutation.

Figure 2

AdPEO pedigrees and shared chromosomal regions on 4q34-35. We have previously described the clinical features of families A (11) as well as B and C (10). The common manifestation of the disease was chronic progressive external ophthalmoplegia, and the age of onset for the symptoms was <45 years. The neuromuscular symptoms were largely confined to eye and facial muscles in families A, C, D, and F, whereas patients from families B and E also had generalized muscle weakness. Peripheral neuropathy, endocrinological abnormalities, or symptoms from the central nervous system, described in some adPEO families (5–8), were not present. Families A to E originate from Romagna County of Italy, suggesting that there may be common ancestry and one founder mutation. Informed consent was obtained from all family members, and total DNA was extracted from lymphoblasts, cultured fibroblasts, or 10 to 150 mg of muscle biopsy sample (40). The individuals with distinct clinical symptoms and/or deletions of mtDNA, detected by Southern blot hybridization, are indicated with black symbols. The white symbols indicate clinically investigated individuals of age >45 years with no clinical symptoms. The individuals marked with question marks have not been clinically investigated or are <45 years old. Haplotypes of chromosome 4q34-35 adPEO region were constructed as described (11), with the indicated 4q DNA markers. The allelically identical part of the haplotype, and consequently the segregation of the A114P mutation, is indicated with a black bar. In families D to F the individuals of whom DNA samples were available are marked with an asterisk. The A114P mutation segregated with the disease in families A to E, with the exception of subject A/402, an asymptomatic carrier of the ANT1 mutation (49 years). Because this subject's muscle sample was not available, his disease status could not be determined. The only patient in family F had the V289M mutation.

Figure 3

The heterozygous A114P and V289M mutations and sequence conservation of ANT1. (A) DNA sequence around codon 114 (left) and 289 (right) in two patients. The heterozygous G→C and G→A missense mutations are marked with arrows. (B) Conservation of ANT1/AAC2 amino acid sequence in different species. The mouse ANT1 is 95% identical and AAC2 is 54% identical with human ANT1. (C) A114 of ANT1 (arrow) is highly conserved, and V289 (arrow) relatively conserved between species. The sequence between amino acids 113 and 125 of ANT1 is fully conserved in AAC2, theS. cerevisiae homolog of ANT1. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Table 1

PEO family and patient material and controls analyzed for ANT1 mutations. We analyzed ANT1 for the presence of the A114P and V289M mutations in Italian adPEO families, in adPEO and recessive PEO (arPEO) families from other European countries or the United States, and in sporadic Italian PEO patients with multiple mtDNA deletions. Control samples were Italian or Finnish, either analyzed individually or from pooled samples of Finnish control subjects. Samples were analyzed by PCR and subsequent sequencing or solid-phase minisequencing (37). The detected genotypes were as follows: AA, alanine/alanine; AP, alanine/proline at amino acid position 114; VV, valine/valine; and VM valine/methionine at amino acid position 289 of ANT1.

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Because of the previous linkage of families B and C to the chromosome 3 locus (10), we confirmed the clinical information on the family members. Upon reexamination, subject B/308 was found to be healthy, although he had been previously recorded as an adPEO patient. Family C was small, and its informativity in the linkage calculations was low. After updating of the phenotypic information, the analyses of the pedigree data with DNA markers from 3p14-21 failed to show significant lod (logarithm of the odds ratio for linkage) scores in this region (highest new multipoint lod score, 2.85) (19). Further, haplotype analyses did not support the existence of an adPEO locus on chromosome 3. These data emphasize the importance of accurate clinical diagnoses in restricted study samples.

Human cells could not be used to evaluate functional consequences of the ANT1 mutation. No disease phenotype has been identified in cultured cells of adPEO patients (20, 21);ANT1 is not expressed in cultured cells, even in myoblasts (20); and apoptosis is induced when wild-typeANT1 is overexpressed (22). Therefore, we introduced the A114→Pro mutation into the fully conserved site (A128) (Fig. 3C) of the major adenine nucleotide translocatorAAC2 gene of the yeast Saccharomyces cerevisiae. We transformed two different yeast strains lacking functional AAC2 with constructs encoding wild-type AAC2 or mutant AAC2, or with the single-copy vector only (23). On glucose medium, all the transformants grew equally well, because the anaerobic energy production is not dependent on AAC2 function (Fig. 4A). On glycerol medium, the cells use the respiratory chain for energy production, and the mutant A128P transformants showed defective growth, as did the vector-only control, whereas the wild-type AAC2 transformants formed single colonies with equal efficiency as on glucose medium (Fig. 4, A and B). This finding indicates that the A128P mutation on AAC2, corresponding to the A114P mutation in human ANT1, affects oxidative respiration. Analysis of the mtDNA in the mutant A128P transformants showed neither large-scale rearrangements nor depletion, suggesting that the growth defect is caused by an ADP/ATP transport defect (24) (Fig. 4C).

Figure 4

Functional consequences of the A128P mutation of AAC2, S. cerevisiae homolog of ANT1. The S. cerevisiae haploid yeast strains were as follows: DNY1 (MATa aac1::LEU2 aac2::HIS3 his3-11,15 trp1-1 ura3-1 can1-100 ade2-1 leu2-3,112) (27), and VG1-5A (MATα ade ura3 trp1 op1) (41). DNY1 is an aac1 aac2 double-deletion strain, and strain VG1-5A has an op1 mutation inAAC2 gene, both resulting in lack of growth in nonfermentable carbon sources. The aerobic growth defect caused by the double mutation is similar to that of aac2 mutant only (42). (A) Growth in glucose medium (SCD-Ura) when strains were transformed with vector control (pSEYc58) wild-type AAC2 (pSEYc58AAC2) or the mutant AAC2 (pSEYc58aac2A128P) construct. All the transformants grew equally well. (B) Growth on glycerol medium (SCG-Ura). Strains transformed with vector control were unable to grow on glycerol, and those expressing the mutant AAC2 showed a clear defect in growth on glycerol compared with the transformants expressing the wild-typeAAC2. The growth of the transformants was tested also as patches first grown on glucose and then replicated onto glycerol. A distinct growth defect was observed for the AAC2A128Pmutant compared with the wild-type allele (43). The difference in growth efficiency on glycerol was apparent on the first day after replication. On the second day the mutant cells started to grow, but the difference in growth rate was still observed. In transformation of a haploid wild-type yeast strain with a single-copy AAC2 mutant plasmid, we could not detect a growth defect (43). (C) MtDNA analysis of vector (V), wild-type AAC2 (W), and mutant AAC2 (M) transformants. Southern hybridization analysis of Acc I restriction–digested DNA was carried out with a yeast mtDNA-specific oligonucleotide as a probe (24). MtDNA on each lane was quantified by densitometry using the hybridization signal of a single-copy nuclear gene, MSO1, as an internal control (43). Neither large-scale rearrangements nor depletion of mtDNA in the mutant AAC2 transformants was detected.

On the basis of the structural modeling of the yeast AAC2, A114P is likely to be located either in the third transmembrane domain of ANT1 (25, 26), or just adjacent to it, in the loop joining the second and third transmembrane domains in the intermembrane space (27). The V289M mutation affects the sixth transmembrane domain (25–27). A simulation analysis of the secondary structure of human ANT1 suggests that the A→P substitution at position 114 may cause an additional bend in the polypeptide, disrupting the local α helix (28). The V289M mutation is also predicted to modify the α helix. Because patients with dominant PEO carry one wild-type and one mutant allele, defective ANT1 dimers would form in two out of three dimerization events.

No defects of the ANT1 gene have previously been reported in humans, although ANT1 deficiency and reduced transcript levels have been described in a patient with lactic acidosis and myopathy (29). Mice with targeted inactivation ofANT1 show exercise intolerance mimicking mitochondrial myopathy, as well as hypertrophic cardiomyopathy (30). The muscle in these mice exhibits dramatic proliferation of mitochondria and reduced rates of mitochondrial ADP-stimulated respiration (30), and both muscle and heart DNA exhibit multiple deletions of mtDNA (31). Although the dominant mutation in humans differs from the gene inactivation in mice, the disease phenotypes are similar, except that humans do not have cardiac symptoms.

Our results suggest that in yeast the A128P mutation disrupts ADP/ATP translocation, because no acute instability of mtDNA was detected. However, in humans the ANT1 defect causes secondary accumulation of mtDNA mutations in postmitotic cells by a still unknown mechanism. A defect in the cytoplasmic thymidine phosphorylase in MNGIE syndrome with multiple mtDNA deletions suggests that disturbed intramitochondrial deoxynucleoside triphosphate (dNTP) pools may have a role in mtDNA deletion formation (32), possibly by increasing the error rate of the mitochondrial γ-polymerase. In postmitotic cells, the short mutant mtDNA may have a replicative advantage over the wild-type mtDNA, possibly resulting in accumulation of mutant mtDNA. Current knowledge does not support dATP as a physiological substrate for ANT (12). However, mammalian mitochondria contain enzymes required to reduce ADP to form dADP, which is phosphorylated to form dATP for DNA synthesis (33,34), so it is conceivable that ANT regulates intramitochondrial dATP concentrations. Several additional mechanisms may modify the adPEO pathogenesis caused by ANT1 defect: (i) the structural defect in ANT1 may affect MPTP opening; (ii) misfolding of ANT1 may expose the protein to oxidative lesions and cause its premature age-related inactivation (35); and/or (iii) dysfunction of ANT1 may increase oxidative stress within mitochondria (31).

The mutant mtDNA is likely to participate in the pathogenesis of adPEO. The proportion of mutant mtDNA in patients increases slowly with age and follows the disease severity (8). In addition, the progression, symptoms, and mutant mtDNA amount of adPEO resemble closely those associated with sporadic PEO with single mtDNA deletions, a disease known to be caused by the mutant mtDNA.

Finally, it is of interest that adPEO is mediated by a dominant mechanism, in contrast to other mitochondrial disorders due to nuclear gene defects, which are caused by loss-of-function mutations. Our finding may provide new insight into the pathogenesis of mitochondrial disorders and form the basis for future studies on mtDNA stability and the function of ANT.

  • * These authors contributed equally to this work.

  • To whom correspondence should be addressed. E-mail: anu{at}


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