Pathophysiology of the MELAS 3243 Transition Mutation
1997; Elsevier BV; Volume: 272; Issue: 43 Linguagem: Inglês
10.1074/jbc.272.43.27189
ISSN1083-351X
AutoresAdrian Flierl, Heinz Reichmann, Peter Seibel,
Tópico(s)RNA and protein synthesis mechanisms
ResumoSingle base substitutions of the mitochondrial genome are associated with a variety of metabolic disorders. The myopathy, encephalopathy, lactic acidosis, stroke-like episodes syndrome, most frequently associated with an A to G transition mutation at position 3243 of the mitochondrial tRNALeu(UUR)gene, is characterized by biochemical and structural alterations of mitochondria. To investigate the pathophysiology of the mutation, we established distinct Epstein-Barr virus-transformed B-cell lines for analyses that harbored 30–707 of the mutated genome. Interestingly, neither an alteration of the processing of primary transcripts nor a general impairment of individual mitochondrial protein subunit synthesis rates could be observed. Nevertheless a marked decrease of cytochrome-c oxidase activity and reduced content of mitochondrial encoded subunits in the assembled respiratory complex IV was recorded on the cell line harboring 707 mutated mtDNA. Quantitative analysis of incorporation rates of the amino acid leucine into newly synthesized mitochondrial proteins, representing the functionality of the tRNALeu(UUR) in protein biosynthesis, revealed a specific decrease of this amino acid in distinct mitochondrial translation products. This observation was supported by a variation in the proteolytic fingerprint pattern. Our results suggest that the malfunctioning mitochondrial tRNALeu(UUR) leads to an alteration of amino acid incorporation into the mitochondrially synthesized subunits of the oxidative phosphorylation system, thus altering it's structure and function. Single base substitutions of the mitochondrial genome are associated with a variety of metabolic disorders. The myopathy, encephalopathy, lactic acidosis, stroke-like episodes syndrome, most frequently associated with an A to G transition mutation at position 3243 of the mitochondrial tRNALeu(UUR)gene, is characterized by biochemical and structural alterations of mitochondria. To investigate the pathophysiology of the mutation, we established distinct Epstein-Barr virus-transformed B-cell lines for analyses that harbored 30–707 of the mutated genome. Interestingly, neither an alteration of the processing of primary transcripts nor a general impairment of individual mitochondrial protein subunit synthesis rates could be observed. Nevertheless a marked decrease of cytochrome-c oxidase activity and reduced content of mitochondrial encoded subunits in the assembled respiratory complex IV was recorded on the cell line harboring 707 mutated mtDNA. Quantitative analysis of incorporation rates of the amino acid leucine into newly synthesized mitochondrial proteins, representing the functionality of the tRNALeu(UUR) in protein biosynthesis, revealed a specific decrease of this amino acid in distinct mitochondrial translation products. This observation was supported by a variation in the proteolytic fingerprint pattern. Our results suggest that the malfunctioning mitochondrial tRNALeu(UUR) leads to an alteration of amino acid incorporation into the mitochondrially synthesized subunits of the oxidative phosphorylation system, thus altering it's structure and function. Mitochondrial encephalomyopathies are often associated with a variety of alterations of the mitochondrial DNA (1Wallace D.C. Lott M.T. Brown M.D. Hupuonen K. Torroni A. Cuticchia A.J. Human Gene Mapping: A Compendium. John Hopkins University Press, Baltimore1995: 910-954Google Scholar). The investigation of potential pathogenic mutations in the mitochondrial genome has revealed a complex relationship between patient's genotype and the clinically defined multisystemic disorders (2Wallace D.C. Annu. Rev. Biochem. 1992; 61: 1175-1212Crossref PubMed Scopus (1213) Google Scholar). One of the best characterized diseases is the MELAS 1The abbreviations used are: MELAS, myopathy, encephalopathy, lactic acidosis, stroke-like episodes; OXPHOS, oxidative phosphorylation; EBV, Epstein-Barr virus; oligo, oligonucleotide; COX, cytochrome c oxidase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]glycine; RFLP restriction fragment length polymorphism. syndrome (mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes) (3Pavlakis S.G. Phillips P.C. DiMauro S. De Vivo D.C. Rowland L.P. Ann. Neurol. 1984; 16: 481-488Crossref PubMed Scopus (1102) Google Scholar). Characterized by biochemical and morphological abnormalities of muscle mitochondria, the MELAS syndrome has been linked to multiple mitochondrial DNA alterations (4Tanaka M. Ino H. Ohno K. Ohbayashi T. Ikebe S. Sano T. Ichiki T. Kobayashi M. Wada Y. Ozawa T. Biochem. Biophys. Res. Commun. 1991; 174: 861-868Crossref PubMed Scopus (77) Google Scholar, 5Kobayashi M. Morishita H. Sugiyama N. Yokochi K. Nakano M. Wada Y. Hotta Y. Terauchi A. Nonaka I. J. Pediatr. 1987; 110: 223-227Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 6Goto Y. Nonaka I. Horai S. Nature. 1990; 348: 651-653Crossref PubMed Scopus (1818) Google Scholar). Although mutations in protein genes have been reported to be associated with the disease, single base replacements in the tRNALeu(UUR) gene have been identified to play the key role in developing this devastating disorder. Among the tRNALeu(UUR) mutations, the pathogenic mechanism of the A to G transition mutation at position 3243 is discussed controversially in the literature. Embedded in the middle of a tridecamer sequence necessary for the formation of the 3′ ends of the 16 S ribosomal RNA, a severe impairment of 16 S transcription termination has been demonstrated in vitro (7Hess J.F. Parisi M.A. Bennett J.L. Clayton D.A. Nature. 1991; 351: 236-239Crossref PubMed Scopus (215) Google Scholar). The molecular defect in patients presenting the mutation could be the inability to produce the correct type and quantity of rRNA relative to other mitochondrial transcripts. Cytoplasmic transfer experiments, performed with mitochondria harboring the 3243 mutation and fused to a cell line devoid of endogenous mtDNA, showed a decreased synthesis rate of mitochondrial translational products in steady-state levels and the appearance of a novel RNA species (termed RNA 19), derived from transcription of the 16 S rRNA/tRNALeu(UUR)/ND1 gene (8Schon E.A. Koga Y. Davidson M. Moraes C.T. King M.P. Biochim. Biophys. Acta. 1992; 1101: 206-209PubMed Google Scholar). In a similar approach performed on a mutation at position 3302 in the tRNALeu(UUR) gene, mitochondrial RNA processing appears to be tissue-specific; while an unprocessed RNA 19 was detectable in muscle cells, this transcript was not detected in fibroblasts, implying that a tissue-specific mitochondrial RNA processing contributes to the generation of RNA 19 (9Bindoff L.A. Howell N. Poulton J. McCullough D.A. Morten K.J. Lightowlers R.N. Turnbull D.M. Weber K. J. Biol. Chem. 1993; 268: 19559-19564Abstract Full Text PDF PubMed Google Scholar). In contrast, RNA transfer hybridization experiments on transformed cells harboring the 3243 mutation revealed no significant change in the steady-state level of the two rRNA species encoded upstream of the termination motif and of the mRNA species encoded downstream to the mutation (10Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (453) Google Scholar). Thus, the suggested pathogenic mechanisms seems to be inconclusive. To investigate the pathophysiology of the 3243 mutation, we established distinct EBV-transformed B-cell lines that harbored 30–707 of the mutated genome. Interestingly, neither an alteration of quality nor quantity of the primary transcripts could be observed. In addition, our experiments carried out on mitochondrial translation revealed no impairment of overall mitochondrial protein synthesis rate, although specific OXPHOS complexes exhibited a marked decrease of enzyme activity and assembly of mitochondrial subunits of the cell line harboring 707 mutated mtDNA. Quantitative analysis of incorporation of the amino acid leucine, monitoring the functionality of the tRNALeu(UUR) in protein biosynthesis, revealed a specific decrease of this amino acid in mitochondrially encoded and synthesized protein subunits. This observation was supported by heteroplasmic variations in the proteolytic fingerprint pattern of these subunits in the assembled OXPHOS-complexes. Our results are leading us to the hypothesis that the crucial step in molecular pathogenicity of MELAS is an alteration of the translation process at the mitochondrial ribosomes, induced by the malfunctioning mitochondrial tRNALeu(UUR). These alterations are leading to reduced amino acid leucine incorporation into the mitochondrially synthesized subunits of the oxidative phosphorylation system, thus altering their structure and function. Cell culture reagents were purchased from Life Technologies, Inc. Additional materials used in molecular biological studies were obtained from Perkin-Elmer (PCR), U.S. Biochemical Corp. (DNA sequencing), Boehringer Mannheim (Northern hybridization, RFLP analyses, reverse transcription), or Amersham Buchler (radiochemicals). Human B-lymphoblastoid cell lines were established by isolation of lymphocytes from blood of patients presenting with MELAS syndrome and controls. The EBV-transformed lymphocytes (11Reedman B.M. Pope J.H. Moss D.J. Int. J. Cancer. 1972; 9: 172-181Crossref PubMed Scopus (17) Google Scholar) were grown in glucose-rich media (RPMI 1640), supplemented with 107 fetal calf serum, 50 ॖg/ml uridine, and 100 ॖg/ml pyruvate. Standard enzymatic assays for OXPHOS enzymes were performed as described previously (12Taylor R.W. Birch Machin M.A. Bartlett K. Turnbull D.M. Biochim. Biophys. Acta. 1993; 1181: 261-265Crossref PubMed Scopus (53) Google Scholar, 13Taylor R.W. Birch-Machin M.A. Lowerson S. Sherratt H.S. West I.C. Bartlett K. Turnbull D.M. Biochem. Soc. Trans. 1993; 21: 804-807Crossref PubMed Scopus (3) Google Scholar). Cells were also used for polarographic assays to determine the respiratory capacity of mitochondria (14Moreadith R.W. Fiskum G. Anal. Biochem. 1984; 137: 360-367Crossref PubMed Scopus (176) Google Scholar). Briefly 1 × 107 cells were either resuspended in cell culture medium or prepared by digitonin treatment according to Granger and Lehninger (15Granger D.L. Lehninger A.L. J. Cell Biol. 1982; 95: 527-535Crossref PubMed Scopus (320) Google Scholar). 5 × 106 cells were resuspended in the appropriate assay buffer, and mitochondrial respiration was determined using a small scale Clarke-type oxygen electrode (Biolytic MS1;RE K1–1N) (16Nakamura M. Nakamura M.A. Okamura J. Kobayashi Y. J. Lab. Clin. Med. 1978; 91: 568-575PubMed Google Scholar, 17Gabridge M.G. J. Clin. Microbiol. 1976; 3: 560-565PubMed Google Scholar). As reference enzyme for oxidative phosphorylation, cytochrome-c oxidase activity was additionally assayed by evaluating the turnover number using reversed Eadie-Hofstee plotting (18Zimmermann P. Kadenbach B. Biochim. Biophys. Acta. 1992; 1180: 99-106Crossref PubMed Scopus (18) Google Scholar). Total DNA was extracted from 1 × 106cultured cells using a standard SDS/proteinase K protocol (19Seibel P. Degoul F. Bonne G. Romero N. Francois D. Paturneau-Jouas M. Ziegler F. Eymard B. Fardeau M. Marsac C. Kadenbach B. J. Neurol. Sci. 1991; 105: 217-224Abstract Full Text PDF PubMed Scopus (59) Google Scholar). Total RNA was isolated from 1 × 107 cells by guanidinium thiocyanate-phenol:chloroform extraction (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (65695) Google Scholar). PCR amplification of the mitochondrial DNA fragments encompassing the 3243 point mutation was performed using the oligonucleotides displayed in TableI. tRNALeu, 1 and 2; pre-tRNALeu (displaying the unprocessed primary transcript): oligonucleotides 3 and 2. Cycling conditions applied for amplification were 94 °C, 30 s; 38 °C, 30 s; 72 °C, 60 s; total of 35 cycles.Table ISequence and localization of the oligonucleotides used for PCR and Northern hybridization experiments in the mitochondrial genomeNo.Oligonucleotide sequence (5′–3′)Nucleotide no.Orientation 1CTATAGTTAAGATGGC3230–3240Forward 2GATCCTGGTGTTAAGAAGA3304–3294Reverse 3CACAAGATGGTGCAGC3010–3020Forward 4AACGATCAGAGTAGTGGTATTTCA4009–4032Reverse 5GGAGTGGGTTTGGGGCTAGG1696–1686Reverse 6GCTACACCTTGACCTAACGTCT1340–1318Reverse 7TTATGCGATTACCGGGCTCTG3270–3240Reverse 8TTTCACTGTAAAGAGGTGTTG8367–8347Reverse 9CCATCTTAACAAACCCTGTT3240–3220Reverse10TTGGACGAACCAGAGTGTAGC1611–1592Reverse11TTTGGGCTACTGCTCGCAGTG3721–3701ReverseNucleotide numbers are according to Anderson et al. (47Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7901) Google Scholar). Open table in a new tab Nucleotide numbers are according to Anderson et al. (47Anderson S. Bankier A.T. Barrell B.G. de Bruijn M.H. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier P.H. Smith A.J. Staden R. Young I.G. Nature. 1981; 290: 457-465Crossref PubMed Scopus (7901) Google Scholar). Sequencing of patient and control tRNA and protein coding genes was performed by cycle sequencing according to the manufacturers' instructions. DNA genotypes were assessed by quantitative RFLP analysis. For this purpose, the amplified DNA fragments were purified and subjected to one further PCR cycle with the corresponding 5′-end-labeled oligonucleotides to eliminate the interference of heteroduplex DNA on quantitative restriction enzyme cleavage. The amount of mutated DNA was detected by RFLP analysis using the restriction endonuclease BstNI. Quantitative evaluation of the heteroplasmic mitochondrial DNA was carried out on excised DNA bands from agarose gels and subsequent scintillation counting. A series of wild-type/mutant DNA mixtures served as standard. cDNA synthesis of mitochondrial RNA was carried out according to the method described by Janke and Päabo (21Janke A. Päabo S. Nucleic Acids Res. 1993; 21: 1523-1525Crossref PubMed Scopus (117) Google Scholar) using the reverse oligonucleotide for first strand DNA synthesis. Purification of RNA was achieved by DNase I treatment prior to reverse transcription. cDNA was amplified by adding the corresponding forward oligonucleotides and performing a PCR amplification for 38 cycles according to the conditions described above. Qualitative and quantitative evaluation of the amplified cDNA was carried out as described for DNA genotyping. Incubation of DNase-treated RNA with RNase A prior to reverse transcription served as control and led to no PCR amplification product. Total cellular RNA was isolated from 1 × 107 cells and quantitated by spectrophotometry and enzyme-linked antibody assay (dip-stick, Invitrogen). 2 ॖg of RNA were denatured by glyoxal/dimethyl sulfoxide (22Bantle J.A. Hahn W.E. Cell. 1976; 8: 139-150Abstract Full Text PDF PubMed Scopus (173) Google Scholar) and separated on 1–47 agarose (Seakem, Nusieve; FMC Bioproducts). RNA was transferred to positively charged nylon membranes by vacuum blotting (23Olszewska E. Jones K. Trends Genet. 1988; 4: 92-94Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Hybridization was performed according to standard conditions (24Meinkoth J. Wahl G. Anal. Biochem. 1984; 138: 267-284Crossref PubMed Scopus (976) Google Scholar) using 5′-labeled oligonucleotides as displayed in Table I as follows: detection of 28 S rRNA (nuclear) (25Gonzalez I.L. Gorski J.L. Campen T.J. Dorney D.J. Erickson J.M. Sylvester J.E. Schmickel R.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7666-7670Crossref PubMed Scopus (280) Google Scholar), oligo 4; detection of 16 S rRNA (mitochondrial), oligo 5; detection of 12 S rRNA (mitochondrial), oligo 6; detection of tRNALeu, oligo 7; detection of tRNALys, oligo 8; detection of 16 S rRNA/tRNALeu primary transcript, oligo 9; detection of 12 S rRNA/tRNAVal primary transcript (26Montoya J. Gaines G.L. Attardi G. Cell. 1983; 34: 151-159Abstract Full Text PDF PubMed Scopus (229) Google Scholar), oligo 10; NADH dehydrogenase subunit 1, oligo 11. 5 × 107 cells were harvested and incubated for 30 min in starvation medium. The starvation medium was specifically formulated using the RPMI 1640 minus medium (deficient in arginine, cysteine, glutamine, leucine, and methionine) (Life Technologies, Inc.) according to the conditions described by the supplier, thereby depleting the amino acids that will be subsequently used to label the proteins. To shut down cytoplasmic protein synthesis, 130 ॖg/ml cycloheximide were added, and incubation was continued for an additional 10 min. The labeling reaction was initiated by adding the following amino acids: for 35S labeling a mixture of 707l-[35S]methionine, 307l-[35S]cysteine; >1000 Ci/mm(Pro-MixTM, Amersham Corp.) was used. 3H labeling was performed by adding 700 kBq/ml eitherl-[5-3H]leucine (120–190 Ci/mmol) orl-[4,5-3H]lysine orl-[2,3,4,5,6-3H]phenylalanine. The labeling reactions were performed for 30 min (only using the35S-amino-acids to measure the rate of protein synthesis) or for 3 h (using also 3H-labeled amino acids to evaluate their rates of incorporation) at 37 °C, followed by a 20-min chase with standard medium, containing 130 ॖg/ml cycloheximide and the 4-fold concentrations of the depleted amino acids. After washing the cells twice in ice-cold phosphate-buffered saline, the labeled cells were processed for subsequent analysis. For denaturing SDS-PAGE (27Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10824) Google Scholar), 1 × 107 cells were resuspended in a 10-fold volume of 10 mm bis-tris/HCl, pH 7, 5.0 mm MgCl2, 1.0 mmphenylmethylsulfonyl fluoride, and 1.0 ॖg/ml aprotinin. After addition of 50 units/ml DNase I, the suspension was kept at 25 °C for 1 h, dissolved in detergent buffer (750 mmaminocaproic acid, 50 mm bis-tris/HCl, pH 7.0, and 17 laurylmaltoside), and centrifuged for 30 min at 100,000 ×g. Protein concentration was determined according to a Lowry assay kit following the manufacturer's protocol (Pierce). 100–150 ॖg of protein (approximately 4 × 106 dpm) were mixed with loading buffer (27Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10824) Google Scholar), incubated at 40 °C for 10 min, and used for 12.57 T/37 C SDS-PAGE on a 1.5 × 120 × 230-mm vertical SIGMA electrophoresis unit. For native PAGE separation of mitochondrial OXPHOS complexes, 5 × 107 cells were resuspended in isolation buffer (28Bourgeron T. Chretien D. Amati P. Rotig A. Munnich A. Rustin P. Neuromuscul. Disord. 1993; 3: 605-608Abstract Full Text PDF PubMed Scopus (27) Google Scholar) and homogenized with a tight fitting glass-Teflon potter. After two centrifugation steps at 2,500 and 15,000 × g the mitochondrial pellet was resuspended in 750 mm aminocaproic acid, 50 mmbis-tris/HCl, pH 7.0, and 17 laurylmaltoside and centrifuged as described above. The supernatant was supplemented with Coomassie Blue G-250 and loaded on a 7–167 linear gradient gel system (29Schagger H. Cramer W.A. von Jagow G. Anal. Biochem. 1994; 217: 220-230Crossref PubMed Scopus (1051) Google Scholar). The separated mitochondrial proteins were electroblotted on 0.1-ॖm nitrocellulose membranes by semi-dry blotting (Schleicher & Schüll) in a discontinuous buffer system (30Schagger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1937) Google Scholar). After denaturing SDS-gel electrophoresis and subsequent electroblotting as described above, the individual protein subunits were localized by autoradiographic detection (24 h exposure to Kodak Biomax MR film) and isolated by excision. For scintillation counting, the nitrocellulose membranes were dissolved in 2 ml of acetonitrile and resuspended in 8 ml of scintillation solution (Insta-Fluor; Canberra Packard). The fractions were analyzed for their 3H and 35S content by spectral scintillation counting. The relative proportions of incorporated amino acids were calculated by using the following calibration: reference spectra of each single radioisotope were recorded under identical experimental conditions, and spectrum-specific channels for 35S and 3H were defined. An experimental correction factor was applied on 35S radiation recorded within the 3H window. The proteolytic digestion pattern of the mitochondrial subunits was performed by a modification of the protocol of Cleveland and colleagues (31Cleveland D.W. Fischer S.G. Kirschner M.W. Laemmli U.K. J. Biol. Chem. 1977; 252: 1102-1106Abstract Full Text PDF PubMed Google Scholar). After separation of whole cell lysates in the first dimension, mitochondrial subunits were excised and incubated for 10 min in 200 mm Tris-Cl, pH 8.3, 0.17 SDS, 107 glycerol. The SDS gel for separation in the second dimension was prepared as described but using preparative combs and 0.057 SDS in the stacking gel and cathode buffer. 25 ॖg of endoproteinase Glu-C (Boehringer Mannheim) was dissolved in 200 mm ammonium carbonate, 0.0257 SDS, 107 glycerol, pH 8.9, 0.00017 bromphenol blue. After loading, electrophoresis was performed until the endoproteinase had entered the gel. The excised gel fragments were positioned in the sample slots between the gel plates and overlaid by another portion of the endoproteinase solution containing 35 ॖg of endoproteinase Glu-C. Electrophoresis was resumed until the marker reached the end of the stacking gel. After a pause of 30 min, electrophoresis was again resumed and continued to its end. The cleavage pattern was visualized by autoradiography of the blotted gel as described above. To elucidate the pathogenic mechanism underlying the A to G transition mutation in the mitochondrial tRNALeu(UUR) gene at position 3243, we established EBV-transformed lymphoblastoid cell lines from patients carrying heteroplasmic populations of the mutated genome. After initial screening, two stable cell lines were obtained, harboring 30 and 707 mutated DNA. These were subjected to biochemical and genetic analyses. While enzymatic activities and polarographic studies of cells presenting with 307 mutant genome showed no significant differences in activity (data not shown), cells harboring 707 mutated DNA exhibited significant reduction in cytochrome-c oxidase, with other OXPHOS enzyme activities lying at the lower limit of standard deviation (see TableII). These data were supported by polarographic studies carried out on permeabilized cells; whereas respiration capacity from cells with 307 mutation rate were almost indistinguishable from control cells, a 207 reduction in oxygen consumption was recorded for permeabilized cells harboring 707 mutated genome (see Fig. 1).Table IIEnzyme kinetics (n = 10) of mitochondrial reference enzymes (units/g protein) measured in whole cell extracts of patient and control using standard assays as referred to under 舠Materials and Methods.舡Enzymes707 patient, ± S.D.Control, ± S.D.units/gunits/gNADH dehydrogenase41.2 ± 0.348.0 ± 4.0NADH cytochrome c reductase8.1 ± 1.810.6 ± 3.2Succinate dehydrogenase4.8 ± 0.55.4 ± 0.0Succinate cytochrome c reductase7.7 ± 0.89.8 ± 1.5Cytochrome c oxidase2.5 ± 1.15.8 ± 1.0Citrate synthase5.3 ± 0.95.9 ± 1.1 Open table in a new tab To prove whether malfunctioning enzymes are responsible for the observed abnormalities, we decided to measure the turnover number of cytochrome-c oxidase as a marker for enzyme integrity. The analysis of cytochrome-c oxidase activity under different concentrations of cytochrome-c revealed a significantly decreased turnover number of the enzyme at low substrate concentrations in cells presenting with 707 mutated genotype (see Fig.2). To exclude that an additional mitochondrial DNA variation could contribute to this effect, the mitochondrial encoded COX genes were sequenced (see 舠Experimental Procedures舡). By comparing the obtained sequence to the published sequence, no variation was identified that could account for the enzymatic alterations. Hence, the altered enzyme integrity must be related to the mutation in the tRNALeu(UUR) gene itself. To determine whether the mutation in the tRNALeu(UUR) gene influences the transcript level of mutated and wild-type tRNA in heteroplasmic cells, we analyzed the degree of heteroplasmy on DNA, tRNA, and primary RNA transcript level by RFLP analysis (see 舠Experimental Procedures舡). The quantitative determination of the proportion of mutated versus wild-type nucleic acids in patient's cells revealed corresponding levels so that for each cell line the amount of mutated DNA (genotype) matched its transcript level (see Fig. 3 a). This was observed for the primary transcripts as well as for their processed counterparts. Hence, neither a malfunctioning processing of primary transcripts nor a difference in stability of the mutatedversus the wild-type tRNA could be observed. To verify that the ratio of mitochondrial to nuclear transcripts was unaltered, mitochondrial and nuclear expression levels were characterized by Northern analyses. A difference in mitochondrial RNA contents was not detectable for primary transcripts of rRNAs or mRNAs, or processed ribosomal RNA, or mature tRNAs (Fig.3 b). There was no detectable accumulation of a precursor-like abnormal primary transcript (termed RNA 19) as has been described for ρ° cells with a high percentage of altered mtDNA (32King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell Biol. 1992; 12: 480-490Crossref PubMed Scopus (413) Google Scholar). Although levels of mutant and wild-type transcripts showed no alteration, further analyses were extended onto the mitochondrial translation process. The reduced enzyme activities could be caused by two effects: a reduction in the abundance of enzymes or by malfunctioning components of the complexes. Thus, mitochondrial protein labeling experiments were carried out in the presence of [35S]methionine/[35S]cysteine as described under 舠Experimental Procedures.舡 Evaluation of the protein synthesis rates of cells affected by 707 mutation expressed no differences in the amounts of mitochondrial subunits (see Fig.4 for laser densitometric scanning of the protein banding pattern). This result was underlined by experiments utilizing a 30-, 60-, and 180-min pulse. Neither condition applied resulted in an alteration of our primary result. Hence, neither the rate of synthesis (represented by a 30-min pulse) nor the accumulation of peptides in stable complexes (as monitored by the 180-min pulse) revealed significant differences. Hence, at least up to levels of 707 mutated genotype, the overall protein synthesis rate of individual subunits seems not to be affected. To prove the functionality of the mutated tRNALeu(UUR)during protein synthesis, a co-labeling of mitochondrial proteins was carried out in the presence of [35S]methionine/[35S]cysteine and [3H]leucine. This allowed us to evaluate the leucine and methionine/cysteine content of distinct translation products by measuring 35S and 3H radiation using defined energy windows of a scintillation counter (see 舠Experimental Procedures舡). While patient's and control's mitochondrial translation products displayed similar apparent molecular weights on SDS-PAGE, incorporation of 3H (representing leucine content) was more abundant in translation products from controls, when compared with those from the MELAS cell lines. Furthermore, the decrease in 3H incorporation was more abundant in cells harboring 707 mutated genome than in cells exhibiting only 307 mutant genotype (see Fig. 5). No traces of premature terminated translation products were detected. Although reduction in 3H content was most extensively seen in the translation products of ND3 and ND6, it was only moderately developed in the ND4L translation product (see Fig. 5). To analyze if the altered peptides participate in the assembly of the OXPHOS complexes, we utilized blue native gel electrophoresis to separate assembled OXPHOS complexes prior to SDS-PAGE analysis. These qualitative and quantitative analyses of assembled OXPHOS complexes were carried out by using mitochondrial fractions derived from digitonin-permeabilized cells. No differences in the relative amount of the individual OXPHOS enzyme complexes between the more severely affected MELAS cell line and wild-type cells could be observed. After separation of the assembled mitochondrial subunits by second dimension SDS-PAGE, no differences in the relative amount of nearly all mitochondrially encoded subunits were detectable. However, an altered pattern of mitochondrial encoded subunits of respiratory complex IV could be observed in the mutated cells (see Fig.6). Analysis of mitochondrial encoded and co-labeled smaller subunits isolated from this gel system also showed a significant [3H]leucine reduction in the 707 cell line relative to controls, monitoring assembly of altered subunits in the OXPHOS complexes. To prove that the defect was tRNALeu(UUR)-specific and to evaluate the possibility of conservative substitution of the UUR-encoded leucine by phenylalanine, encoded by the related UUN codon, we carried out identical experiments using 3H-labeled phenylalanine in conjunction with similar amounts of [35S]methionine/[35S]cysteine in the more severe affected MELAS cell line. Experiments were also performed by using the tritiated amino acids phenylalanine, lysine, and proline, encoded by the codons UUN, AAR, and CCW. Analysis of these co-labeling experiments was performed with the abundant mitochondrial subunits COX I and COX II, also pre
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