The Diabetes-associated 3243 Mutation in the Mitochondrial tRNALeu(UUR) Gene Causes Severe Mitochondrial Dysfunction without a Strong Decrease in Protein Synthesis Rate

Abstract

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNALeu(UUR) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNALeu(UUR), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed. Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNALeu(UUR) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNALeu(UUR), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed. maternally inherited diabetes and deafness mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes myoclonus epilepsy with ragged red fibers Dulbecco's modified Eagle's medium Nucleotide substitutions in the mitochondrial tRNA genes are important factors in the pathogenesis of several multisystem disorders including mitochondrial encephalomyopathies and maternally inherited diabetes and deafness (MIDD)1(1Goto Y. Nonaka I. Horai S. Nature. 1990; 348: 651-653Crossref PubMed Scopus (1765) Google Scholar, 2Shoffner J.M. Lott M.T. Lezza A.M. Seibel P. Ballinger S.W. Wallace D.C. Cell. 1990; 61: 931-937Abstract Full Text PDF PubMed Scopus (1283) Google Scholar, 3Van den Ouweland J.M.W. Lemkes H.H.P.J. Ruitenbeek W. Sandkuijl L.A. de Vijlder M.F. Struijvenberg P.A.A. van de Kamp J.J.P. Maassen J.A. Nat. Genet. 1992; 1: 368-371Crossref PubMed Scopus (1061) Google Scholar, 4Wallace D.C. Annu. Rev. Biochem. 1992; 61: 1175-1212Crossref PubMed Scopus (1196) Google Scholar, 5Larsson N.G. Clayton D.A. Annu. Rev. Genet. 1995; 29: 151-178Crossref PubMed Scopus (410) Google Scholar, 6Grossman L.I. Shoubridge E.A. Bioassays. 1996; 18: 983-991Crossref PubMed Scopus (95) Google Scholar). A point mutation in the structural gene for a tRNA may be expected to influence protein synthesis either quantitatively or qualitatively. A strong reduction in mitochondrial protein synthesis, in particular in the proteins with a high lysine content, has been reported in the case of the 8344 mutation in the tRNALysgene of myoclonus epilepsy with ragged red fibers (MERRF). This reduction is thought to be caused by the premature termination of mitochondrial translation on lysine codons and the subsequent formation of truncated polypeptides (7Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar). Mitochondrial protein synthesis has also been found decreased in the case of the 3243 mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) (8Chomyn 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 (447) Google Scholar, 9King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar). Therefore, the general view has emerged that mutations in tRNA genes cause mitochondrial dysfunction by lowering mitochondrial protein synthesis and, as a consequence, the amount of mitochondrially encoded respiratory chain enzymes. Indeed, mitochondrial respiration is severely reduced in these cases (7Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar, 8Chomyn 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 (447) Google Scholar, 9King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar). However, the large variation in clinical phenotypes that associate with tRNA mutations is difficult to explain solely by the lowered respiration rate. MIDD accounts for 1–2% of the diabetic population in Europe and Japan and is clinically characterized by an early middle age onset of diabetes and sensorineural hearing loss, progressive insulin secretory defect, absence of islet cell antibodies, and absence of obesity (10Kadowaki T. Kadowaki H. Mori Y. Tobe K. Sakuta R. Suzuki Y. Tanabe Y. Sakura H. Awata T. Goto Y. N. Engl. J. Med. 1994; 330: 962-968Crossref PubMed Scopus (546) Google Scholar,11Maassen J.A. Kadowaki T. Diabetologia. 1996; 39: 375-382Crossref PubMed Scopus (164) Google Scholar). As a first step in understanding the molecular mechanisms caused by the 3243 mutation, we have established mitochondrial transformants (cybrids) carrying either the wild-type adenine or the mutant-type guanine at position 3243 but having otherwise identical mitochondrial DNA sequences (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). Care was taken to ensure that all cell lines contained high and comparable mitochondrial DNA copy numbers. The mutant-type cells displayed a severe reduction of cellular respiration and respiratory chain activity, which has provided proof for the pathogenicity of the 3243 mutation in MIDD (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). In the present work, the analysis of these cybrids has been extended toward mitochondrial tRNA and protein metabolism in order to understand better the molecular mechanisms leading from the 3243 mutation to impaired respiration and MIDD. The mitochondrial transformants (cybrids) used in this study had been generated earlier by the transfer of mitochondria from two genetically unrelated diabetes patients (V and A) into mitochondrial DNA-less Bρ°-3 cells (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). Patient V displays the phenotype of maternally inherited diabetes and deafness. Patient A suffers from Alport-like syndrome, which is characterized by progressive kidney disease, sensorineural hearing loss, and maternal inheritance (13Jansen J.J. Maassen J.A. van der Woude F.J. Lemmink H.A. van den Ouweland J.M. 't Hart L.M. Smeets H.J. Bruijn J.A. Lemkes H.H. J. Am. Soc. Nephrol. 1997; 8: 1118-1124PubMed Google Scholar). Diabetes also develops after transplantation and immunosuppressive therapy with steroids but is overshadowed by the kidney disease. Therefore, MIDD and Alport-like syndrome have in common maternal inheritance, sensorineural deafness, and diabetes, with renal failure as a prominent characteristic for Alport-like syndrome. From both donors we obtained fully wild-type cybrids (W6 and W7 from patient V; W20 from patient A) as well as mutant-type cybrids (M48 and M50 from patient V; M12, M26, and M30 from patient A) (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). Cybrids, 143B osteosarcoma cells and Bρ°-3 cells were all cultured in Dulbecco's modified Eagle's medium containing 4.5 mg/ml glucose and 110 μg/ml pyruvate (DMEM) supplemented with 50 μg/ml uridine and 10% fetal bovine serum (complete DMEM). For long-term culturing (maximum ∼3 months), the cybrids were cultured in DMEM supplemented with 10% dialysed fetal calf serum to preserve a high level of mitochondrial DNA. The mitochondrial DNA copy numbers of the cybrids used were all comparable with that of 143B cells. Cybrid cells were never cultured for longer than about 3 months. After this period a fresh vial of cells from the stock, stored in liquid nitrogen, was used. The rate of oxygen consumption by ∼5 × 106 exponentially growing cells in 2 ml of complete DMEM but without glucose was measured at 37 °C using a Clark-type electrode as described (14King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1463) Google Scholar). Total RNA (20 μg), isolated from exponentially growing cells by the guanidinium thiocyanate method (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar), was subjected to electrophoresis through a 1% agarose-formaldehyde gel, transferred onto Hybond N+ membranes and hybridized overnight at 42 °C with 32P-labeled probes specific for the tRNALeu(UUR) gene (nucleotides 3245–3861) or the tRNAPhe gene (nucleotides 245–706), and washed with several changes of 1× SSC, 0.5% SDS at 42 °C (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). tRNA bands were visualized by autoradiography and quantified using PhosphorImager detection and ImageQuant analysis. The degree of in vivo aminoacylation of tRNAs was determined by high-resolution electrophoresis and subsequent Northern analysis as described (17Varshney U. Lee C.-P. RajBhandary U.L. J. Biol. Chem. 1991; 266: 24712-24718Abstract Full Text PDF PubMed Google Scholar). To prevent deacylation of tRNA during isolation, RNA was isolated under acidic conditions by the guanidinium thiocyanate method (15Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar) but using 0.15 m sodium acetate, pH 4.0, instead of pH 7 buffers throughout. The final RNA preparation was dissolved in 10 mm sodium acetate, pH 4.0. [35S]Methionine incorporation into mitochondrially encoded proteins was analyzed on SDS-polyacrylamide gels essentially as described (18Chomyn A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar). In brief, ∼5 × 105 exponentially growing cells in a 3.4-cm dish were washed twice with methionine-free DMEM and incubated for 30 min at 37 °C with Promix (a 2:1 mixture of [35S]methionine and [35S]cysteine from Amersham Pharmacia Biotech; 10 μCi/μl, 1,000 Ci/mmol) in 0.75 ml of methionine-free DMEM supplemented with 10% dialysed fetal bovine serum and emetine (100 μg/ml), which was added 15 min before the addition of label. The cells were washed three times with phosphate-buffered saline and finally dissolved in 100 μl of SDS sample buffer. Samples (40 μg) were analyzed on 15% SDS-polyacrylamide gels. Mitochondrial translation products were assigned according to Chomyn et al. (18Chomyn A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar). Protein concentration was determined by the bicinchoninic acid assay (Pierce). In the case of [3H]leucine incorporation, [35S]methionine was replaced byl-[4,5-3H]leucine (10 μCi/0.75 ml on a 3.4-cm dish; 68 Ci/mmol) in leucine-free complete DMEM. After the specified incubation period, the ∼5 × 105 cells were washed three times with phosphate-buffered saline and dissolved in 0.5 ml of 0.1 m NaOH. 100-μl aliquots were immediately withdrawn in duplicate, added to 100 μl of cold 20% trichloroacetic acid, and heated for 10 min at 90 °C. The precipitate was collected on a glass fiber filter, washed, and counted as described (19Janssen G.M.C. Maassen J.A. Möller W. Spedding G. Ribosomes and Protein Synthesis. Oxford University Press, New York1990: 51-68Google Scholar). The significance of the differences between wild-type and mutant-type incorporation was estimated by linear regression analysis using SPSS 7.0 software (Chicago). The protein degradation rate of individual mitochondrial proteins was estimated from pulse-chase experiments (20Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). For pulse values, ∼5 × 105 cells (pretreated with chloramphenicol) were incubated for 2 h at 37 °C with Promix and the reversible cytoplasmic protein synthesis inhibitor cycloheximide (200 μg/ml) instead of emetine and washed three times with phosphate-buffered saline. For chase values, the pulsed cells were washed with complete DMEM instead of phosphate-buffered saline and subsequently cultured for 48 h in complete DMEM with two daily changes of medium. The decay rate was from min and the amount of in an individual protein pulse and were and using a analysis for that analysis of the decay was using was essentially as described H. T. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In brief, cellular protein was on a 15% SDS and transferred to a the proteins were visualized using subunits and and Pharmacia S.W. Methods Enzymol. PubMed Scopus Google Scholar) were by a incubation at °C of μg) (20Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar) with μg) and protein (10 Pharmacia in 0.5 ml of 10 mm pH mm and 1% The were washed twice with the and twice with mm pH and were in SDS sample buffer. The of the proteins was analyzed by SDS-polyacrylamide electrophoresis by mitochondrial transformants containing wild-type or mutant-type mitochondrial DNA have been generated and characterized earlier (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). were cultured on to and preserve high and comparable mitochondrial DNA copy numbers. cybrids displayed an reduced rate of respiration and a reduction of individual respiratory chain (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). Northern analysis that the level of tRNALeu(UUR) in the mutant-type cells was reduced to of the level found in wild-type cybrids or the 143B the amount of tRNAPhe this of cybrids The of tRNALeu(UUR) to tRNAPhe was estimated to be for the mutant-type and for the wild-type was in the level of and which an and the was in the mutant-type cells comparable with the results described for the 3243 cybrids M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar). To the 3243 mutation also the degree of aminoacylation of we the of mitochondrial and tRNALeu(UUR) in wild-type RNA was isolated under acidic conditions to prevent deacylation and analyzed on an acidic by Northern (17Varshney U. Lee C.-P. RajBhandary U.L. J. Biol. Chem. 1991; 266: 24712-24718Abstract Full Text PDF PubMed Google Scholar). aminoacylation results in a lowered of this method the and of both The in vivo degree of aminoacylation of tRNALeu(UUR) was found to be reduced from leucylation in wild-type cells to in all of the mutant-type cybrids 2 In a level of aminoacylation in both of cells. Therefore, when into the reduced steady state level of the amount of for mitochondrial protein synthesis is low in mutant-type cells and is estimated to be of the wild-type all mitochondrial a of may be expected that a low level of the rate of mitochondrial at when is a for mitochondrial protein Therefore, we estimated the rate of mitochondrial protein synthesis by the incorporation of [35S]methionine into protein in the of the cytoplasmic protein synthesis we not a severe decrease in the rate of [35S]methionine incorporation into mitochondrially encoded proteins Rather, the rate of mitochondrial protein synthesis of of the mutant-type cybrids of was comparable with that of the wild-type The which was as a a decrease in mitochondrial translation 3 (7Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar, A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar). not in cybrids a between of a particular protein and the of codons in J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar). the synthesis of was in mutant-type cybrids mutant cells displayed but protein bands by in which may degradation products of the mutant mitochondrial The cybrids from the MIDD patient were from that of the patient with Alport-like syndrome, with the of M12, which a reduction of protein synthesis rate. the rate of translation does not seem to be seriously in the 3243 mutation may the of mitochondrial protein synthesis by the incorporation of than at Therefore, we measured the rate of [3H]leucine incorporation into mitochondrial protein as a first of the degree of at codons of the mitochondrial incorporation was measured at cell we have that the rate of mitochondrial protein synthesis on cell a of protein synthesis as as the cells the mitochondrial two genes with as the for codons for the between wild-type and mutant-type cybrids is expected to be in the order at when complete at incorporation was but in wild-type cells at all of the cell analyzed Therefore, the of at codons The rate of mitochondrial protein degradation was estimated from pulse-chase experiments (20Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). After with to the mitochondrial proteins after of the (20Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar), the cybrids were for 2 h with [35S]methionine in the of the reversible cytoplasmic protein synthesis inhibitor The decay of during the period was subsequently determined for individual protein The of mitochondrially encoded proteins was found to be but in the wild-type cybrids the decay of individual mitochondrial proteins are in a that mitochondrial protein be analysis a decreased steady state level of the mitochondrially encoded proteins subunits and that of the mitochondrial proteins and were both found to be comparable in wild-type cybrids As may be the mitochondrially encoded proteins were not present in the mitochondrial DNA-less Bρ°-3 cells. the specific reduction of mitochondrially encoded proteins to the rate of protein The gene of the 2 and is in the synthesis of for Therefore, mutation in mitochondrial DNA may be expected to mitochondrial respiration in the first Indeed, in the case of mitochondrial DNA respiration is reduced in mutant of the of mitochondrial DNA mutation. The clinical phenotype to these For which is caused by a mutation in the is a severe with a low MIDD with the mutation as a the mitochondrial DNA mutation clinical phenotypes in L.I. Shoubridge E.A. Bioassays. 1996; 18: 983-991Crossref PubMed Scopus (95) Google Scholar). The is for the 3243 mutation in the which may to Alport-like syndrome, or progressive (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). This in pathogenesis may be by the of nuclear or mitochondrial factors that the and of the 3243 mutation the and thereby the tRNA also to be in than translation S. H. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), be that the tRNA gene instead have generated cell lines carrying the 3243 mutation from patients with MIDD and Alport-like syndrome in a The mitochondrial from the patient with MIDD has been with the nucleotide found at position 3243 den Ouweland J.M.W. Lemkes H.H.P.J. Ruitenbeek W. Sandkuijl L.A. de Vijlder M.F. Struijvenberg P.A.A. van de Kamp J.J.P. Maassen J.A. Nat. Genet. 1992; 1: 368-371Crossref PubMed Scopus (1061) Google Scholar). Therefore, may be that the between wild-type and mutant-type cybrids is the A-to-G transition at position to on cybrids from and patients (7Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google 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 (447) Google Scholar, A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar, J.A. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), mutant-type cybrids a in cellular respiration without a strong decrease in the rate of protein The for mutant-type cybrids from Alport-like syndrome and also for of the 3243 cybrids isolated in H. laboratory of In this be that of cybrids with low copy numbers of mitochondrial DNA displayed a decrease of mitochondrial protein synthesis den Ouweland J.M.W. A New of Diabetes with a Mitochondrial Scholar). Therefore, we to ensure that all cell lines contained a high and comparable amount of mitochondrial DNA (12van den Ouweland J.M.W. Maechler P. Wolheim C.B. Attardi G. Maassen J.A. Diabetologia. 1999; 42: 485-492Crossref PubMed Scopus (87) Google Scholar). we have generated cybrids with severe mitochondrial dysfunction without a strong decrease in protein synthesis is that a decrease in mitochondrial protein synthesis is not a necessary prerequisite for mitochondrial Therefore, into the that a decrease in mitochondrial protein synthesis is a necessary prerequisite for loss of respiration and of Our that the 3243 mutation the of the mitochondrially encoded which may be to at codons of the mitochondrial a has been to in containing 3243 mutant mitochondrial DNA A. H. Seibel P. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). is important to that cybrids not a mutation in the of the gene as found by and A. N. D. Nat. Genet. 18: PubMed Scopus Google Scholar). The low level of tRNALeu(UUR) may be by for degradation as a of decreased toward or the In this has been that a tRNA is never but is to of these three M.P. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). the is be that the synthesis of the tRNALeu(UUR) is impaired at the level W. Scopus Google Scholar). has been in that a level of is low to the synthesis of mitochondrial proteins in cybrids (7Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar). However, with a level of we not such the levels of tRNALeu(UUR) and are comparable in wild-type cells M.P. Attardi G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the low level of be with the high rate of protein the of an rate of D. H. J. Mol. Biol. PubMed Scopus Google Scholar), which to an of 50 at the synthesis of a complete protein of × The decrease in may the rate of at at by a of but by A. U. W. PubMed Scopus Google Scholar). the at to at the the to the complete containing on 10 be elevated to × 50 (10 × which to a 10% reduction of the rate of protein synthesis at the The 3243 mutation degradation of mitochondrial proteins and we have not that the of these proteins is at the in mutant-type two obtained results both the decreased of these which at an of their The pulse-chase experiments an elevated degradation rate the may also be from the low steady state levels in the absence of a strong decrease of de protein synthesis and of isolated mitochondrial proteins as to in the are in the pathogenesis of MIDD. are to H. of for G. Attardi of for of cybrids and J. of for mitochondrial and 't Hart University for in analysis.