Human Molecular Genetics, 2000, Vol. 9, No. 7 1041-1048
© 2000 Oxford University Press
Sequential deletion of C-terminal amino acids of the E1
component of the pyruvate dehydrogenase (PDH) complex leads to reduced steady-state levels of functional E12ß2 tetramers: implications for patients with PDH deficiency
Department of Biochemistry, University of Toronto, and Metabolism Research Programme, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada and 1Departments of Neurosciences and Paediatrics, University of California—San Diego, San Diego, CA, USA
Received 5 November 1999; Revised and Accepted 1 February 2000.
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ABSTRACT |
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Human pyruvate dehydrogenase (PDH) complex deficiency is an extremely heterogeneous disease in its presentation and clinical course. We have characterized novel mutations that affect the C-terminal portion of the PDH-E1
-coding sequence. Although the molecular defects underlying these mutations are different, both effectively produce a stop codon prematurely three amino acids from the C-terminus. The clinical and biochemical consequences of these mutations are unusual in that the affected individuals are very long-term survivors with PDH complex deficiency despite having low (<20%) activity in skin fibroblasts. These findings prompted us to investigate the C-terminus of E1 in greater detail. We constructed and expressed a series of PDH-E1 deletion mutants in a cell line with zero PDH complex activity due to a null E1 allele. Sequential deletion of the C-terminus by one, two, three and four amino acids resulted in PDH complex activities of 100, 60, 36 and 14%, respectively, compared with wild-type E1 expressed in PDH complex-deficient cells. The immunodetectable protein was decreased by the same amount as the activity, suggesting that the stability and/or assembly of the E12ß2 heterotetramer might depend on the intactness of the PDH-E1 C-terminus. In addition, we compared the somatic and the testis-specific isoforms of E1 and concluded that they are biochemically equivalent. |
INTRODUCTION |
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The pyruvate dehydrogenase (PDH) complex is the link between the glycolytic pathway and the tricarboxylic acid cycle and catalyzes the irreversible decarboxylation of pyruvate to acetyl-CoA. The PDH complex is a multienzyme system composed of multiple copies of three catalytic components, E1 (E1
and E1ß), E2 and E3, and an additional component, protein X, which is thought to play a role as an E2–E3-binding polypeptide (1–3). The activity of the PDH complex is regulated by a PDH-specific kinase and a PDH-specific phosphatase, which phosphorylate/dephosphorylate the E1 component of the E1 subunit. PDH also utilizes four co-factors: thiamine pyrophosphate (TPP), which binds non-covalently to the E1 subcomponent; lipoic acid, which binds covalently to a lysine residue located on the E2 subcomponent; FAD, which is bound by E3; and NAD+, which is reduced by E3. TPP is probably bound by elements of both E1 and E1ß subunits, so that each 2ß2 heterotetramer has two TPP molecules, one each bridging E1 and E1ß to form an active site (4).
PDH deficiency due to defective E1 is a well defined inborn error of metabolism that is clinically very heterogeneous. It is a major cause of primary lactic acidosis and Leigh disease in both male and female children, often accompanied by cerebral atrophy or microcephaly (1,5). In males, it may also present as a milder carbohydrate-sensitive, intermittent ataxia syndrome (1). Whereas there are two E1 genes, one encoded on the X chromosome and one on chromosome 4, mutations in the somatically expressed X-linked E1 gene are responsible for producing the E1 deficiencies associated with PDH complex defects (1,6–8). There is no expression of the chromosome 4 gene in tissues other than the testis (8). Suggestions that the testis isoform could substitute for the X-linked coded E1 protein if suitably expressed have been evaluated using a bacterial expression system, with some success (9). The testis E1 gene product will integrate to produce a fully functional PDH complex able to undergo phosphorylation and dephosphorylation, as does the complex made with the X-linked E1 gene product (9). In this study, we have used human skin fibroblasts with no endogenous E1 activity (the 
0 cell line), as a vehicle to express both the somatic and the human testis-specific E1 isoforms and compare their activities.
In the past, there have been reports that some mutations within the E1-coding region that affect the C-terminus lead to a significant decrease in E1- as well as E1ß-immunoreactive material (10–12). Two of these cases are female patients, and the biochemical and protein expression data are hard to evaluate since one normally expressing E1 allele is present and cell cultures are a mosaic (10). In the case of PDH deficiency in a 10-year-old male with relatively mild symptoms of exercise intolerance and lactic acidemia, the E1 mutation resulted in the addition of 33 amino acids with a residual PDH complex activity of 27.4% (12). In these cases, little systematic investigation has been carried out to determine whether the decreased amount of activity is due to the mutation itself, decreased mRNA, decreased protein import into mitochondria or increased mitochondrial degradation of imported mutant protein.
Here we present results from three patients with mutations in the C-terminal portion of the E1-coding region, who are very long-term survivors with PDH complex deficiency. We then compare these results with those of a study in which four C-terminal amino acids of the subunit of E1 were deleted sequentially and the resulting E1 variants were expressed in a 0 cell line.
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RESULTS |
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Case reports
Patients 1 and 2.
These are twin brothers born in 1959 who were normal until 2 years of age when they started to have episodes of weakness, ataxia and hyperventilation. They were labeled as having cerebral palsy in their early childhood. One twin appears to be more adversely affected than the other, though both have persevered with education at a school for the disabled. Although they have learning difficulties, they are not mentally retarded. A recent admission for one twin (G.F.) after a collapse followed by dystonic movements revealed hypodensities in the internal capsule and basal ganglia on CT scanning. During this crisis, he had an elevated blood lactate but this soon returned to normal. In cultured skin fibroblasts from the patients, overall PDH complex activity was 12.2 and 12.4% for the native complex (patients 1 and 2, respectively) and 13.8 and 14.6% for the dichloroacetate (DCA)-activated complex (patients 1 and 2, respectively). In cultured lymphoblasts from the patients, overall PDH complex activity was 25.7 and 22.8% for the native complex (patients 1 and 2, respectively), and 32.5 and 25.7% for the DCA-activated complex (patients 1 and 2, respectively).
Patient 3.
This patient, now 39 years old, first diagnosed as PDH deficient by John Blass (13) many years ago, was seen initially at 17 months for intermittent ataxia and dysarthria. He continued to have 5–6 attacks per year until his teens, when the frequency decreased to 1–2 episodes per year. Attacks are usually precipitated by viral illness, stress or anxiety and are associated with high blood pyruvate levels (0.4 mM). In addition to his intermittent symptoms, he has constant coarse bilateral hand tremor, dysarthria and progressive Dupuytren’s contractures in both hands. He also has persistent appendicular and truncal ataxia which worsens during periods of metabolic decompensation. Vertigo, particularly pronounced when viewing rippling water, occurred in recent ataxic attacks. At ages 34 and 37 years, he suffered acute psychotic episodes requiring psychiatric in-patient care and antipsychotic medications, with paranoia, anxiety, memory loss and mutism. He has also been severely depressed. Despite his episodic illness, the patient attended university, obtaining a degree in computer engineering. He works as a computer programmer. He has been treated with intermittent acetazolamide, intermittent prednisone and persistently with 300 mg/day of thiamine. In cultured fibroblasts from the patient, overall PDH complex activity was 14.7% for the native complex and 22.8% for the DCA-activated complex.
Western blot analysis showed lowered PDH-E1 and -ß immunoreactivity for all three patients, with ~15 and 30% of levels of the control present in fibroblasts and lymphoblasts, respectively (Fig. 1).
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Characterization of the mutations
Patients 1 and 2 have an AAGT insertion at 1159 and patient 3 has a C1163T change. Both mutations effectively produce a stop codon prematurely a short distance from the C-terminus (Fig. 2).
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Creation of the
0 cell line for E1 expressionWe transformed a human skin fibroblast cell line derived from a female patient with a mutation that nullified one of the PDH-E1
alleles (see Materials and Methods). After transformation and cloning of individual foci, two types of clone were identified: those that had normal PDH complex activity and those that had zero PDH complex activity, renamed 0. Both types as well as the parental cell line (see Materials and Methods) were probed with anti-total PDH complex antibody by western blotting. As shown in Figure 3, neither E1 nor E1ß subunits were expressed in the 0 cell line; however, the expression of the E2 component of the PDH complex remains unaltered. Clones with normal PDH complex activities also had normal expression of the PDH complex subunits. The parental cell line, however, showed a substantial decrease in both the E1 and E1ß protein levels. Densitometry analysis of protein peaks reveals that the amount of each of the E1 subunits was ~10% of the control. This correlates with the 7.5 ± 2.3% residual activity in the parental cell line.
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Expression of C-terminus deletion mutants of E1
To investigate the importance of the C-terminus of PDH-E1
for the stability and/or assembly of the E12ß2 heterotetramer, we constructed a series of PDH-E1 deletion mutants in which each mutant was sequentially shorter by one amino acid from the C-terminus. cDNA constructs corresponding to these E1 amino acid variants (
1, 2, 3 and 4) as well as the testis-specific isoform were subcloned into a mammalian expression vector and transfected into the 0 cell line. All PCR-generated E1 cDNA variants were digested with KpnI–XhoI, and subcloned into the mammalian expression vector pcDNA 3.1. The individual clones were sequenced to confirm that there were no PCR artifacts generated. The error-free clones were used in the transfection experiments.
The 0 cell line was transfected with cDNAs encoding the wild-type E1, the testis-specific E1 as well as four different C-terminus deletion mutants of E1: 1, 2, 3 and 4. Stably expressing clones were selected, subcloned and analyzed for expression of the E1 variant proteins by the total PDH complex activity assay (Table 1), as well as by the binding of antibody directed against pig heart total PDH complex (Fig. 4). Levels of exogenous E1 mRNA expression were examined by quantitative PCR using phosphoglycerate kinase (PGK) mRNA as an internal control (Fig. 5). This method was chosen to differentiate between the endogenous and exogenous E1 mRNA levels due to the fact that, although the 0 cell line presents with a complete loss of enzyme activity and immunoreactive E1 protein, the transcription and message production of the endogenous E1 remain unaltered.
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Analysis of PDHC activity assays
Complete recovery of activity on post-transfectional wild-type E1
cDNA expression in 0 cell lines was not achieved (Table 1), and rates of only 55% compared with control were recorded. Expression of the testis-specific E1 as well as the 1 variant resulted in a comparable restoration of activity: ~55%. When rates for 2, 3 and 4 were recorded and averaged, they were 33, 20 and 8%, respectively (Table 1). Therefore, a specific pattern emerged from this part of the study, in which total PDH complex activity decreased as the C-terminal residues were removed one by one from the E1 protein.
Analysis of expression of variant E1 proteins
To compare the rates of total PDH complex activities with the levels of E1 protein expression, western blotting was performed using antibodies directed against total PDH complex. The levels of E1 expression for each clone were standardized to the corresponding levels of E2 (the core protein) by densitometry analysis and expressed as a ratio E1:E2. The ratio E1:E2 for the SV40-transformed control cell line was taken as 1 (Fig. 4). Results of this experiment mirror results obtained from the activity assays such that progressively lower amounts of immunoreactive E1 protein were detected as the C-terminal E1 residues were removed. The amount of E1 protein expressed for each mutant also correlates with the corresponding activity of the complex, perhaps indicating that the specific activities of mutant complexes are unaltered. The expressed levels of E1ß progressively decrease as well, implying that E1ß is incapable of autonomous existence without E1 (1,14,15). The amount of protein detected for the testis-specific and wild-type E1 were comparable, further supporting the hypothesis that these isoforms are interchangeable within the PDH complex.
Analysis of mRNA levels for exogenous E1 variants
Results for the quantitative PCR performed on clones expressing wild-type, 1 and 4 PDH-E1 are shown in Figure 5. Densitometric analysis of band intensities showed that when compared with the internal control (PGK, upper band), the levels of exogenous mRNA transcribed from the cytomegalovirus (CMV) promoter of the transfected vectors were similar in each cell line. Comparable results were obtained for the remaining PDH-E1 clones (data not shown). Therefore, these findings show that lower protein levels for E1 deletion mutants were not caused by lower levels of mRNA transcription.
Import of E1 variants into the mitochondrial matrix
To investigate whether lower levels of immunoreactive E1 deletion mutant proteins were due to deficient import into mitochondria, all PDH-E1 variant proteins were synthesized in vitro and added to freshly prepared rat heart mitochondria. A mitochondrial matrix protein, MnSOD, was used to assess the import competence of the system (data not shown). Results for this experiment are shown in Figure 6. The 49 kDa precursor proteins were imported into mitochondria isolated from rat heart, being processed to the mature size 42 kDa protein. By densitometric analysis, it was established that despite small differences in overall amount adhering to mitochondria, by comparing the amount of precursor E1 (p-E1) with that of mature E1 (m-E1), there was no difference in precursor processing between wild-type E1 and any one of the investigated E1 deletion variants.
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DISCUSSION |
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We have shown that three patients with a long-lived mild phenotype of PDH complex deficiency display a strong correlation between residual PDH complex activity in fibroblasts and lymphoblasts and the levels of the E1
and the E1ß subunits as seen by immunoblot. E1 subunit levels of 12–15% seen in fibroblasts correlated with residual activities of the PDH complex of ~15%, and residual E1 levels of 25% seen in lymphoblasts correlated with enzyme activities of 26–32% for the PDH complex. These patients had E1 mutations that effectively removed the three C-terminal amino acids (SerValSer) from the protein, exposing a lysine as the N-terminal residue. For the majority of patients investigated with PDHC-E1 deficiency, residual rates in fibroblasts and lymphoblasts are the same, whereas rates in heart, kidney and liver can vary in the same patient (1). Thus, the 2-fold difference in activity and protein seen in lymphoblasts versus fibroblasts was due to different residual levels of mutant E1 in the complex presumably caused by differential rates of degradation. This conclusion that the missing C-terminal amino acids led to increased degradation of a potentially functionally active protein E1 component was investigated further by making mutant cDNA constructs coding for subunits missing one, two, three and four amino acids and expressing them in a 0 cell line totally devoid of PDH complex activity. The residual activity of the PDH complex again decreased with increasing number of amino acids removed, correlating strongly with the amount of immunotitratable E1 and E1ß subunits in the complex, suggesting no change in specific activity of E1 (Fig. 4). This supports a mechanism in which truncations of E1 appear to bestow instability either on the E12ß2 tetramer or, alternatively, on the whole assembled complex. The fact that E2 levels remain the same while E1 and E1ß are depleted mitigates against a mechanism involving the whole complex. Similarly, differential degradation of E1 mRNA species and possible failure of import of truncated E1 protein were ruled out by examining mRNA levels and import, respectively (Figs 5 and 6). Missense mutations of E1 with no change in protein length may also present in males as carbohydrate-sensitive ataxia (1). In this case, the change in activity may be due to E1 protein degradation or more usually due to a change in specific activity of the E1 component (1,14–16).
The present study showed that it is possible to complement the PDH enzyme defect caused by a faulty PDH-E1 gene by transfecting the cells with the normal PDH-E1 cDNA. Although normal levels of activity were never achieved, in stably transfected populations significantly higher values were recorded than had been reported previously by others for this type of transfection (17). It is known that the E1 subunit alone has no demonstrable enzymatic activity and requires assembly with E1 in order to form an active 2ß2 heterotetramer. Otherwise, the proteins degrade. Examples of the instability of individual subunits of heterotetramers have been seen in patients with PDH deficiency (18,19), and branched-chain -keto acid dehydrogenase deficiency (20,21). Therefore, the overexpressed E1 proteins may degrade at a time when there is no E1 protein available due to uncoordinated expression (22,23).
The influence of thiamine on patients with PDH complex deficiency has been controversial, with many patients having been treated from the time of diagnosis in infancy (1,24). TPP is thought to be bound by elements of both E1 and E1ß subunits; each 2ß2 heterotetramer has two TPP molecules, one each bridging E1 and E1ß to form an active site (4). The patients here represent the two ends of the spectrum in that one pair of sibs were totally untreated because of late diagnosis as adults, whereas the other patients with the same mutation were treated from an early age. It would seem that the treated patients have fared better than the untreated patients. Patients 1, 2 and 3 belong to a group of relatively long-lived, mildly affected males who have the presentation originally referred to by us and others as carbohydrate-sensitive ataxia (25,26). There are two further patients from the Maritime provinces of Canada who have the 1159 AAGT ins mutation (19). These unrelated males are both now 13 years of age, attend special school and have bouts of ataxia, which are carbohydrate and stress induced. So far, these patients have no detectable central nervous system lesions and have been given thiamine supplements from an early age. Our conclusion, therefore, is that this group of patients with apparently low residual fibroblast activities of PDH complex nevertheless have mild phenotypes because the specific activity of E1 is not altered by C-terminal truncation. Overall residual activity is governed by tissue-specific degradation mechanisms allowing the brain to have the 30–40% normal activity necessary for normal function (1).
Despite its importance, the molecular mechanisms governing the tight tissue-specific regulation of the testis-specific isoform of the PDH complex E1 subunit remain poorly understood (27). Even more limited data have been published on the biochemical aspects of this isoform. Results obtained in this study show for the first time that the expression of the testis-specific E1 isoform in a mammalian cell line produces a fully functional PDH complex, supporting previous in vitro findings (9). This shows that should a mechanism for inducing somatic expression of the testis-specific E1 isoform be found, correction of X-linked E1 defects might be feasible.
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MATERIALS AND METHODS |
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Enzymology and immunological analysis
Cultured skin fibroblasts were grown from skin biopsies in
-minimal essential medium (-MEM) culture medium. The activity of the PDH complex in the native and DCA-activated state was determined in fibroblast extract by the method of Sheu et al. (28). Western blotting was performed on mitochondrial extracts prepared by the method of Pitkanen et al. (29), using a polyclonal antibody against porcine heart PDH complex raised in rabbit (30).
RNA and cDNA preparation
Total RNA was extracted from cultured skin fibroblasts using TRIzol reagent (Total RNA Isolation Reagent) from Gibco-BRL (Burlington, Canada). First strand cDNA synthesis was carried out using total cellular RNA (20 µg) and an E1-specific oligonucleotide aG17' (5'-TCTAGAATTCGTACAAACTGCATGCAATTAC-3') with M-MLV reverse transcriptase (Gibco-BRL).
Amplification of DNA
DNA was amplified by PCR with cDNA as a template, 250 µM of each deoxynucleotide (dATP, dCTP, dGTP and dTTP), 10 µl of 10x PCR buffer (Gibco-BRL), 1.5 mM MgCl2, oligonucleotide primer (1 µg each) and 2 U of Taq polymerase (Gibco-BRL), in a total reaction volume of 100 µl. PCR amplification (31) of the cDNAs extracted from primary cell lines was carried out as described previously (19). Briefly, the total coding sequence of the E1 subunit was amplified in two overlapping fragments, subcloned into pSP65 T-A cloning vector (Clontech, Palo Alto, CA) and sequenced using a series of -specific primers. DNA was prepared from cultured skin fibroblasts of patients and control cell lines by a modified version of the method of Miller et al. (32). After 30 cycles of amplification under the conditions specified, the amplified fragments were visualized on ethidium bromide-stained gels, extracted and subcloned into pSP65. All DNA sequencing was performed by the Sanger dideoxy chain termination method (33) on double-stranded templates using a T7 polymerase sequencing kit (Pharmacia, Quebec, Canada). To make E1 deletion mutants, 5 µl of the cDNA reaction mixture was used for PCR using E1-specific forward primer Kpn-F (5'-TTTGGTACCTTGTGAGGAGTCGCCGCTGC-3') and a series of reverse primers, each containing an XhoI restriction site:
Xho-R (5'-TTTCTCGAGGAGAACACTGTCTGGTAGCC-3') for wild-type E1 cDNA;
E1-1-R (5'-TTTCTCGAGTTAGACTGACTTAAACTTGATCCACTG-3') for 1 cDNA;
E1-2-R (5'-TTTCTCGAGTTARGACTTAAACTTGATCCACTGATTC-3') for 2 cDNA;
E1-3-R (5'-TTTCTCGAGCTTAAACTTGATCCACTGATTGGC-3') for 3 cDNA;
E1-4-R (5'-TTTCTCGAGAAACTTGATCCACTGATTGGCACC-3') for 4 cDNA.
The last four primers were designed so that a series of E1 cDNA clones was made that were sequentially shorter by three nucleotides. On translation, they produce a series of E1 proteins sequentially shorter from the C-terminus by one amino acid.
The PCR conditions used were: 94°C for 1 min, 60°C for 1 min and 72°C for 1.5 min for 35 cycles. The PCR product was then subcloned into pCR 2.1 (Invitrogen, Carlsbad, CA) using a TA Cloning kit (Invitrogen).
Construction of the testis-specific variant of E1
Since the testis-specific gene is intronless, its sequence was obtained from genomic DNA by PCR. PCR primers were designed with 5' restriction sites for easy cloning: forward primer Ts-F (5'-TTTGGTACCTGCCATCTACAGCACTCCGT-3') and reverse primer Ts-R (5'-TTTCTCGAGCCTCCTTGAGTTGAGAACAC-3'). The PCR conditions used were: 94°C for 30 s, 58°C for 30 s and 72°C for 1 min 30 s. The testis-E1 was also first subcloned into pCR 2.1 to find an error-free clone [in comparison with the sequence published by Dahl (34)] and then recloned into the KpnI–XhoI sites of pcDNA 3.1.
DNA sequence analysis
Plasmid clones containing PCR product cDNAs were sequenced with the T7 Sequencing kit according to the manufacturer’s recommendations (Amersham Pharmacia Biotech). The reaction mixture was separated on an acrylamide gel. The entire coding region of the patient’s cDNA was sequenced with the primers described (35).
Cloning of E1 variants into the expression vector
Wild-type and mutant clones were digested with KpnI–XhoI and subcloned into the KpnI–XhoI site of pcDNA 3.1 (Invitrogen).
Construction of 0 (PDH complex-deficient) cell line
A human female skin fibroblast cell line with a 7 bp deletion at base 931 (exon 10) in one of the E1 alleles was transformed with SV40 large T antigen using a vector containing a large T antigen DNA (courtesy of Dr John Dick, HSC, Toronto) by transfection. Transfection was carried out using the Superfect reagent (Qiagen, Mississuaga, Canada) according to the manufacturer’s specifications on cells 18–22 h after splitting at a confluence of 60–80% in six-well plates (35 mm). A 2 µg aliquot of column-purified (Qiagen) plasmid DNA was combined with 8 µl of the Superfect reagent in 150 µl of serum-free -MEM. Cells were split 24–36 h after transfection at a ratio of one 35 mm plate to five 100 mm plates. After 10–14 days, transformed foci were cloned and assayed for PDH complex activity. Clones that had zero PDH complex activity, named 0, were chosen for further characterization. In order to confirm the absence of the E1 protein, western blot analysis was performed on mitochondria from the 0 cell line, the control cell line and the parental cell line. Twice the amount of protein was used for the 0 cell line than for the control or the parental cell lines to ensure that the experiment was not performed at the antibody’s limit of immunoreactivity.
Expression of wild-type and mutant E1 variants in the 0 cell line
All transfections were performed as described above using the 0 cell line and Qiagen column-purified DNA (either wild-type, mutant or vector alone), which was linearized with PvuI. Two days post-transfection, cells were plated into 100-mm plates and selection was applied at a concentration of 0.2 mg/ml G418 for 14–20 days until visible colonies could be detected. These colonies were cloned using cloning rings, and PDH complex activity was determined.
Quantitative PCR
RNA for quantitative PCR was isolated from transfected human skin fibroblasts using a total nucleic acid isolation kit (Qiagen). First strand cDNA synthesis was performed as described above using oligo(dT) as a primer. A negative control was designed so that first strand synthesis was carried out without MLV reverse transcriptase and used later in the PCR to ensure that no plasmid DNA contamination gave false-positive results. PCR was performed using a set of target primers as well as a set of internal control primers targeted against human PGK (both sets of primers were present in the same PCR). Primer sequences were target primers: pcDNA 3.1F (5'-TTAATACGACTCACTATAGGGAG-3' dihydrolipoamide acetyltransferase core) and pcDNA 3.1R (5'-AACTAGAAGGCACAGTCGAGG-3'); and internal control primers: PGK-For (5'-CAGTTTGGAGCTCCTGGAAG-3') and PGK-Rev (5'-TGCAAATCCAGGGTGCAGTG-3').
PCR was carried out as described above using the following set of conditions: 94°C for 30 s, 59°C for 30 s, 72°C for 45 s in a total volume of 50 µl. The following numbers of cycles were performed: 18, 20, 22, 24, 26, 28 and 30. One PCR tube was set up for each set of cycles. Samples were run on a 2% agarose gel, in sequential order of the number of cycles, and the amount of PCR product was quantitated by densitometry.
Cell culture
Untransfected SV40-transformed human skin fibroblasts were grown in -MEM supplemented with 15% fetal bovine serum (FBS). Transfected SV40-transformed human skin fibroblasts were grown in -MEM supplemented with 15% FBS together with 0.2 mg/ml G418. Increased thiamine concentration in the medium was achieved whenever necessary by addition of 0.1 mg per 10 ml of medium.
Mitochondrial import studies
Mitochondria were isolated from rat hearts by the method of Takahashi and Hood (36). Full-length cDNAs encoding wild-type and variant E1 as well as human MnSOD were subcloned into pGEM 3Z. Proteins were synthesized with the TNT-coupled reticulocyte lysate system (Promega, Madison, WI) using [35S]methionine as radioactive label (Amersham Pharmacia Biotech). Mitochondria were incubated with lysate containing the translated radiolabeled precursor proteins at 30°C for various periods of time, as indicated below. Final import reactions consisted of 40 µl (120 µg) of mitochondria, 5 µl of reticulocyte lysate and 2 µl of translation reaction. Glutamate was added as an additional respiratory substrate at a final concentration of 33 mM. Following incubation, mitochondria were recovered by centrifugation and washed twice with the import buffer. Pellets were resuspended in 15 µl of import buffer and 15 µl of 2x loading buffer [10% glycine (v/v), 80 mM SDS, 62.5 mM Tris–HCl pH 6.8, 5% 2-mercaptoethanol (v/v) and 10% Coomassie blue]. Samples were denatured and electrophoresed through a 12.5% SDS–polyacrylamide gel at 80 V. Gels were fixed [30% methanol (v/v), 3% glycerol (v/v)], treated for 30 min with Amplify (Amersham Pharmacia Biotech), dried and exposed to film (Bio-Max; Kodak, Rochester, NY) at –80°C. Autoradiograms were quantified using densitometry.
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ACKNOWLEDGEMENTS |
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We thank Dr Ives of St John’s, Newfoundland, for the cell lines of patients 1 and 2, and MRC Canada for the financial support of this research project.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 416 813 5989; Fax: +1 416 813 8700; Email: bhr@sickkids.on.ca
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REFERENCES |
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