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Human Molecular Genetics Advance Access originally published online on July 25, 2007
Human Molecular Genetics 2007 16(20):2472-2481; doi:10.1093/hmg/ddm203
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The A3243G tRNALeu(UUR) mutation induces mitochondrial dysfunction and variable disease expression without dominant negative acting translational defects in complex IV subunits at UUR codons{dagger}

George M.C. Janssen1,*,{ddagger}, Paul J. Hensbergen2,{ddagger}, Frans J. van Bussel1, Crina I.A. Balog2, J. Antonie Maassen1, André M. Deelder2 and Anton K. Raap1

1 Department of Molecular Cell Biology and 2 Biomolecular Mass Spectrometry Unit, Department of Parasitology, Leiden University Medical Centre, Post Zone S1-P, Einthovenweg 20, PO Box 9600, 2300RC Leiden, The Netherlands

* To whom correspondence should be addressed. Tel: +31 71 526 9263; Fax: +31 71 526 8270; E-mail: G.M.C.Janssen{at}lumc.nl

Received April 26, 2007; Revised June 1, 2007; Accepted July 22, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in the mitochondrial tRNALeu(UUR) gene are associated with a large variety of human diseases through a largely undisclosed mechanism. The A3243G tRNALeu(UUR) mutation leads to reduction of mitochondrial DNA (mtDNA)-encoded proteins and oxidative phosphorylation activity even when the cells are competent in mitochondrial translation. These two aspects led to the suggestion that a dominant negative factor may underlie the diversity of disease expression. Here we test the hypothesis that A3243G tRNALeu(UUR) generates such a dominant negative gain-of-function defect through misincorporation of amino acids at UUR codons of mtDNA-encoded proteins. Using an anti-complex IV immunocapture technique and mass spectrometry, we show that the mtDNA-encoded cytochrome c oxidase I (COX I) and COX II exist exclusively with the correct amino acid sequences in A3243G cells in a misassembled complex IV. A dominant negative component therefore cannot account for disease phenotype, leaving tissue-specific accumulation by mtDNA segregation as the most likely cause of variable mitochondrial disease expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitochondrial translation critically determines the activity of the oxidative phosphorylation cascade. All the RNAs required for mitochondrial translation, 22 tRNAs, 2 rRNAs and the mRNAs for 13 oxidative phosphorylation (OXPHOS) subunits, are encoded on the mitochondrial DNA within the mitochondrial matrix. Nucleotide substitutions in the mitochondrial tRNA genes are important factors in the pathogenesis of maternally inherited diabetes and deafness (MIDD; OMIM 520000 [OMIM] ), neurodegenerative disorders and heart failure (1–5). The A3243G substitution in the tRNALeu(UUR) gene (MTTL1; OMIM 590050 [OMIM] .0001) causes MIDD in most carriers, but may also display the classic phenotype of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS; OMIM 540000 [OMIM] ) (6–8), in which it was originally discovered (1). The A8344G mutation leads to myoclonic epilepsy and ragged red muscle fibers (MERRF; OMIM 545000 [OMIM] ) syndrome. All three syndromes are associated with a strong reduction of mitochondrial respiration and OXPHOS activity. This loss-of-function, however, is in essence a recessive condition, thus leaving no potential to explain the large variation in clinical phenotypes in terms of mitochondrial translation. Within this context, it seems a priori more plausible that a tRNA mutation would lead to dominantly acting, qualitative translation defects like amino acid misincorporation or premature translation termination products (9) (see Fig. 1 for an outline). Each type of tRNA mutation may then in principle direct the synthesis of a different set of aberrant translation products, thereby expanding the diversity of effects and the potential to explain variation in clinical phenotypes. A dominant effect also needs to be assumed to explain the clinical phenotype of MIDD patients, because the relevant tissue, pancreatic ß cells, contains only moderate levels of mutation load (~30%), far from the ~75% threshold levels required for mitochondrial dysfunction. In line with a gain-of-function scenario is also that different types of mtDNA mutation lead to distinct nuclear gene-expression patterns (10). Next to misincorporation, a dominant effect may also arise from unprocessed RNA19 which, when incorporated into mitochondrial ribosomes, may interfere disproportionately with mitochondrial translation, thereby causing the phenotypic changes (8,11). Finally, a gain of function may be reached when the affected tRNALeu(UUR) is redirected to a function unrelated to translation (12–15) like the control of GCN4 expression (16).


Figure 1
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  		Figure 1. Scheme depicting dominant negative and recessive models for explaining variable disease expression. Cells with a quantitative translation reduction (7,8,12,22–25) probably respond via recessive mode on the left, whereas translation competent cells seem to lose most of their mtDNA-encoded OXPHOS subunits through enhanced degradation (12–14). The residual aberrant OXPHOS subunits may act in dominant negative mode in specific tissues. The same scheme may also explain variable disease expression between mutations in different tRNA genes.

 
A point mutation in a tRNA gene may in principle influence protein synthesis either quantitatively or qualitatively (17). At the molecular level, the A3243G mutation has been shown to lead to reduced levels of the tRNALeu(UUR), decrease in aminoacylation and absence of the normal modification with 5-taurinomethyl group at the wobble base (12,13,18–20). The biochemical effects on the mtDNA-encoded OXPHOS subunits are also evident. Their level is severely decreased, leading to low OXPHOS and mitochondrial respiratory activity (7,8,12,21). However, the underlying mechanism by which this low level is reached is still a matter of discussion (5). A point mutation leading to a low level of charged tRNALeu(UUR) may be expected to affect protein synthesis in a quantitative way. Indeed, mitochondrial translation has been found decreased in several A3243G cybrid cells (7,8,12,22–25). In contrast, another class of translation-competent cybrid cells displayed low levels of mtDNA-encoded proteins and severe mitochondrial dysfunction without affected translation rates (7,12,14). T3271C tRNALeu(UUR) cybrids were also found to be translation competent (26). Thus, reduced mitochondrial translation is not requisite for reduction of mtDNA-encoded proteins and loss of respiration. This situation tempted several laboratories to propose that the primary defect should be found in the qualitative aspects of translation such as misincorporation of amino acids (12–14) (Fig. 1). In support of this view is that misincorporation induced by an editing-defective tRNA synthetase causes protein misfolding and neurodegeneration (27). To directly test the misincorporation hypothesis, we isolated cytochrome c oxidase (complex IV) from affected cells by an immunocapture procedure and extensively analyzed the mtDNA-encoded subunits COX I and COX II by mass spectroscopy. We show here that the mtDNA-encoded subunits exist only with the correct amino acid sequence and exclude thereby dominantly acting qualitative translation defects. We suggest tissue-specific mutation accumulation by segregation to underlie variable disease expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Immunoprecipitation and mass spectrometric characterization of cytochrome c oxidase
When using complex IV immunocapture beads to immunoprecipitate cytochrome c oxidase from mitochondrial solubilisates of wild-type VW7 cybrids, a limited number of ~10 distinct protein bands were found upon SDS–PAGE and Coomassie blue staining (Fig. 2). Intensities were comparable suggesting the presence of a complex with equimolar subunit composition. Protein bands were subjected to in-gel digestion and analyzed by nanoLC-iontrap MS/MS (Table 1). The protein at 25 kDa, which was also present in the absence of mitochondrial input was identified as IgG light chain. Six protein bands from the VW7 immunoprecipitate were identified as one of the 13 subunits of cytochrome c oxidase, i.e. COX I, COX II, COX IV, COX Va, COX Vb and COX VIc, including the ~16 kDa band which contained COX IV. We identified COX IV as the target of the immunocapture antibody, because the 16 kDa COX IV band was also immunoprecipitated from an SDS denatured mitochondrial fraction (Fig. 2). Of the three mtDNA-encoded subunits of cytochrome c oxidase, we unequivocally identified COX I and COX II but not COX III. However, COX III is a very hydrophobic subunit which behaves as a faintly stained band close to COX II (http://www.mitosciences.com/ms401.html) and may be easily missed. The two remaining bands were assigned to subunits I and II of cytochrome c reductase, which forms a supercomplex with cytochrome c oxidase (28). We thus identified almost exclusively subunits of the cytochrome c oxidase/reductase supercomplex, except some signal from the abundant mitochondrially localized Tom 40 and flotillins. Therefore we conclude that the immunocapture antibody yields a relatively pure cytochrome c oxidase/reductase supercomplex enabling direct comparison of the Coomassie blue stained protein bands. Figure 3 presents a schematic overview of the interactions in bovine heart cytochrome c oxidase for the six COX subunits investigated here.


Figure 2
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  		Figure 2. Immunopurified cytochrome c oxidase using anti-complex IV immunocapture beads. Cytochrome c oxidase was immunoprecipitated from a crude mitochondrial protein extract from wild-type (VW7), A3243G mutant (VM50) and mtDNA-deficient ({rho}0) cells. For identification of the target of the immunocapture antibody, an SDS-denatured wild-type mitochondrial fraction was treated similarly (SDS). Immunocaptured proteins were subjected to SDS–PAGE and stained with colloidal Coomassie blue. Visible protein bands were in-gel digested with trypsin and chymotrypsin and identified by LC-MS/MS. m, All-Blue Standard (Bio-Rad) marker proteins 10, 15, 20, 25, 37, 50, 75, 100, 150 and 250 kDa respectively; input, 100 µg total mitochondrial fraction from VW7 cells; COX, cytochrome c oxidase; CR, cytochrome c reductase; astersik, assigned to COX IV; the faintly stained band migrating just above COX II was also assigned to COX II.

 


Figure 3
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  		Figure 3. Schematic representation of the interactions within bovine heart cytochrome c oxidase. This model is based on the crystal structure data of (34). For clarity, only the subunits that have been analyzed are shown here. COX IV (bold) is the target for the immunocapture antibody, COX I and COX II (underlined) are the mtDNA-encoded subunits.

 


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  		Table 1. Protein identifications in anti-complex IV immunoprecipitates

 
In VM50 and {rho}0 immunoprecipitates, most protein bands were found decreased or even absent, whereas only COX IV and COX Va remained comparable in these cells. COX I and COX II were also identified at lower intensities in the VM50 immunoprecipitate, whereas COX Vb and COX VIc bands were undetectable. As expected, the mtDNA-encoded COX I and COX II were absent in {rho}0 immunoprecipitates. Here, three pronounced additional bands were observed and assigned to COX IV (bands marked with asterisk in Fig. 2). Cytochrome c reductase subunits were also found decreased in mutant immunoprecipitates.

Western blot analysis
The above results were confirmed by western blot analysis of the immunoprecipitates using antibodies specific for subunits of cytochrome c oxidase (Fig. 4). Subunits COX IV and COX Va remained at a comparable level in mutant-type cells. As anticipated, the mtDNA-encoded subunits COX I and COX II showed a strong decrease in A3243G cybrid cells and are absent in the mtDNA-less {rho}0 immunoprecipitate. There is no antibody available against human COX III, hampering its detection.


Figure 4
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  		Figure 4. Western blot analysis of anti-complex IV immunoprecipitates (IP) and mitochondrial fractions (MITO). Immunoprecipitate (IP) and total mitochondrial fraction (MITO) from VW7, VM50 and {rho}0 cells were subjected to SDS–PAGE and subsequent western blotting using specific antibodies against 6 subunits of COX.

 
The nuclear-encoded subunits COX Vb and COX VIc were absent in the immunoprecipitate of both mutant cell lines. In order to assess the presence of COX subunits in the mitochondrion, mitochondrial fractions were also directly subjected to western blot analysis. Comparable ratios were observed among the three mitochondrial fractions (Fig. 4), except in the case of COX Vb which was clearly present in the mitochondrial fraction of VM50 cybrids, but not detectable in the respective immunoprecipitate.

Recently it was shown that mutations at positions 3243 and 3271 abolish the proper modification to 5-taurinomethyluridine at the wobble position of tRNALeu(UUR) (20). The absence of this modification leads in vitro to a specific ~5-fold decrease of translation of UUG codons and normal translation of UUA in vitro. Our in vivo results at the protein level indicate that in A3243G cybrids the level of COX I is more severely affected than that of COX II, both in immunoprecipitates and in mitochondrial fractions. This is unexpected in light of the taurine modification hypothesis which predicts the reverse since COX II, in contrast to COX I, contains a UUG-encoded leucine (Table 2) inducing a codon-specific translational defect in vitro (20).


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  		Table 2. Leucine codon usage of human mitochondrial COX genes

 
Detailed analysis of COX peptide fragments by MS/MS
The mass spectrometric data obtained from the mtDNA-encoded subunits COX I and COX II from VM50 cells were subjected to a detailed analysis in order to detect potential misincorporation of amino acids at the leucine codons translated by tRNALeu(UUR). We therefore focused our analysis on peptides containing UUA- and UUG-encoded leucine residues. In the case of a significant rate of misincorporation, one may expect the emergence of novel UUR-encoded peptides having another amino acid at the position of the UUR-encoded leucine.

COX I contains seven UUR-encoded leucine residues, none of them encoded by UUG but all encoded by UUA (Table 2). Within the tryptic/chymotryptic digest of COX I of both VW7 and VM50 (Table 3), we found a peptide with m/z 547.7 (M+2H)2+ corresponding to the peptide TVYPPL132AGNY of COX I (amino acid 127–136), which contains a UUA-encoded leucine (Fig. 5A). MS/MS analysis of this peptide confirmed the identity of the peptide and clearly demonstrates that VM50 cells are able to correctly decode UUA codons into leucine.


Figure 5
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  		Figure 5. MS/MS analysis of COX-I- and COX-II-derived peptides containing UUR-encoded leucine. COX I and COX II bands from immunoprecipitates (A and B) and mitochondrial extracts (C) were in-gel digested with a combination of trypsin and chymotrypsin or trypsin alone. Extracted peptides were analyzed by LC-MS/MS. UUA-encoded leucine is in bold and UUG-encoded leucine is in bold and underlined. (A) Identification in both wild-type (VW7) and A3243G mutant (VM50) cells of a COX-I-derived peptide TVYPPL132AGNY (m/z 547.7, (M+2H)2+) with leucine-132 encoded by UUA. (B) MS/MS analysis of a COX-II-derived peptide containing the unique UUG-encoded leucine-160. Identification of the correct leucine at the UUG-encoded position in COX II from both the VW7 and VM50 immunoprecipitates of the peptide MMITSQDVL160HSW (m/z 740.2, (M+2H)2+). (C) MS/MS analysis of a COX-II-derived tryptic peptide MMITSQDVL160HSWAVPTL168GL170K (m/z 754.0, (M+3H)3+) containing three UUR-encoded leucines from the crude mitochondrial fraction of VW7 and VM50 cells. Total crude mitochondrial extracts from wild-type (VW7), A3243G mutant (VM50) and mtDNA-less ({rho}0) cells were separated by SDS–PAGE. The material migrating at the position of COX II was in-gel digested with trypsin and analyzed by LC-MS/MS. No COX-II-derived signal could be obtained from the mtDNA-less extracts.

 


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  		Table 3. Relevant peptides obtained from COX I and COX II

 
COX II contains five UUR-encoded leucines, one of which is encoded by UUG, providing a rigid test of codon-specific translational defects as predicted by the taurine modification hypothesis (Table 2). This UUG-encoded peptide with an m/z value of 740.2 (M+2H)2+ was identified both in VW7 and VM50 immunoprecipitates and corresponds to the peptide MoxMoxITSQDVL160HSW (amino acid 152–163) of COX II (Fig. 5B). Upon manual inspection of the MS/MS data, no single peptide could be recognized with a sequence deviating from MoxMoxITSQDVL160HSW at the UUG-encoded leucine 160.

Specific attention was also paid to phenylalanine-containing peptides because it has been postulated that A3243G mutant tRNALeu(UUR) may decode the phenylalanine codons UUU and UUC and appearance of leucine at phenylalanine-encoded positions (13). Again, A3243G cybrid cells do contain the correctly translated phenylalanine in the tryptic/chymotryptic peptides LVPLMIGAPDMAFPR (amino acid 82–96 of COX I) and FLEPGDLR (amino acid 127–134 of COX II) (Table 3), with no indications for aberrant peptides containing leucine instead of phenylalanine.

In addition to the analysis of specific peptides, we also manually inspected all the MS/MS spectra of both COX-I- and COX-II-derived peptides from VM50 immunoprecipitates in an attempt to find peptides originating from any misincorporation at the UUR-encoded positions. Thus, the spectrum of each individual peptide abundant enough to meet the pre-set threshold levels of the mass spectrometer for subsequent MS/MS analysis was manually compared to the sequences of COX I and COX II in order to find misincorporated COX-derived peptides in VM50 immunoprecipitates. However, no single aberrant COX I or COX II peptide could be identified using this unbiased approach.

Analysis of anti-COX-II immunoprecipitate and total input mitochondrial protein
The possibility exists that when COX II contains a misincorporated amino acid it may not be assembled anymore in a complex with COX IV. Therefore, COX II was directly precipitated with an anti-COX-II antibody from the SDS-solubilized mitochondrial fraction of VM50 cells. Although this antibody proved ineffective for precipitating the whole complex of cytochrome c oxidase, upon denaturation with SDS a ~20 kDa band was observed migrating at the position of COX II, among several other protein bands (not shown). COX II could be readily recognized and identified by MS/MS in this ~20 kDa band. Because two UUA and one UUG-encoded leucine are situated within the tryptic fragment MMITSQDVL160HSWAVPTL168GLK170 (amino acid 152–171 of COX II) (Table 3), the putative COX II band was in-gel digested using trypsin alone. Despite the high complexity of the anti-COX-II immunoprecipitate, this peptide was identified with no indication for aberrant peptides. We also identified a C-terminal peptide of COX II, IFEMGPVFTL (amino acid 218–227), indicating that mitochondrial protein synthesis in VM50 cybrids is able to synthesize full-length COX II.

Finally, we applied a mitochondrial lysate directly to SDS–PAGE and mass spectrometric analysis to exclude the small possibility that COX II with misincorporated amino acid is no longer recognized by the monoclonal anti-COX-II antibody. Although in this case we have to deal with an even higher complexity of the sample due to the relatively large number of background proteins, the proteins at the position of COX II were in-gel digested with trypsin and analyzed by LC-MS. The tryptic peptide MoxMoxITSQDVL160HSWAVPTL168GL170K (amino acid 152–171) could again be readily detected in the VW7, but also VM50 cells (Fig. 5C). As expected, COX-II-derived peptide was not found in the {rho}0 sample (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Extensive studies on the consequences of the A3243G mutation on the tRNALeu(UUR) itself have revealed serious impairments at the level pre-tRNA processing, half-life, base modification and aminoacylation (12,13,18,19,29–32). Yet, their downstream effect on the mtDNA-encoded proteins could only be studied by indirect methods such as (35S)methionine incorporation and western blot analysis (7,8,12,14,22–25). This was largely due to the difficulty in obtaining the affected OXPHOS complexes in a form suitable for direct mass spectrometric analysis. With our cybrid cells, two-dimensional blue native PAGE did not provide sufficient resolution in the first dimension as the protein patterns were rather smeared. Classical immunoprecipitation, using anti-COX I, COX II and COX IV antibodies non-covalently attached to protein G beads, were also not successful in isolating complex IV. The availability of an antibody capable of precipitating complex IV in pure form (33), however, enabled us to compare its constituent subunits in wild-type and mutant cells for the first time in more detail. We have chosen for immunoprecipitation of complex IV, because cytochrome c oxidase is a very well characterized enzyme. It is composed of only 13 subunits (34), and its activity is critically sensitive to the A3243G mutation in our cybrids (~3% residual complex IV activity versus 20 and 30% for complex I and III, respectively) (21). Complex I, for instance, is less abundant than complex IV (28) and contains ~40 nuclear-encoded subunits complicating the identification and mass spectrometric analysis of mtDNA-encoded ND subunits (33). Importantly, the leucine codon usage among the three mitochondrial COX genes provided the unique possibility to examine the effect of taurine modification deficiencies on mitochondrial translation in vivo because only COX II contains a single UUG-encoded leucine with COX I and COX III serving as internal reference (Table 2).

Misincorporation generally involves misacylation of the tRNA by a non-cognate aminoacyl-tRNA synthetase according to established rules defining tRNA identity elements in bacteria (35). In the case of misacylation, it would occur in competition with leucylation, and only a sub-population of the mutated tRNA would therefore be expected to be esterified by an amino acid different from leucine. Therefore, in the absence of direct in vitro mischarging experiments of tRNALeu(UUR) by the various mitochondrial synthetases, it is difficult to predict the non-cognate amino acid as well as its expected misincorporation rate during translation of UUR codons. Alternatively, misincorporation may also involve misreading of the non-cognate UUY phenylalanine codons at unknown frequency by A3243G tRNALeu(UUR) due to absence of taurine modification (13).

We have characterized relevant COX-derived peptides with UUR-encoded leucines by MS/MS analysis using three different approaches: (i) upon immunocapture with anti-COX IV, (ii) upon immunoprecipitation with anti-COX II and (iii) directly from mitochondrial lysates in order to avoid specific loss of hypothetical aberrant peptides during the immunoaffinity-based procedures. None of them was able to determine peptides with deviant m/z, clearly showing that A3243G cybrids do not contain aberrant mtDNA-encoded proteins at a detectable level. In line, no deviant molecular mass or isoelectric property was found among the (35S)methionine-labeled mitochondrial proteins of A3243G cybrids (7,8,12,25). Furthermore, no misacylation could be detected in isolated A3243G and T3271C tRNALeu(UUR) (13).

Mutations at position 3243 and 3271 prevent the proper 5-taurinomethyl modification of the uridine at the wobble position of tRNALeu(UUR) (13,20,36,37). Removal of this taurine modification from wild-type tRNALeu(UUR) by a molecular surgery procedure leads to a specific ~5-fold decrease of UUG translation, although the translation of UUA remains unaffected on defined synthetic mRNAs in vitro (20,38). The consequences, however, of this specific reduction have not been examined in vivo. Does stalling at UUG lead to reduced but correct translation, or does mistranslation occur at these positions? The latter seems unlikely, given the correct incorporation of the unique UUG-encoded leucine-160 in the COX-II-derived peptide MMITSQDVL160HSW without any indication of mistranslation in the A3243G mutant cell. In line with stalling at UUG due to absence of the taurine modification is the reduced COX II level. Not in line with this prediction is that COX I which has no UUG codon is virtually absent (Figs 2 and 4). Therefore, a UUG-specific translation reduction with indirect effects on UUA-coded subunit COX I is implicated in vivo.

The assembly of cytochrome c oxidase is critically obstructed in A3243G cybrids and {rho}0 cells. From the IP (Fig. 2) and its western blot analysis (Fig. 4), it is seen that the mtDNA-encoded subunits COX I and COX II and the nuclear-encoded COX Vb and COX VIc are strongly reduced or absent in A3243G and {rho}0 cells. In the latter, the mtDNA-encoded subunits COX I, II and III are completely absent and a distinct set of COX IV degradation products were observed. Only subunits COX IV and COX Va were found in amounts approaching that of the wild-type cybrid cell. According to crystal structure of bovine heart cytochrome c oxidase (34), these two subunits are found in close contact with each other.

Absence of subunits COX Vb and COX VIc in immunoprecipitates of both mutant cells is in agreement with the notion that they do not directly interact with subunit COX IV but require the presence of subunits COX I and COX II, respectively (Fig. 3) (34). The binding of subunits COX I and COX IV is the first event in the assembly of cytochrome c oxidase in wild-type cells (39). Presumably, in mutant cells COX IV binds COX Va without preceding assembly with COX I. The observed reduction of cytochrome c reductase subunits (Fig. 2) indicates a decrease in the supercomplex composed of cytochrome c oxidase and cytochrome c reductase (28). Obviously, a residual cytochrome c oxidase depleted of COX I, COX II, COX Vb and COX VIc is insufficient to bind cytochrome c reductase and thus serves the efficiency of the ‘respirasome’ through substrate channeling (28,40).

The mitochondrial extracts showed roughly the same subunit stoeichiometry as the immunoprecipitates, except COX Vb which was only moderately decreased in A3243G mitochondria. Thus, although COX Vb is present in the mitochondrion, it fails to be assembled into the complex of COX IV and COX Va. This confirms that in human cells there is no direct interaction between COX Vb and the complex of COX IV and COX Va. Subunit COX Vb has also been found decreased in A3234G mitochondrial extracts during a comparative proteomics approach (41). In addition, a comparable decrease of subunits COX Vb and COX VIc has been observed in total cell extracts from {rho}0 cells using the same monoclonal antibodies (42). Translational repression of these nuclear-encoded subunits by a feed-back signal from inactive mitochondria seems unlikely, given that the synthesis of nuclear-encoded OXPHOS subunits is not preferentially inhibited in cells arrested in mitochondrial translation. Instead, an increase in their turnover rate was observed (43). Therefore it is feasible that the nuclear-encoded subunits become susceptible to the protein-degrading machinery because they have a difficulty in finding a suitable binding partner. Exceptions are COX IV and COX Va, which are found together in a complex presumably less amenable to breakdown, although some breakdown products were observed in mutant cells.

In conclusion, our results favor a scheme in which variable disease expression is mediated by a recessive loss-of-function model following tissue-specific mutation accumulation and loss of OXPHOS activity (the left part of Fig. 1). In essence, we did not find any evidence for misincorporation into mtDNA-encoded proteins in A3243G cells, thereby excluding dominant negative acting translational defects. All results indicate the correct incorporation of leucine at UUR codons, thereby leaving unexplained the lowered levels of mtDNA-encoded proteins in translation-competent cybrid cells. Obviously we cannot exclude that rapid degradation of mistranslated proteins underlies their apparent absence. But if this were so, then a loss-of-function scenario would still prevail and variable disease expression should also stem from tissue-specific accumulation of mutant mtDNA above threshold values. Indeed, both classes of cybrid cells, translation-defective (7,8,12,22–25) and translation-competent (7,12,14), are characterized by recessive loss-of-function of respiration and OXPHOS activity. The loss-of-function hypothesis is also supported by mouse models where a loss-of-function induced by mtDNA gene inactivation in specific tissues determines the clinical phenotype: Hart-specific inactivation induces dilated cardiomyopathy (44), whereas beta cell-specific inactivation causes a diabetes phenotype (45). For an mtDNA mutation to become expressed, high mutation loads are required indeed. This condition seems amply met in the case of muscle tissues of MELAS and MERRF patients (46–48). In pancreatic beta cells of MIDD patients, only moderate to low mutation levels have been found (49–51), but the total cell mass is reduced suggesting accumulation of A3243G to high levels followed by apoptosis (51). In accord with this reasoning is the finding that apoptosis is induced by loss of mtDNA in mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase gamma POLG (52). This notion together with the present results which show no indication of a dominantly acting translational defect leads us to conclude that phenotypic variability of mtDNA disease seems to arise primarily from tissue-specific mutant mtDNA accumulation through as yet enigmatic mtDNA segregation mechanisms (5).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
The mitochondrial transformants (cybrids) used in this study have been generated earlier by the transfer of mitochondria from fibroblasts from an A3243G MIDD patient's mitochondrial DNA-less 143B {rho}0 cells (21). Cybrids containing either the wild-type (VW7; respiration 82 ± 34 fmol O2/h/cell) or the A3243G mutant-type (VM50; respiration 7.2 ± 4.8 fmol O2/h/cell) tRNALeu(UUR) gene and the {rho}0 cells (mtDNA undetectable; respiration<0.5 fmol O2/h/cell) were all cultured in Dulbecco's modified Eagle medium (DMEM) containing 4.5 mg/ml of glucose and 110 µg/ml of pyruvate supplemented with 50 µg/ml uridine and 10% fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere of 5% CO2/95% air and subcultured twice a week via trypsin treatment to a cell plating density of 10–15% confluence. Before each experiment, cells were tested for the presence of the correct type respectively the absence of mtDNA using a PCR-RFLP assay (detection sensitivity ~1%) (2). mtDNA copy number of the cybrid cells was determined using real time PCR relative to the ß globin gene and found to be ~3000 (53).

Preparation of partially purified mitochondria
Mitochondria were essentially prepared by the method of Chomyn (54). Briefly, the cells from five 14 cm dishes (~2x107 cells) were washed twice with PBS, harvested by trysinization and washed twice with 10 ml aliquots of PBS by low-speed centrifugation for 5 min at 900g in a table-top centrifuge at ambient temperature, and once with an ~1.5 ml aliquot of ice-cold 10 mM HEPES pH 7.4, 0.25 M sucrose by centrifugation for 5 min at 4000g in a pre-cooled Eppendorf centrifuge. Next, the cells were re-suspended in 1 ml of the same buffer and maintained cold during the whole procedure. Cells were disrupted on ice by 20 strokes in a pre-cooled Potter–Elvehjem tube. The crude mitochondrial fraction was obtained from this homogenate by serial centrifugation for 5 min at 4000g twice (leaving the pellets) and 45 min at 10 000g (saving the pellet) in an Eppendorf centrifuge. The crude mitochondrial pellet was re-suspended in 100 µl of 20 mM Tris–Cl pH 7.4, 0.25 M sucrose, 2 mM EDTA and stored at –30°C. The yield, as determined by a bicinchoninic acid protein assay (Pierce), was ~0.2–0.4 mg of total mitochondrial protein per dish.

Immunoprecipitation of cytochrome c oxidase
Cytochrome c oxidase was isolated from the crude mitochondrial fraction by immunoprecipitation using anti-complex IV immunocapture antibody beads (mouse monoclonal anti-bovine cytochrome c oxidase IgG1, cross-linked to protein G agarose) from Mitosciences (Eugene). The mitochondrial fraction (1 mg of protein) was solubilized by the addition of a 10% n-dodecyl-ß-D-maltopyranoside stock to an end concentration of 1%. Next, 40 µl anti-complex IV capture resin (equilibrated with 50 mM Tris–Cl pH 7.5) was added and incubated overnight at 4°C. The beads were washed three times with PBS. The immunocaptured cytochrome c oxidase complex was directly dissolved in SDS–PAGE sample buffer at ambient temperature and analyzed on a 13% SDS–PAGE gel. Proteins were visualized by extensive staining (three refreshments of staining solution for maximal staining) with a colloidal Coomassie Brilliant Blue solution (SimplyBlue SafeStain, Invitrogen). To assess the target of the immunocapture antibody, the same procedure was performed using VW7 mitochondrial fraction, but pre-incubated for 15 min at 0°C with 0.25% SDS to disrupt the complex, and diluted out to<0.05% SDS before the antibody beads were added. Immunoprecipitation with anti-COX II was performed using 1 mg of mitochondrial protein (previously denatured with 0.25% SDS), 50 µg of anti-COX II (clone 12C4) and 25 µl of protein G beads (Pharmacia).

In-gel digestion and mass spectrometry
Protein bands were excised from SDS–PAGE gels, reduced, alkylated and in-gel digested using trypsin (modified, sequencing grade, Promega) as previously described (55). For additional chymotryptic digestion, 125 ng of chymotrypsin (Sequencing grade, Roche) and 10 mM CaCl2 were added, followed by overnight incubation at 25°C. After digestion, peptides were collected using two rounds of extraction with 20 µl of 0.1% TFA and stored at –20°C prior to analysis by mass spectrometry. Samples were injected onto a nano-LC system (Ultimate, Dionex, Amsterdam, the Netherlands) equipped with a peptide trap column (Pepmap 100, 0, 3 i.d. x 1 mM) and an analytical column (Pepmap 100, 0.075 i.d. x 150 mM). The mobile phases consisted of (A) 0.04% formic acid/0.4% acetonitrile and (B) 0.04% formic acid/90% acetonitrile. A 45 min linear gradient from 0 to 60% B was applied at a flow rate of 0.2 µl/min. The outlet of the LC system was coupled to an HCT ion-trap mass spectrometer (Bruker Daltonics, Bremen) using a nano-electrospray ionisation source. The spray voltage was set at 1.2 kV and the temperature of the heated capillary was set to 165°C. Eluting peptides were analyzed in the data-dependent MS/MS mode over a 400–1600 m/z range. The five most abundant fragments in each MS spectrum were selected for MS/MS analysis by collision-induced dissociation. Mass spectra were evaluated using the DataAnalysis 3.1 software package (Bruker Daltonics, Bremen, Germany). For protein identification, MS/MS spectra were searched against the human IPI database using the Mascot search algorithm (Matrixscience, London, UK), allowing mass tolerances of 1.5 Da for MS and 0.5 Da for MS/MS. One missed cleavage site was allowed for tryptic peptides and two missed cleavage sites for tryptic+chymotryptic peptides. Carbamidomethylcysteine was taken as a fixed modification and oxidation of methionine as a variable modification.

Western blot analysis
Crude mitochondrial fractions (50 µg of protein) and immunoprecipitates (1/14th of the 1 mg input) were analyzed by western blotting using antibodies directed against the different subunits of cytochrome c oxidase (COX I, clone 1D6; COX II, 12C4; COX IV, 10G8; COX Va, 6E9; COX Vb, 16H12; COX VIc, 3G5) in the dilution suggested by the manufacturer (Molecular Probes). Anti-yeast COX III, clone DA5, designated as ‘human mitochondrial reactivity’ did not recognize human COX III.


   ACKNOWLEDGEMENTS
 
Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} This paper is dedicated to the memory of the late Professor Wim Möller (1930–2005), a remarkable man, molecular biologist at Leiden University and pioneer of ribosome structure and function. Back

{ddagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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