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Human Molecular Genetics Advance Access originally published online on June 1, 2006
Human Molecular Genetics 2006 15(13):2157-2169; doi:10.1093/hmg/ddl141
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Respiratory chain supercomplexes set the threshold for respiration defects in human mtDNA mutant cybrids

Marilena D'Aurelio1, Carl D. Gajewski1, Giorgio Lenaz2 and Giovanni Manfredi1,*

1 Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY 10021, USA and 2 Dipartimento di Biochimica, G. Moruzzi, Universitá di Bologna, Bologna 40126, Italy

* To whom correspondence should be addressed at: Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 525 E. 68th Street, A-505, New York, NY 10021, USA. Tel: , +1 2127464605; fax: +1 212 7468276; Email: gim2004{at}mail.med.cornell.edu

Received April 26, 2006; Accepted May 24, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mitochondrial DNA (mtDNA) mutations cause heterogeneous disorders in humans. MtDNA exists in multiple copies per cell, and mutations need to accumulate beyond a critical threshold to cause disease, because coexisting wild-type mtDNA can complement the genetic defect. A better understanding of the molecular determinants of functional complementation among mtDNA molecules could help us shedding some light on the mechanisms modulating the phenotypic expression of mtDNA mutations in mitochondrial diseases. We studied mtDNA complementation in human cells by fusing two cell lines, one containing a homoplasmic mutation in a subunit of respiratory chain complex IV, COX I, and the other a distinct homoplasmic mutation in a subunit of complex III, cytochrome b. Upon cell fusion, respiration is recovered in hybrids cells, indicating that mitochondria fuse and exchange genetic and protein materials. Mitochondrial functional complementation occurs frequently, but with variable efficiency. We have investigated by native gel electrophoresis the molecular organization of the mitochondrial respiratory chain in complementing hybrid cells. We show that the recovery of mitochondrial respiration correlates with the presence of supramolecular structures (supercomplexes) containing complexes I, III and IV. We suggest that critical amounts of complexes III or IV are required in order for supercomplexes to form and provide mitochondrial functional complementation. From these findings, supercomplex assembly emerges as a necessary step for respiration, and its defect sets the threshold for respiratory impairment in mtDNA mutant cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The respiratory chain is the major source of the transmembrane electrochemical gradient utilized in mitochondria to generate ATP. In mammals, mitochondria contain multiple copies of a circular DNA molecule (mtDNA), which encodes for 13 protein components of the respiratory chain and a full complement of tRNAs and rRNAs necessary for mitochondrial protein translation (1).

The structural models explaining the supramolecular organization of the respiratory chain have changed over the past decades. The initial one was the ‘solid-state’ model proposed by Chance and Williams (2). This model has later been replaced by the ‘liquid-state’ one, where functionally active individual complexes are free to diffuse laterally in the lipid bilayer of the inner membrane (3). In the ‘random collision’ model (4,5), mitochondrial electron transfer depends on the random contacts between independent complexes and small diffusible molecules, such as coenzyme Q and cytochrome c. Only recently, the development of blue native gel electrophoresis has allowed for the identification of supramolecular structures containing multimers of respiratory chain complexes, in yeast and mammalian mitochondria, which are reminiscent of the original solid-state model (6–8). Furthermore, the architecture of mammalian respiratory chain supercomplexes has been determined by a combined electrophoretic and single-particle image-analysis approach (9). Flux control ratio studies on bovine mitochondria demonstrated that the respiratory chain is functionally organized in partial supercomplexes (10). To date, however, it remains unclear to what extent the oxidative function of the respiratory chain is dependent upon the formation of supercomplexes.

In humans, mutations in mitochondrial genes cause metabolic disorders biochemically characterized by respiratory chain defects (11). Pathogenic mtDNA mutations often coexist with wild-type (WT) mtDNA within the same cells, a condition called mtDNA heteroplasmy. In this case, the degree of respiratory chain impairment depends on the relative proportions of mutant and WT mtDNA, with a clear threshold effect, which varies among cell types, based on the metabolic requirements for ATP. WT mtDNA can complement the loss of function of mutant mtDNA up to a certain threshold. To complement the deleterious effects of the mutations, WT mtDNA, mtRNA and mtDNA-encoded proteins must be able to diffuse within mitochondria. A more complex situation occurs when different mtDNA species are segregated within different mitochondria, because in this case, complementation requires exchange of factors across the boundaries of mitochondrial membranes. In mammalian cells, mitochondria are organized in a dynamic network, which is subject to a constant reshaping process modulated by fusion and fission events (reviewed in 12). This reshaping process is likely to provide the mechanism whereby molecules are exchanged among mitochondria, allowing for functional complementation.

Cell hybridization models have been used to confirm the hypothesis that mitochondria can fuse to provide complementation. When cells containing homoplasmic levels of mutant mtDNA (i.e. 100% mutant mtDNA) and severely compromised respiratory function are fused with cells containing WT mtDNA, respiration is restored in the resulting hybrid cells (13). Furthermore, the fusion of two cell lines, each containing a distinct homoplasmic mt-tRNA mutation, resulted in restored respiration, suggesting that tRNAs were exchanged among mitochondria (14).

To date, functional complementation between mitochondria-containing mutations in polypeptide–coding genes has not been explored. To address this issue, we have investigated complementation between two human cell lines, each containing a homoplasmic mtDNA mutation in a gene encoding a subunit of distinct respiratory chain complexes. Both mutations result in a complete loss of their respective complexes. We have characterized the biochemical properties of protein–protein complementation in hybrids derived from fusion of these mutant cell lines. We have studied the direct effects of protein mutations on the assembly of individual respiratory chain complexes and on supermolecular complex formation, as well as the secondary effects on the assembly and function of complexes that are not directly affected by the mutations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Fusion of cybrids containing two distinct mutant mtDNA results in hybrids with restored respiratory chain functions
Hybrid clones were obtained by fusion of two human osteosarcoma cybrid lines, one containing a homoplasmic stop-codon mutation in the cytochrome c oxidase subunit 1 gene (MTCO1), denoted as COX1-MT, and the other a frame-shift mutation in the gene encoding cytochrome b (MTCyB), denoted as CyB-MT. Both parental cybrids completely lacked mitochondrial respiration (15,16). Each cybrid line was engineered with a specific antibiotic resistance (i.e. hygromycin and puromycin, respectively), allowing for the double-selection of hybrid clones (nuclear complementation). Upon fusion, hybrids contained a mixture of variable proportions of the two mutant mtDNAs. In addition, because mtDNA recombination occurs between the two mtDNA alleles, new molecules of WT mtDNA are formed (17). Therefore, in most clones, the sum of the two mutant alleles does not account for 100% of the mtDNA molecules (Figs 4–6).

To assess whether selective growth pressure has a significant impact on the frequency of hybrid clones, cybrid fusion was performed under different growth conditions. The clone frequency in non-selective medium, containing glucose, pyruvate and uridine, reflects the efficiency of nuclear complementation. The clone frequency in selective media lacking pyruvate and uridine (18), or containing galactose instead of glucose as the major carbon source (19), reflects the frequency of both nuclear and mitochondrial complementation, because these selective conditions only allow for survival of clones with restored mitochondrial respiratory chain activity. There is a small but statistically significant difference in the number of hybrid clones obtained in non-selective and mitochondrial selective conditions (Fig. 1A). This indicates that the majority of the hybrids that survived the nuclear selection can also survive under mitochondrial selection and suggests that cybrid fusion results both in nuclear and mitochondrial complementation. In all of the following experiments, we have studied hybrid clones that are able to survive in selective medium. Because in the literature there are a few examples of nuclear suppression of mtDNA defects (20,21), as a control, COX1-MT cybrids were also fused with cells devoid of mtDNA ({rho}0 cells) and resistant to neomycin. As expected, the resulting COX1-MT-{rho}0 hybrids obtained by hygromycin and neomycin selection did not yield any viable clone under mitochondrial selective conditions (not shown), suggesting that the hybridization procedure per se does not afford survival under mitochondrial selection.


Figure 1411
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  		Figure 1. Mitochondrial functional complementation. (A) Average number of surviving hybrid clones in three different growth conditions: non-selective medium A (black bar), selective medium B (light gray bar) and selective medium C (dark gray bar), as described in Materials and Methods. Error bars represent SD. *indicates statistically significant differences (P<0.05, estimated by unpaired two-tailed Student's t-test) compared with non-selective medium. (B) Growth in galactose medium (medium B). 1x105 cells (indicated by the dashed line) plated in triplicate for each cell line were counted after 3 days of selection in medium B. The value for hybrid clones is the average number of cells for 12 individual hybrid clones each counted in triplicate. Measurement of oxygen consumption in intact cells in the absence (C) or in the presence (D) of the uncoupling agent FCCP. The value for hybrid clones is the average rate of respiration in 14 individual clones. Error bars indicate SD. WT, WT cybrid; COX1, 100% COX1-MT; CyB, 100% CyB-MT. Statistically significant differences between the WT average value and the average value in hybrid clones estimated by unpaired two-tailed Student's t-test are indicated: *P<0.05; **P<0.005.

 
Next, we investigated the growth of hybrid clones in galactose medium, as an indirect estimate of oxidative phosphorylation function. On average, the number of hybrid cells counted after 3 days of growth in galactose is ~60% lower than WT cells (Fig. 1B). The wide variability suggests that individual clones have varying levels of respiratory chain recovery. As expected, both parental cybrids fail to grow in galactose medium. Oxygen consumption measurements confirm that mitochondrial respiration is restored in hybrid cells. However, on average, coupled respiration in intact cells is lower in hybrid cells than in WT controls, with a wide variation (Fig. 1C). Cell respiration uncoupled with FCCP, which generates the maximal respiratory chain activity in intact cells, follows a similar trend (Fig. 1D). As expected, parental cybrids have no respiratory activity, either in coupled or in uncoupled conditions (Fig. 1C and D).

Correlation between mtDNA mutation levels, specific enzyme activities and respiration in hybrid cells
In Figure 2A, the relative abundance of each of the two mutant mtDNA species is correlated with cell respiration. Because the proportions of each heteroplasmic mutant mtDNA species varied in the same clone over time, the levels of both mutations have been measured after each experiment. From the tri-dimensional plot, it is apparent that a number of clones have respiration similar to WT cells. These clones contain variable proportions of the two mutant mtDNAs, but in each clone, these proportions were below 40 and 90% for COX1-MT and CyB-MT, respectively. All the clones with proportions of mutant mtDNA above these levels had defective respiration. The 40% threshold for COX1-MT was in agreement with the one that we had previously reported in cybrids containing only COX1-MT mtDNA (16). Because the threshold for CyB-MT had not been established previously, we have looked at the correlation between the proportion of CyB-WT and cell respiration. The plot in Figure 2B shows hybrid clones with high proportions of COX1-WT, >60% (i.e. above the threshold for respiratory defect), and variable proportions of CyB-WT. The data points were interpolated with the equation Y=BmaxxX/(Kd+X), where X is the percentage of CyB-WT, Y the value of oxygen consumption, Bmax the horizontal asymptote of the curve and Kd the percentage of CyB-WT, which allows for 1/2 Bmax. On the basis of this interpolation, 5% of CyB-WT allows for 50% of the maximal respiration, suggesting that the respiratory threshold for CyB-MT is very high, close to 90%. The hybrid clones with defective respiration coincided with those with defective growth in galactose (Fig. 1B). It is likely that hybrids with very high proportions of mutant mtDNA, close to 100%, are unable to complement and do not survive the metabolic selection, which may explain why a decrease in clone numbers is observed under these conditions (Fig. 1A).


Figure 1412
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  		Figure 2. Mitochondrial threshold for respiration and specific enzymatic activities. (A) Rates of coupled respiration as a function of the proportions of CyB-MT and COX1-MT in 14 hybrid clones, parental cybrids 100% CyB-MT and 100% COX1-MT, and WT cybrids. Note that in the tridimensional graph, respiratory rates are represented by the height of each individual pin. (B) Plot of the rate of coupled respiration as a function of the relative proportions of CyB-WT in 13 hybrid clones and the parental 100% CyB-MT. The data are fitted by a single rectangular hyperbole, two parameters, with equation Y=BmaxxX/(Kd+X); Y is the rate of respiration, Bmax=17.08, the horizontal asymptote, the maximum of respiration, X the percentage of CyB-WT and Kd=5.01 the percentage CyB-WT which corresponds to 1/2 Bmax. Respiratory chain complex III (III), complex IV (IV) and the mitochondrial matrix citrate synthase (CS) activities measured spectrophotometrically using specific substrates and inhibitors on isolated mitochondria from 16 individual hybrids clones. Values are averages of two independent mitochondria preparations each measured in duplicate. (C) Plot of III/CS activities ratio as a function of the relative proportion of CyB-MT. (D) Plot of IV/CS activities ratio as a function of the relative proportion of COX1-MT. In both (C) and (D), data are fitted by linear regression curves as shown by the relative equations and R2 values.

 
To study the correlation between mutation loads and activities of respiratory chain complexes, we measured complex IV- and complex III-specific activities using exogenous substrates. There is no threshold for these specific activities, because they both show an inverse linear correlation with the proportions of their respective mutations (Fig. 2C and D). This suggests that both mutations affect the respective complex activities by decreasing the total amount of assembled active enzymes, without decreasing their intrinsic kinetic parameters. It also excludes the possibility that CyB-MT affects complex IV activity and vice versa, because in this case, the correlations would not be linear.

Respiratory chain complex assembly in human cybrid cells
To understand the effects of the mtDNA mutations on the composition of the respiratory chain, we have investigated the assembly of its complexes by blue native gel electrophoresis. We first determined the appropriate conditions to solubilize mitochondrial membranes while preserving intact respiratory chain complexes. We tested two detergents at various concentrations in WT cybrid cells. First, increasing lauryl maltoside from 0.01 to 0.4% results in an increase of the amounts of individual complexes detectable by western blot (Fig. 3A, left lanes). At the same time, it results in a decrease of the bands migrating higher than complex I, presumably corresponding to supercomplexes. In addition, increasing the concentration of lauryl maltoside causes a faster migration of complex IV, possibly resulting from removal of ancillary proteins or conformational changes. Lauryl maltoside concentrations higher than 1% result in disassembly of complex I (data not shown). Secondly, digitonin concentrations between 0.33 and 5.0% allow for the detection of abundant high migrating bands, whereas the amounts of individual complexes are lower with lauryl maltoside. No difference is apparent among samples treated with various concentrations of digitonin (Fig. 3A). On the basis of these results, we chose to use 0.4% lauryl maltoside for the detection of individual respiratory chain complexes and 0.3% digitonin for the detection of supercomplexes.


Figure 1413
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  		Figure 3. Assembly and structural organization of the respiratory chain complexes. (A) Respiratory chain complexes and supercomplexes from WT cybrid resolved by blue native gel electrophoresis followed by western blot and detected with a mixture of monoclonal antibodies: anti-39 KDa subunit for complex I, anti-70 KDa subunit for complex II, anti-core 2 for complex III and anti-COX1 for complex IV. Different percentages of lauryl maltoside and digitonin were used for mitochondrial solubilization. A mixture of high molecular weight purified proteins was used as a molecular mass marker: ferritin dimer, 880 KDa; thyroglobulin, 669 KDa; ferritin monomer, 440 KDa; catalase 232 KDa; lactate dehydrogenase, 140 KDa; albumin, 66 KDa. (B) Mitochondrial complexes and supercomplexes from WT cybrids, solubilized with 0.33% digitonin, resolved in five individual lanes by blue native gel electrophoresis, blotted and separately probed with antibodies: anti-39 KDa subunit for complex I, anti-core 2 for complex III, anti-COX 1 for complex IV, anti-70 KDa subunit for complex II and anti-ß subunit for complex V. (C) Effect of decreasing digitonin concentrations on complex II in WT and parental mutant cybrids. Solubilization of complex II, whose assembly is unaffected by the mtDNA mutations, is similar in WT and mutant cells. I, II, III, IV and V indicate the positions of complexes I–V. III2, dimeric complex III; I+III2, complex I associated with dimeric complex III; III2+IV, dimeric complex III associated with monomeric complex IV; I+III2+IV(1–4), supercomplex containing complex I, dimeric complex III and monomeric (1), dimeric (2), trimeric (3) and tetrameric (4) complex IV. ‘Insol.’ indicates non-solubilized mitochondrial proteins.

 
In lauryl maltoside-treated samples, the apparent molecular weight of the major band detected with anti-complex III core2 antibody is ~460 KDa, suggesting that complex III is in a homodimeric form (III2). Anti-subunit I of complex IV (COX1) antibody detects a major band of ~200 KDa, indicating that complex IV is mostly resolved in its monomeric form. A higher migrating band, reacting with both core2 and COX1 antibodies, is detected at ~600 KDa. Second dimension, denaturing, electrophoresis shows that this band corresponds to a supercomplex composed of complex III and complex IV (III2+IV, not shown). Such a III2+IV supercomplex has been previously demonstrated in mammalian cells (22). The relative proportion of this III2+IV complex appears to increase with progressive solubilization with lauryl maltoside (Fig. 3A), suggesting that the interactions between the components of complex III and complex IV are strong and not completely disrupted by the solubilization.

To confirm that the high migrating bands observed in the digitonin-treated samples correspond to supercomplexes containing multimers of the various respiratory chain enzymes, the same WT cybrid sample was loaded side-by-side in four adjacent wells. After electrophoresis and blotting, each lane was cut and probed with an antibody against subunits of different complexes. As expected, using antibodies against complexes I, III and IV, at least one band migrating at a common position can be detected (band 1 in Fig. 3B), whereas complex V and complex II migrate at different positions because they are not part of the same supercomplex. In addition to the common supercomplex band, COX1 antibodies also reveal the 200 KDa monomeric complex IV plus three high migrating bands (bands 2–4 in Fig. 3B), which presumably correspond to supercomplexes containing variable numbers of complex IV, as previously reported by Schagger (7). These three higher migrating bands are better detected with the COX1 antibody than with antibodies against the other complexes, possibly because complex IV subunits are the most abundant or because epitopes for the other complexes may be partially masked in these high molecular weight structures.

In order to ensure that digitonin solubilizes mitochondrial membranes similarly in WT and mutant cells, a digitonin titration was performed in control and in 100% mutant COX1 or CyB parental cybrids. We looked at complex II by blue native western blot, because this complex is not affected directly or indirectly by either mutation. As expected, decreasing digitonin below 0.3% had similar effect on the solubility of complex II in all cell lines (Fig. 3C).

The assembly of the respiratory chain complexes is defective in hybrid cells
The amounts of assembled respiratory chain complexes in hybrid clones were estimated using blue native gel electrophoresis of mitochondrial proteins solubilized with 0.4% lauryl maltoside. Compared with WT cybrids, mutant hybrids show loss of complexes III and IV, which worsens with increasing CyB-MT and COX1-MT loads, respectively (Fig. 4A). In addition, the supercomplex III2+IV is reduced when compared with WT cells in hybrid clones containing levels of mutant mtDNA above the threshold for defective respiration (40 and 90%, for COX1-MT and CyB-MT, respectively). Interestingly, in some clones, there is a loss of complex I, which is not genetically affected by the two mutations. Complex I defect is most pronounced in the clones with the highest proportions of either COX1-MT (51%) or CyB-MT (96%). Complex II is unaffected by the mtDNA mutations and, therefore, was used to normalize the levels of the other complexes in Figure 4. We also studied the levels of assembled complexes in the parental homoplasmic mutant cybrids (Fig. 4B). In COX1-MT cybrids, there is a complete lack of complex IV and supercomplex III2+IV, whereas complex I is decreased by ~30%. In CyB-MT cybrids, there are no complexes III and III2+IV, and complex I is severely decreased by ~90%. Complex III is not decreased in COX1-MT cybrids and complex IV is not decreased in CyB-MT cybrids, suggesting that the assembly of complexes III and IV are independent from each other. Note that, in CyB-MT and COX1-MT homoplasmic cybrids, the levels of complexes IV and III, respectively, appear higher than in WT cells. This is because, unlike WT cells, the supercomplex III2+IV cannot form in mutant cybrids, and each complex is resolved in an individual band. In-gel complex I activity is reduced in the hybrid clones and in the parental mutant cybrids when compared with WT cells, consistent with the decrease of assembled complex I (Fig. 4C and D). To confirm that cells containing high levels of COX1-MT mtDNA have reduced complex I, we also studied heteroplasmic cybrid COX1-MT cells. Clones with mutation levels >90% have a clear reduction of complex I, whereas a clone with 33% mutant mtDNA shows normal complex I content (Fig. 4E).


Figure 1414
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  		Figure 4. Defective respiratory chain complex assembly in hybrid clones and parental mtDNA mutant cybrids. Mitochondrial respiratory chain complexes from (A) hybrids (B) and WT cybrid and from 100% COX1-MT and CyB-MT cybrids solubilized with 0.4% lauryl maltoside, resolved by blue native gel electrophoresis, blotted and sequentially probed with antibodies: anti-39 KDa subunit for complex I, anti-core 2 for complex III, anti-COX1 for complex IV and anti-70 KDa subunit for complex II. Relative proportions of CyB-MT and COX1-MT are indicated at the bottom for each hybrid clone. Note that the proportions of the two mutant mtDNA species do not add up to 100%, because hybrid clones contain WT molecules originating from mtDNA recombination (17). The amounts of respiratory complexes normalized by the amount of complex II (II) and expressed as percentage of WT cells as estimated by densitometry of the western blot bands are indicated below each lane. (C) Complex I in-gel activity in the same samples, as in (A). (D) Complex I in-gel activity in the same samples, as in (B). After resolving complex I by blue native gel electrophoresis, a specific NADH-dependent colorimetric assay was performed and its intensity estimated by densitometry. Complex I activity is indicated below each lane as a percentage of WT cybrid cells. (E) Blue native western blot solubilized and detected as described in (A). Complex I content is reduced in cybrids containing ≥90% COX1-MT mtDNA.

 
Recovery of supercomplex assembly correlates with recovery of mitochondrial respiratory function in hybrid cells
The proportion of mutant mtDNA correlates with the levels of their respective enzymatic activities (Fig. 2C and D). However, they are not linearly correlated with mitochondrial respiration (Fig. 2A). Therefore, we have investigated whether respiration is dependent on the presence of respiratory chain supercomplexes. Mitochondrial membranes are solubilized with 0.33% digitonin to preserve supercomplex integrity. The high molecular weight bands, corresponding to a multimeric structure composed of complexes I+III2+IV(1–4), are clearly detectable in the representative clones c and d shown in Figure 5A, which have very low amounts of individual III2 and IV. In these clones, the proportions of both mutations are below the threshold for respiratory defect (Fig. 2A). Conversely, hybrid clones a and b, in which CyB-MT mtDNA is above the threshold (i.e. >90%, Fig. 2A and B), have very reduced levels of supercomplexes I+III2+IV(1–4). As a comparison, WT cybrids show the presence of supercomplexes as well as individual complexes III2, IV and III2+IV (Fig. 5A). Homoplasmic CyB-MT cybrids with no complex III2 have a complete lack of supercomplexes, but contain individual complex IV. In contrast, hybrid clone e, which contains COX1-MT mtDNA above the respiratory defect threshold (i.e. >40%), has no individual complex IV, but a large amount of III2 (Fig. 5A), suggesting that all the available complex IV is part of a supercomplex, whereas most of complex III is in its dimeric form. In this clone, the reduced amount of complex IV available is sufficient for the formation of I+III+IV with stochiometry 1:2:1 or 1:2:2, but not of supercomplexes with complex IV stochiometry higher than 2. This is not readily apparent in the blot in Figure 5A (long exposure), but it is better demonstrated in Figure 5B, where gel running conditions are optimized to resolve the high molecular weight structures, and clone e shows a marked reduction in supercomplex content when compared with WT.


Figure 1415
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  		Figure 5. Respiratory chain supercomplexes in hybrid clones. (A) Respiratory chain complexes and supercomplexes from WT cybrids, hybrid clones, 100% CyB-MT cybrids and 91, 99 and 100% COX1-MT cybrids were solubilized with 0.33% digitonin, resolved by blue native gel electrophoresis (5–13% polyacrylamide gradient gel) and detected with a mixture of antibodies: anti-39 KDa subunit for complex I, anti-70 KDa subunit for complex II, anti-core 2 for complex III and anti-COX1 for complex IV. Relative proportions of CyB-MT and COX1-MT are indicated for each cell line. (B) Mitocondrial supercomplexes from WT cybrids, hybrid clones, 100% CyB-MT cybrids and 100% COX1-MT cybrids solubilized with 0.33% digitonin, resolved by blue native gel electrophoresis (5–8% polyacrylamide gradient gel) and detected with the antibody anti-COX 1. (C) Mitochondrial complexes and supercomplexes subunits from COX1-MT cybrids (left) and WT cybrids (right) solubilized with digitonin and resolved by first dimension blue native gel electrophoresis (as in A), followed by separation by second dimension, denaturating, gel electrophoresis of gel strips placed side by side and detection with a mixture of antibodies: anti-30 KDa subunit of complex I and anti-core 2 subunit of complex III. Note that in this experiment, complex IV is detected using anti-COX IV instead of COX1 antibodies to avoid overlap with the subunits of complexes I and III.

 
Cybrids containing very high proportions of COX1-MT (>90%) and no CyB-MT mtDNA (16) lack complex IV and have no detectable supercomplex I+III2+IV(1–4), whereas individual complex III2 and a partially formed supercomplex (I+III2) are detectable (Fig. 5A, right lanes). A second, denaturing, dimension western blot of COX1-MT cybrids confirms the composition of this partial supercomplex, because it shows the presence of subunits of complex III (core2) and I (30 KDa), but not of a subunit of complex IV (COX IV; Fig. 5C). In this second dimension, WT cells show broad bands detected by antibodies against complexes I, III and IV, in correspondence to the migration points of the multiple forms of the supercomplexes in the first dimension, confirming the presence of all three complexes in these structures.

To further investigate how respiration correlates with the amounts of individual respiratory chain complexes and with the assembly of supercomplexes, we assayed in parallel the levels of each individual complex, the presence of supercomplexes and respiration in hybrid clones containing increasing proportions of CyB-MT mtDNA. The relative amount of complex III (i.e. III2 and III2+IV) in samples solubilized with lauryl maltoside is shown in Figure 6A (top figure). We find a linear inverse correlation between the proportion of CyB-MT mtDNA and complex III content (Fig. 6B). This result was expected on the basis of the linear inverse correlation between the proportion of CyB-MT mtDNA and complex III activity shown in Figure 2C. However, the correlation between complex III content and respiration is not linear (Fig. 6C). Instead, we observe a hyperbolic curve similar to that describing the correlation between the proportion of CyB-MT mtDNA and respiration (Fig. 2B). This type of correlation curve suggests that a respiration defect occurs only when complex III content falls below ~10% of normal. In the clones tested, a decrease in respiration appears to be associated with a marked decrease of supercomplex content (Fig. 6A bottom figure). Clones with CyB-MT mtDNA <90% have normal respiration and abundant supercomplex (right lanes), whereas clones >90% have reduced respiration and very low or undetectable supercomplex (left lanes).


Figure 1416
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  		Figure 6. Supercomplex levels are restored in hybrid clones with full respiratory complementation. (A) Top figure: hybrid clones containing variable proportions of mutant mtDNA solubilized in lauryl maltoside and analyzed by blue native gel western blot as in Figure 4A. The relative amount of each complex normalized by complex II and expressed as a percentage of WT is indicated below each lane. Bottom figure: the same samples are solubilized with digitonin to detect supercomplexes. Respiration rates are indicated below each lane and expressed as a percentage of WT cells. The relative proportions of each mutant mtDNA species in the clones analyzed are shown at the bottom. (B) Complex III (i.e. the sum of III and III+IV) content is inversely correlated with the proportion of CyB-MT mtDNA. Solid diamonds represent clones studied in Figure 4A, whereas open diamonds represent clones studied in Figure 6A. (C) Correlation plot between complex III content and respiration expressed as percentages of WT cells in clones studied in Figure 6A. The data are fitted by a single rectangular hyperbole, as in Figure 2B, with Kd=1.8 and Bmax=111.7.564.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Functional complementation between mitochondria-containing pathogenic tRNA mutations has been demonstrated previously (13,14,23), although one report suggests that it may be a rare event (24). Here, we demonstrate complementation resulting from exchange of mtDNA-encoded polypeptides among mitochondria. We show that complementation of respiratory chain function occurs in hybrid clones containing a mixture of two pathogenic mutations in polypeptide-coding genes, COX1 and CyB. Metabolic selection in conditions that only allow for survival of cells with respiratory chain function (i.e. uridine and pyruvate deprivation or galactose medium) decreases the number of hybrid clones only by 25% (Fig. 1A). This indicates that complementation of mtDNA proteins is a frequent event following cell hybridization and that diffusion of molecules within fused organelles is very efficient. Rapid and efficient exchange of proteins in the matrix of fused mitochondria has been clearly demonstrated, and membrane potential is necessary for this process (25). The parental cybrids used in this study have no residual respiratory chain function. However, it is likely that they can maintain a mitochondrial membrane potential sufficient for fusion by intramitochondrial hydrolysis of glycolytic ATP (26).

Although all selected hybrid clones show restored respiratory functions, the degree of complementation is very variable (Fig. 1B–D). The level of the recovery of respiration depends on the proportions of WT COX1 and CyB genes present in each hybrid clone. The correlation between mutation load and respiration shows a clear threshold effect, which differs for the two mutations: hybrid cells are able to tolerate a much higher proportion of mutant CyB than mutant COX1 before showing a decrease in respiration [Fig. 2A and (16)]. When looking at specific enzymatic activities of individual complexes, measured with exogenous electron donors and acceptors (Vmax), activities of complexes III and IV show an inverse linear correlation with the proportions of the respective mutations (Fig. 2C and D). As expected, the content of assembled individual complexes inversely correlates with the proportion of mutant mtDNA (Fig. 6B). On the basis of these findings, we conclude that in human cells, complexes III and IV can be assembled independently from each other, as previously demonstrated in yeast (6), and that their specific enzymatic activities are correlated to the amounts of individual assembled complexes. However, this correlation does not fully explain the threshold effect observed for respiration (Figs 2B and 6C), suggesting that the amounts of individual complexes III are not rate-limiting for respiration, until they decrease below a threshold level.

The difference in the threshold for respiratory defect between CyB-MT and COX1-MT mtDNA [Fig. 2B and (16)] may be explained by a faster turnover of complex IV when compared with complex III. In addition, it is possible that the stoichiometry of the two complexes in the supermolecular structures is different, because of single complex III dimers, but multiple complex IV units appear to be contained in the supercomplex bands detected in Figure 3B.

We show a decrease in the levels of complex I in the hybrid clones containing high levels of CyB-MT or COX1-MT mtDNA (Fig. 4A and C). Complex I stability has been shown to depend on the levels of complex III in various organisms, including bacteria (27) and mammalian cells (8,28,29). Here, we confirm this finding, but we also show for the first time that complex I stability can also be affected by complex IV, in human cells, although high COX1 mutation levels are needed to observe destabilization of complex I (Fig. 4). In addition, the mutation level necessary to observe this phenomenon could vary in different cell types, because muscle cells from patients with nuclear-encoded complex IV defects, which contain only 10% of normally assembled complex IV, do not show a marked reduction of complex I (8,30). These variations could be associated with the different rates of turnover of mitochondrial respiratory chain complexes in different cell types.

It was shown that the synthesis of complex I subunits and their assembly occurs normally in complex III mutant cells, but in the absence of complex III, complex I is rapidly degraded (28). This indicates that complex III may act as a chaperone that stabilizes complex I, and on the basis of our data, complex IV may further stabilize the supercomplex. Because I+IV intermediates are never detected, the interactions between complexes I and IV are probably indirect through complex III. A supercomplex I+III2 can form in the absence of complex IV (Fig. 5A), and a stable supercomplex III2+IV is observed after solubilization with lauryl maltoside (Fig. 4A). These are likely to be supercomplex assembly intermediates, perhaps forming assembly cores for the addition of complex I and complex IV. However, it appears that fully assembled complex I is not necessary for the assembly and stability of complexes III and IV (8), although a partial defect of complex III assembly has been reported in a subset of individuals with mutations of nuclear-encoded complex I subunits (22). Nuclear-encoded assembly factors have been implicated in the formation and stabilization of individual respiratory chain complex IV (31,32); thus, it is possible that similar factors regulate the assembly of supercomplexes, but this possibility still remains to be explored.

In WT cells, individual complexes III2 and IV are detected under mild solubilization conditions with digitonin, suggesting that they are not part of a supercomplex. These individual complexes may have a function in respiration per se, and they may also act as a functional reserve. When the amounts of individual complexes decrease, due to mtDNA mutations affecting their subunits, the reserves become depleted. This has virtually no effect on respiration as long as enough supercomplex can form. Any further decrease in the amounts of complexes III or IV results in a depletion of the supercomplexes and a significant reduction in respiration. These conclusions are based on the correlation observed between the presence of supercomplexes in native western blots and respiratory function. To obtain further confirmation, it would be useful to disrupt the supercomplexes without affecting their individual components and test the effects on mitochondrial respiration. Unfortunately, this could only be achieved by knocking down a hypothetical ‘supercomplex assembly factor’, whose existence is purely speculative at this stage.

Figure 7 shows a schematic model of supercomplex assembly and stabilization in WT and mtDNA mutant cells. We propose a semi-solid-state model of the respiratory chain, where respiring supercomplexes exist in a dynamic equilibrium with randomly organized, enzymatically active, isolated complexes. Furthermore, we do not exclude that in the native state (i.e. without any detergent manipulation), other proteins may be associated with the supercomplex, forming structures, with molecular weight even higher than those detected by blue-native electrophoresis.


Figure 1417
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  		Figure 7. Model of respiratory chain complexes structure and assembly in WT and mtDNA mutant cells. (A) In WT mammalian cells, respiratory chain complexes are organized in supramolecular structure (supercomplexes) composed of monomeric complex I, dimeric complex III and mono-, di-, tri- or tetrameric complex IV. The supercomplexes coexist with a pool of partially assembled supercomplexes (III2+IV) and individual complexes (dimeric complex III and monomeric complex IV), indicating that a functional ‘solid-state’ model, like the supercomplexes, can exist in equilibrium with randomly organized, enzimatically active, isolated complexes. (B) In 100% CyB-MT cybrids, the complete loss of complex III results in disassembly and degradation of complex I; only complex IV, mostly organized in a monomeric form, remains in the mitochondrial inner membrane. (C) In 100% COX1-MT cybrids, the total loss of complex IV results in a partial decrease of complex I. The residual complex I is completely assembled with complex III; complex III dimers are unaffected by the COX1 mutation. From this model, it is clear that complex III constitutes the structural core to which complexes I and complex IV bind to form a stable supramolecular structure. Complex I, because of its instability, cannot exist as an individual complex.

 
There are several potential advantages to a supramolecular organization of the respiratory chain. For example, it is possible that such structures allow for a more efficient utilization of available substrates and cofactors than in a random collision model. It is also possible that they decrease the probability of electron escape to generate reactive oxygen species (8). In hybrid cells, complex I, the principal source of free radicals through semi-quinone (33), is always associated with complexes III and IV (Fig. 5), possibly to prevent excessive free radical production.

In conclusion, this study of COX1-MT and CyB-MT hybrid human cells provides novel clues for understanding the functional structures underlying mitochondrial respiration and the mechanisms that modulate the biochemical and clinical phenotypes in mitochondrial disorders. We suggest that the presence of supercomplexes sets the threshold for functional complementation. Therefore, the ability to form sufficient levels of supercomplexes emerges as one of the critical steps modulating the expression of mitochondrial diseases associated with mutations in the respiratory chain subunits.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture
Parental COX1-MT and CyB-MT cybrid lines were cultured in Dulbecco's Modified Eagle's medium (DMEM, Invitrogen) containing 4.5 g/l glucose, 110 mg/l pyruvate and 50 µg/ml uridine (medium A) supplemented with 10% fetal bovine serum (FBS, Cellgro).

Cell fusions and nuclear selection of hybrid cells were performed using 50% (w/v) polyethylene glycol (ATCC) and a combination of puromycin (Sigma-Aldrich) and hygromycin (Invitrogen) as described (17) in medium A.

For metabolic selection, 14 days after fusion, hybrid clones were grown in triplicate plates with mitochondrial selection media. Selection medium B contained 4.5 g/l galactose, 110 mg/l pyruvate and 50 µg/ml uridine (all from Sigma-Aldrich). Selection medium C contained 4.5 g/l glucose, no pyruvate and no uridine. Both media B and C were supplemented with 10% dialyzed FBS. One week later (21 days after fusion), the number of surviving hybrid clones was counted in triplicate plates for each of the selection conditions. In addition, a portion of the hybrid clones selected in medium C were collected by the cylinder method (18), cultured in the same medium and used for further studies.

Respiratory chain analyses
Growth of hybrid clones in galactose (medium B) was determined by plating 100x103 cells in 9x35 mm2 plastic Petri dishes. Cells were counted in triplicate daily, for three consecutive days.

Oxygen consumption was measured on intact cells, using pyruvate as substrate, with or without the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 2 µM, Sigma-Aldrich), in a thermostatic oxygraph chamber equipped with a Clark-type electrode (Hansatech) as described (16). The CyB-MT mtDNA threshold for respiration defect was obtained with a non-linear regression fitting curve, using the application program SigmaPlot (Sigmaplot Scientific Graphing Software, Version 9.01).

Enzymatic assays of respiratory chain complex activities were performed on isolated mitochondria obtained from 10 semi-confluent 150 mm dishes (~100–150x106 cells) as described (34). The activities of complex III, IV and citrate synthase were measured by spectrophotometric assays as described (34,35).

For complex I in-gel activity assay, samples were separated by blue native gel electrophoresis and incubated in 2 mM Tris–HCl, pH 7.4, 0.1 mg/ml NADH (Sigma-Aldrich), and 2.5 mg/ml nitrotetrazolium blue (NTB, Sigma-Aldrich). After overnight incubation at room temperature, the stained gel was washed in distilled water and imaged with a digital scanner.

MtDNA analyses
The relative proportions of COX1-MT and CyB-MT mtDNA species were determined in each hybrid clone by PCR-RFLP (restriction fragment length polymorphism) analysis of digested radiolabeled products, as described (17).

Blue native electrophoresis
Mitochondrial membranes were isolated from 2.5x106 cells as described (36). Various concentrations of two different detergents were tested to optimize the solubilization of mitochondrial proteins: lauryl maltoside (Sigma-Aldrich) from 0.01–0.4% (w/v), and digitonin (Sigma-Aldrich) from 0.3–5% (w/v). Ten microliters of sample was loaded on a 5–13% gradient polyacrylamide gel. Electrophoresis was performed as described (36). Transfer of proteins onto a PVDF membrane (BioRad) was carried out overnight at 30 V at 4°C. For immunodetection of protein complexes, monoclonal antibodies (Invitrogen) against the following subunits were used: 39 KDa of complex I, 70 KDa of complex II, core 2 of complex III, subunit I of complex IV and subunit ß of complex V. Native high molecular weight markers were from Amersham Biosciences.

For second dimension, gel electrophoresis, a lane excised from the first dimension native gel was first treated with denaturing buffer containing 15 mM ß-mercaptoethanol and 1% SDS for 30 min and then washed in 1% SDS for 1 h. The gel strip was then electrophoresed on a tricine–SDS–polyacrylamide gel as described (37). For immunodetection of proteins, monoclonal antibodies (Invitrogen) against the following subunits were used: 30 KDa of complex I, core 2 of complex III and subunit IV of complex IV. Quantification of respiratory chain complexes was performed by densitometric analyses of band intensities on digital images of the western blots using the Scion2 image software.


   ACKNOWLEDGEMENTS
 
We thank Dr Anatoly A. Starkov for critical reading of the manuscript. This work was supported by grants from the United Mitochondrial Disease Foundation, Muscular Dystrophy Association (GM), NIH/NINDS K02 NS047306 (GM) and Telethon Italia Fondazione ONLUS (MD).

Conflict of Interest statement. None declared.


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