A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex

Energy transduction in mitochondria involves five oligomeric complexes embedded within the inner membrane. They are composed of catalytic and non-catalytic subunits, the role of these latter proteins often being difficult to assign. One of these complexes, the bc1 complex, is composed of three catalytic subunits including cytochrome b and seven or eight non-catalytic subunits. Recently, several mutations in the human cytochrome b gene have been linked to various diseases. We have studied in detail the effects of a cardiomyopathy generating mutation G252D in yeast. This mutation disturbs the biogenesis of the bc1 complex at 36°C and decreases the steady-state level of the non-catalytic subunit Qcr9p. In addition, the G252D mutation and the deletion of QCR9 show synergetic defects that can be partially bypassed by suppressor mutations at position 252 and by a new cytochrome b mutation P174T . Altogether, our results suggest that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between the catalytic subunits, this role being essential at high temperature.


Introduction
Numerous cellular functions are performed by oligomeric complexes composed of catalytic and non-catalytic subunits and the role of these latter proteins is often difficult to assign. Energy transduction in mitochondria involves five oligomeric complexes that are embedded within the inner membrane. One of these complexes, the bc1 complex is composed of three catalytic and seven or eight non-catalytic subunits (for review,1). The three catalytic subunits, cytochrome b, cytochrome c1 and the Rieske iron-sulfur protein (Rieske) are conserved in the bacterial equivalents of this complex. However, several non-catalytic subunits are present in the mitochondrial bc1 complexes and are often referred to as supernumerary subunits although some are required for the enzymatic activity. In by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Interaction between cytochrome b and Qcr9p.
3 Saccharomyces cerevisiae the bc1 complex contains seven supernumerary subunits that are conserved in the mammalian enzyme. The mammalian bc1 complex presents an additional subunit that has no equivalent in yeast. The mitochondrial bc1 complexes from bovine, chicken and yeast have been crystallized (2,3,4,5) and an analysis of these structures reveals that the general shape, size and topology of the complexes are similar in the three organisms.
The mitochondrial respiratory chain is required for electron transport and oxidative phosphorylation. The bc1 complex transfers electrons from ubiquinol to cytochrome c and translocates protons into the inter-membrane space, the resulting electrochemical gradient is utilized by the ATP synthase to produce ATP in the mitochondrial matrix. The bc1 complex exhibits two site of ubiquinol binding, the Qo site in the inter-membrane side and Qi site in the matrix side. The oxidation of ubiquinol at the Qo site releases two electrons; one is transferred to Rieske, then to cytochrome c1 and finally to cytochrome c. The second electron is transferred to the low potential heme b L , then to the high potential heme b H and to ubiquinone at the Qi site. Genetic analyses in yeast have shown that the absence of a catalytic subunit always leads to a complete block of electron transfer, while the absence of noncatalytic subunits causes various defects in bc1 complex activity (for review, 6). For example, the absence of the supernumerary subunits Qcr7p or Qcr8p (7,8) leads to a complete respiratory deficiency at any temperature while Qcr6p or Qcr9p appears to be essential only at 36°C (9,10). The precise role of these supernumerary subunits and the nature of the functional interactions existing between them and the catalytic subunits still remain to be elucidated.
Cytochrome b plays a crucial role in the activity of the bc1 complex since it harbours the two b hemes and participates in determining the shape of the two ubiquinone fixation sites. This integral membrane protein containing eight trans-membrane segments is the only bc1 subunit encoded by the mitochondrial genome. The sequence alignment between yeast by guest on March 24, 2020 http://www.jbc.org/ Downloaded from Interaction between cytochrome b and Qcr9p.
4 and bovine cytochrome b shows 51% identity and present also a striking structural conservation between the cytochrome b of the two organisms (5).
Recently several mutations in the human cytochrome b gene have been linked to diseases such as cardiomyopathy, exercise intolerance or Leber's Hereditary Optic Neuropathy (for review, 11 and references therein, 12,13,14,15). In particular, one mutation that substitutes a glycine by an aspartate residue at position 251 of cytochrome b (G251D) is associated with a histiocytoid cardiomyopathy (16,17). This mutation is located in the intermembrane space loop connecting the fifth and sixth trans-membrane segments. Analyses on mitochondria purified from patient heart have established a defect in the succinate cytochrome c oxido-reductase activity and in cytochrome b assembly, showing the importance of residue 251 for bc1 function.
The fact that S. cerevisiae is a facultative aerobe, considerably facilitates the study of respiratory deficient mutants. Moreover, the rapid segregation of mitochondrial chromosomes in yeast permits the generation of homoplasmic cells containing only the mutated mitochondrial DNA. On the contrary, wild type and mutated mitochondrial DNAs often coexist within the cells of patients carrying a mitochondrial pathology. Thus, yeast mitochondrial mutants can provide invaluable help to fully understand the consequences of a mitochondrial mutation observed in a patient.
In this paper, we have studied in detail the effects of the histiocytoid cardiomyopathy corresponding mutation in yeast (G252D). This mutation strongly disturbs the respiratory functions of cells grown at high temperature (36°C). We have found that the presence of the aspartate residue at position 252 renders the non-catalytic subunit Qcr9p essential for the activity of the bc1 complex at 28°C and that this defect can be bypassed by a second mutation in the cytochrome b gene (P174T) located in a region close to Rieske. We propose that the supernumerary subunit Qcr9p enhances or stabilizes the interactions between cytochromes b, c1 and Rieske, particularly at high temperature.

Strains, media, genetic methods
All strains have the same nuclear background ade2-1 ura3-1 his3-11,-15 trp1-1 leu2-3,-112 can1-100. The construction of the intron-less mitochondrial genome was described in 18 and that of the ∆qcr9 strain in 19. Yeast genetic methods were described in 20 and 21. Nonfermentable ethanol/glycerol medium is 1% yeast extract, 1% casamino acids, 0,05 M sodium potassium phosphate (pH6.25), 3% ethanol, 3% glycerol. Cytoductions experiments were performed by using intermediate strains with the kar1-1 mutation that delays nuclear fusion (22). Rho° derivatives devoid of mitochondrial genome were constructed by growing the cells in complete medium containing ethidium bromide (40 µg/ml). The rho° derivative recipient and the donor strain cells were mixed on complete liquid glucose medium and shaken for 2.5 hours at 28°C. The mixture of cells was harvested by centrifugation and incubated for one hour at 28°C without shaking. Then, the cells were resuspended and shaken for 2.5 hours at 28°C, diluted and plated onto media selective for cytoductants containing the recipient strain nuclear genome and the donor strain mitochondrial genome.

Construction of the intron-less cytochrome b gene with a glycine codon at position 252.
The CYTB gene was site corrected by a two step PCR strategy, using DNA extracted from the strain CW30 carrying the intron-less mitochondrial genome with the G252D mutation as template. The corrected fragment was cloned at the HincII site of the pJM2 plasmid containing the wild type COX2 gene (23). The resulting plasmid (pYS10) was introduced into mitochondria from the rho° strain DFS160 by biolistic transformation (24). 6 mitochondrial genome of the YST1 strain (∆qcr9 G252D) to give the strain YST2 (∆qcr9 252G). The corrected intron-less mitochondrial genome was also introduced by cytoduction into the rho° derivatives of CW30 and YST6, a G252D strain expressing the c-myc-tagged Qcr9p (19) to give strains CW252 (252G) and YST7 (252G strain expressing Qcr9p-c-myc), respectively. The CYTB sequences of all these strains were verified.

Isolation and genetic characterization of the revertants.
The ∆qcr9 G252D strain (YST1) was subcloned and 10 respiratory deficient subclones were grown in complete liquid medium. 100 µl of each stationary phase cultures were plated on non-fermentable medium supplemented with 0.1% glucose and incubated at 28°C for 1 week.
Nine independent revertants (each from a different liquid cultures) were isolated and called QR1 to QR9. The genetic nature of the suppressor mutations present in the revertants was established according to 21. The revertants and their rho° derivatives were crossed to a ∆qcr9 G252D strain of opposite mating type and the resulting diploids were tested for their ability to grow on glycerol medium at 28°C. Whereas all the diploids resulting from crosses with the rho + revertants were respiratory competent, all diploids resulting from crosses with rho° derivatives of the revertants were respiratory deficient, showing that the suppressor mutations had been lost in the rho° derivatives and thus were located on the mitochondrial genome. The mitochondrial genomes of the revertants QR1, QR2 and QR3 were introduced by cytoduction in the rho° derivative of CW30 to give WQR1, WQR2 and WQR3 respectively.

Isolation of mitochondria and respiratory chain activities.
Mitochondria were purified from cells grown in complete galactose medium (25). The activities of the different respiratory complexes were measured from purified mitochondria at 28°C or 34°C. Ubiquinol cytochrome c oxido-reductase activity was measured as in (26).
Antimycin sensitive NADH cytochrome c oxido-reductase activity was detailed in 27,

The yeast cytochrome b gene encoded by the intron-less mitochondrial genome carries a G252D mutation
Saccharomyces cerevisiae mitochondrial genome contains up to thirteen introns, but an intron-less mitochondrial genome has been constructed (18). We have sequenced the yeast cytochrome b gene encoded by this genome and shown that there is a transition at position 755 of the open reading frame. By comparison to the reference sequence of the yeast introncontaining mitochondrial genome (28), this mutation changes a GGT codon to GAT and leads to the substitution of a glycine by an aspartate at position 252 of the apocytochrome b ORF (G252D). This glycine is conserved in several bacterial cytochromes b (e. g. Rhodobacter capsulatus) as well as in higher eukaryotes such as mammals ( Figure 1). In humans, it has been shown that, the substitution of the homologous glycine 251 by an aspartate leads to an hystiocytoid cardiomyopathy (17). This demonstrates the critical importance of this residue for respiratory function. 8

The G252D mutation affects the biogenesis of the bc1 complex at high temperature.
Using biolistic transformation, we have corrected the G252D mutation by introducing a glycine codon at position 252 of cytochrome b in the intron-less mitochondrial genome and we have compared the respiratory functions of two isonuclear strains that differ only at codon 252 of the cytochrome b gene.
As shown in Figure 2, both strains grow on non-fermentable medium at 28°C (strains 1 and 2), but the doubling time of the G252D mutant was slightly increased compared to the wild type strain. The respiratory growth of the mutant was severely affected at 36°C, leading to a substantially increased doubling time (5h20 versus 2h40). Mitochondria were purified from cells grown at 28°C or 34°C and the respiratory complex activities were measured at 28 and 36°C for each preparation of mitochondria. No differences in the enzymatic activities were ever observed between mutant and wild type mitochondria purified from cells grown at 28°C (data not shown). However, when mitochondria were purified from cells grown at high temperature, the antimycin-sensitive NADH-cytochrome c oxido-reductase (NADH-cytc) and the ubiquinol-cytochrome c oxido-reductase (bc1) activities were diminished in the mutant by approximately 50% of the corresponding wild type level (Table I). This decrease was independent of the temperature used for the activity measurements. The cytochrome c oxidase activity (Cox) is not affected in the mutant. These results suggest that the respiratory growth defect of the mutant at high temperature is due to a deficiency in the bc1 complex biogenesis rather than activity.
A priori, this could be simply due to a defect in the assembly of the catalytic subunits at 36°C. Since an assembly defect generally leads to the degradation of unassembled subunits, In conclusion, the yeast mutant G252D grown at high temperature, is affected in the biogenesis of the bc1 complex leading to a substantial decrease of the enzymatic activity.
However, the accumulation of the three catalytic subunits is not significantly modified.

Interaction between residue 252 of cytochrome b and the supernumerary subunit Qcr9p.
In addition to the three catalytic subunits, the yeast bc1 complex contains seven other subunits apparently not directly involved in the energy transducing mechanism (for review, 1). The 7.3 kDa subunit Qcr9p is one of these supernumerary subunits. The deletion mutant ∆qcr9 exhibits a temperature-dependent respiratory growth (9, 29 and Figure 2, strain 3). The similarity between the respiratory phenotypes of both G252D and ∆qcr9 mutants have led us to measure the accumulation of Qcr9p in presence of the glycine or aspartate residue at position 252 of cytochrome b. As no anti-Qcr9p antibody was available, we have used a Qcr9p tagged at its C-terminus with c-myc epitopes. Whether they carried the wild type or G252D variants of cytochrome b, the QCR9-c-myc strains were both able to grow on a nonfermentable substrate. However, Western blot analysis of mitochondrial proteins showed that the steady-state level of Qcr9p-c-myc was lower in the G252D mutant than in the wild type strain whatever the temperature is ( Figure 4 and not shown). To test if this decreased level of Qcr9p was responsible for the phenotype of the G252D mutant at 36°C, we have over- Altogether, these results suggest either a direct or a long-range interaction between Qcr9p and the residue 252 of cytochrome b.

G252D double mutant
We have purified mitochondria from the ∆qcr9 G252D double mutant and both single mutants and measured the activities of the respiratory complexes at 28°C. Only complex bc1 activity was affected in the mutants. Table II shows that the antimycin-sensitive, NADH cytochrome c oxido-reductase activity at 28°C was wild type in the G252D mutant, decreased by about 30% in the ∆qcr9 mutant and undetectable in the double mutant. This synergetic defect suggests the existence of a genetic interaction between the two mutants. The ubiquinol cytochrome c oxido-reductase activity (data not shown) was also undetectable in the ∆qcr9 In order to determine if this lack of bc1 activity was due to an assembly defect, we measured the amounts of the catalytic bc1 subunits in 28°C grown cells. Cytochrome b and c1 were detected by recording cytochrome spectra after full reduction by dithionite. As shown in Figure 3A, there was only a slight decrease in the amounts of cytochrome b and c1 in the ∆qcr9 strains and the G252D mutation had no additional effect. Immuno-detection experiments, revealed a threefold decrease of the steady-state level of Rieske in the ∆qcr9 strains, as expected from 9. However, the decrease due to the ∆qcr9 mutation was not dramatically modified by the G252D mutation ( Figure 3B and legend). Moreover, and as expected from cytochrome spectra, the accumulation of apo-cytochrome c1 (Cyt1p) was not modified. Altogether, these results suggested that the bc1 activity defect observed at 28°C in the double mutant was not due to a defect in the accumulation of catalytic subunits.
To determine which step of the electron transfer was blocked in the mutants, the endogenous reduction of the respiratory chain cytochromes was investigated by recording the cytochrome absorption spectra without exogenous reducing agent ( Figure 3C Table II, the doubling time of the revertant strains in non-fermentable medium varied from five to six hours at 28°C, the P174T mutation being the least efficient suppressor mutation. All revertants failed to grow on non-fermentable medium at 36°C (Figure 2).
The bc1 complex activity and assembly were analysed at 28°C in the three classes of revertant strains. The antimycin-sensitive, NADH cytochrome c oxydo-reductase activity was partially restored in the three revertants, the bc1 activity in the D252N suppressor strain is restored almost to ∆qcr9 single mutant level (Table II). As shown in Figure 5 In order to examine the respiratory phenotype of the three suppressor mutations in presence of Qcr9p, we have introduced the mitochondrial genomes of the revertants in a wild type QCR9 context by cytoduction. The D252N and D252Y suppressor mutations had no effect on the respiratory phenotype as assessed by cell doubling time, assembly or activity of the bc1 complex compared to the wild type at 28° or 36°C (Table II; Figure 5A, trace 8 and data not shown). The P174T G252D strain which carries two substitutions in cytochrome b, has a slightly longer doubling time, this phenotype being nearly undetectable on plates, and no significant difference in the bc1 complex activity was detected when compared to the wild type (Table II) Qcr9p. Although, the P174T mutation is located in the intermembrane space domain of cytochrome b like the G252D mutation, the two mutations appear rather distant according to the crystal structure (5). This speaks in favour of a long range interaction between these two residues of cytochrome b.

Discussion
In this study, we have shown that the intron-less mitochondrial chromosome from the yeast S. cerevisiae carries a mutation leading to the substitution of a glycine residue by an aspartate at position 252 of cytochrome b. The same mutation has been recently described in a patient with a cardiomyopathy (17) associated with a defect in succinate cytochrome c oxido-reductase activity (16). This G252D mutation has no major effect on respiratory function of yeast cells grown at 28°C, the appropriate growth temperature of yeast but leads to a 50% decrease of the bc1 complex activity at 36°C, which is close to the temperature of the human body.
A growing number of mutations in the human mitochondrial DNA were shown to be responsible for numerous pathologies (for review, 30). Among a dozen mutations mapped to the human cytochrome b gene, only two have been investigated in the yeast system (11). The G34S mutation has been observed in a patient suffering from exercise intolerance (31) and the substitution of the corresponding glycine by aspartate in yeast leads to a total defect of the bc1 complex activity at 28°C (32). The G339E mutation is responsible for a human myopathy and the same mutation totally abolishes the bc1 complex assembly in yeast at 28°C (33,343).
Thus, G252D is the first cytochrome b mutation corresponding to a human pathology that leads to a thermosensitive bc1 activity. This stresses the interest of testing different growth temperatures when using yeast as a model to study mutations implicated in human pathology. Our results suggest the existence of an interaction between cytochrome b residue 252 and Qcr9p, the mutation G252D leading to a decrease in the steady-state level of Qcr9p. However, according to (5), the residue 252 is far from Qcr9p and cannot directly interact with Qcr9p while cytochrome c1 is close to the residue 252 ( Figure 6). Thus, it is tempting to propose that the interaction between the residue 252 and Qcr9p would be a long range interaction via cytochrome c1. Since, the closest amino acid in cytochrome c1 is the lysine 182, the G252D mutation could create an illegitimate electrostatic interaction with this positively-charged amino acid. This illegitimate interaction would affect cytochrome c1 conformation and partially destabilize Qcr9p or affect the insertion of Qcr9p within the membrane. This would lead to a decrease of the bc1 complex activity at high temperature, as in the ∆qcr9 mutant.
Previous studies have shown that Qcr9p interacts with Rieske and cytochrome c1 (5,9,35), therefore we propose that Qcr9p could enhance or stabilize the interactions between these two catalytic subunits. It was previously shown that in the total absence of Qcr9p, the conformation of Rieske and its iron-sulfur cluster insertion is altered (9). Thus, in the ∆qcr9          ∆qcr9 CYTB-G252D -0 5