(Received for publication, August 7, 1996, and in revised form, November 4, 1996)
From the Centre de Génétique Moléculaire du Centre National de la Recherche Scientifique, Laboratoire propre associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, F-91190 France and the ¶ Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
A yeast mutant (cor2-45) in which approximately half of the C terminus of core protein 2 of the cytochrome bc1 complex is lacking due to a frameshift mutation that introduces a stop at codon 197 in the COR2 gene fails to assemble the cytochrome bc1 complex and does not grow on non-fermentable carbon sources that require respiration. The loss of respiration is more severe with this frameshift mutation than with the complete deletion of the COR2 gene, suggesting deleterious effects of the truncated core 2 protein. A search for extragenic suppressors of the nuclear cor2-45 mutation resulted (in addition to the expected nuclear suppressors) in the isolation of a suppressor mutation in the mitochondrial DNA that replaces serine 223 by proline in cytochrome b.
Assembly of the cytochrome bc1 complex and the respiratory deficient phenotype of the cor2-45 mutant are restored by the proline for serine replacement in cytochrome b. Surprisingly, this amino acid replacement in cytochrome b corrects not only the phenotype resulting from the cor2-45 frameshift mutation, but it also obviates the need for core protein 2 in the cytochrome bc1 complex since it alleviates the respiratory deficiency resulting from the complete deletion of the COR2 gene. This is the first report of a homoplasmic missense point mutation of the mitochondrial DNA acting as a functional suppressor of a mutation located in a nuclear gene and the first demonstration that the supernumerary core protein 2 subunit is not essential for the electron transfer and energy transducing functions of the mitochondrial cytochrome bc1 complex.
In the mitochondrial respiratory chain, the cytochrome bc1 complex transfers electrons from ubiquinol to cytochrome c and couples this electron transfer to vectorial proton translocation across the inner mitochondrial membrane (for reviews, see Refs. 1, 2). The structure of this respiratory enzyme is best characterized for yeast, beef, and potato where it consists of 10 or 11 different polypeptide subunits, three of which (cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein) carry redox prosthetic groups, and 7 to 8 polypeptides, which lack redox prosthetic groups (core proteins 1 and 2 and five to six small proteins with molecular masses below 15 kDa). The cytochrome b subunit is encoded by the mitochondrial DNA while all the other subunits of the complex are encoded in the nucleus.
The three subunits carrying redox prosthetic groups have been studied extensively, and their roles in electron transfer and proton translocation have been elucidated. Much less is known about the subunits that do not carry redox prosthetic groups. These are often referred to as supernumerary subunits because they have no counterparts in bacteria such as Paracoccus denitrificans and Rhodospirillum rubrum, where the cytochrome bc1 complex contains only cytochrome b, cytochrome c1, and the Rieske iron-sulfur protein (3, 4). The bacterial and mitochondrial cytochrome bc1 complexes exhibit essentially identical electron transfer and proton translocating activities, suggesting that the supernumerary polypeptides of the mitochondrial enzyme are not directly involved in the energy transducing mechanism (5).
This view has received support with the discovery that the core
proteins of the Neurospora crassa and plant cytochrome
bc1 complexes are
MPPs,1 the proteases that process the
presequences of a number of nuclear encoded precursors of mitochondrial
proteins (6-10; see Refs. 11-13 for reviews). In yeast -MPP and
-MPP are water-soluble proteins located in the matrix and not
membrane bound (14-16). Although they are proteolytically inactive,
the two core proteins of the yeast cytochrome
bc1 complex show structural similarities to MPP
(17, 18). Thus, it is believed that the core proteins and MPP have a
common phylogenetic origin in an ancestral protease and that the
proteolytic activity became detached from the yeast complex after some
gene duplication occurred (6, 12).
The question which remains to be addressed is what are the functions of the proteolytically inactive core proteins of the yeast cytochrome bc1 complex? Yeast mutants with a structural deficiency in either core protein 1 or core protein 2 fail to properly assemble the cytochrome bc1 complex and, consequently, they do not grow or have extremely reduced growth rates on non-fermentable substrates (17, 18). Here we aimed to know if the respiratory capacity can be restored in such a mutant by a second mutation in another gene, thus allowing the assembly of bc1 complex despite a core protein deficiency.
We first describe a mutant in which about half of core protein 2 is lacking due to a frameshift mutation in its gene and show that the assembly of the bc1 complex is blocked in this mutant. We then show that the assembly and activity of the complex are restored by a second mutation located in the mitochondrial DNA which replaces serine 223 by proline in cytochrome b and that this single missense mutation eliminates the need for core protein 2. This is the first report of a homoplasmic missense point mutation of the mitochondrial DNA acting as a functional suppressor of a mutation located in a nuclear gene. This also unequivocally establishes that core protein 2 is not centrally involved in the electron transfer and energy transducing functions of the bc1 complex and emphasizes the question of its biological role.
The genotypes and
origins of the strains used in this study are listed in Table I.
Procedures for MnCl2 mutagenesis (19), random sporulation
and synchronous crosses (20), cytoduction (21), and induction of
rho0/rho cells (22)
were described previously.
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The mutant SM45 was selected as a respiratory deficient
mutant after MnCl2 mutagenesis of the wild-type strain CW30
and shown by crosses to be a nuclear mutation (see "Results").
Genomic DNAs were prepared from the mutant and wild-type strains (23)
and were PCR amplified with two primers flanking the COR2
gene: C1, 5-GCTCGAACGATTAGGACGGG and C2, 5
-CCTTTAGTTTTTCGTTTTGTAC
(the 5
-ends are located, respectively, at nucleotide positions
58 and +1141 from the first base of the initiator AUG codon; see Ref. 18).
PCR was performed from 100 ng of genomic DNA with 25 pmol of each
primer in 50 µl of each 200 µM dNTP, 50 mM
KCl, 10 mM Tris/HCl, pH 8.8, 1.5 mM
MgCl2, and 0.1% Triton X-100, containing 2.5 units of
Taq polymerase (Bioprobe), at 95 °C for 3 min, 55 °C for 30 s, and 72 °C for 1.5 min (for 1 cycle), at 95 °C for
30 s, 55 °C for 45 s, and 72 °C for 1.5 min (for 28 cycles), and at 95 °C for 30 s, 55 °C for 30 s, and
72 °C for 15 min (for 1 cycle). The PCR products were sequenced
twice from separately made PCR products using a primer extension
procedure developed by E. Coissac2 with
primers C1 and C2 and three primers located between C1 and C2: S1,
5
-GCTTTGAAATTAGTCAGAG (+189); S2, 5
-CCGAAGACCAATTGTATGCC (+424); and
S3, 5
-GGTGAAGAAAACAGGG (+675).
Twenty-two independent subclones of the mutant SM45 were grown until stationary phase in 10% glucose, 1% yeast extract, 1% bactopeptone, and 20 mg/liter adenine. About 5 × 108 cells of each culture were plated on 2% glycerol, 1% yeast extract, 1% bactopeptone, and 2% agar. Eleven subclones gave at least one colony after 1 week of incubation at 28 °C. Twenty-two isolates (2 per original subclone) corresponding to at least 11 independent reversion events were retained for further analysis. The revertants were treated with ethidium bromide to eliminate their mitochondrial DNA (rho0) and then back-crossed to the original mutant (strain CB10-2a). With one revertant, called SM45/CB2, all the resulting diploid cells were respiratory deficient, indicating that this revertant was either nuclear recessive or mitochondrial.
The 21 other revertants gave respiratory competent diploids and were
thus nuclear dominant. These were not further analyzed. Two additional
test-crosses proved that the revertant SM45/CB2 was the result of a
mutation located in the mtDNA. First, the original mutant (strain
CB10-2a) was treated with ethidium bromide to eliminate its
mitochondrial DNA (rho0) and then crossed to the
revertant SM45/CB2; the resulting diploid progeny was composed entirely
(barring the spontaneous rho cells occurring
in all yeast cultures) by respiratory competent cells (it would have
been composed of only respiratory deficient cells if the suppressor was
nuclear recessive). Second, the revertant SM45/CB2 was crossed
synchronously to the original mutant (strain CB10-2a) and after
segregation of mitochondrial heteroplasmons, about 50% of the
resulting diploid cells were respiratory competent (mitotic segregation
of respiratory competence would not have been observed if the
suppressor was nuclear recessive).
The suppressor was localized on mtDNA using a previously described
procedure (24). To this end the revertant SM45/CB2 was treated 15 h at 28 °C in 0.4% NaCl containing 10 mg/liter ethidium bromide,
and the resulting rho cells were tested for
the retention or loss of the suppressor mutation by crossing with the
original mutant (strain CB10-2a). Forty rho
clones having retained the suppressor mutation were then analyzed for
the retention or loss of the wild-type alleles of four different mit
mutations previously located on mtDNA (25,
26). As summarized in Table II, the rho
tested
were all able to rescue mit
mutations located
in the cytochrome b gene, whereas only a minor fraction of
them were able to rescue mit
mutations located
in the genes COXI and COXII, indicating that the
suppressor mutation is located in the cytochrome b gene.
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Optical spectra of cytochromes were recorded at liquid nitrogen temperature with a Cary 128 spectrophotometer after reduction of the cytochromes by dithionite of whole cells grown for 1 day at 28 °C on 2% glucose, 1% yeast extract, 1% bactopeptone, and 2% agar (27).
Sequence Determination of the SM45/CB2 RevertantThe cytochrome b mRNA was sequenced by primer extension with avian myeloblastosis virus reverse transcriptase (Amersham Corp.) from total mitochondrial RNA of the SM45/CB2 revertant (28).
Enzyme Assays and Western AnalysisMitochondrial membranes
were isolated using a procedure described previously (29). Yeast were
grown overnight on 2% glucose, 1% yeast extract, 1% bactopeptone,
and 2% agar. The cells were harvested by centrifugation at 4,000 × g for 10 min. They were then washed once with distilled
water and resuspended in 400 mM mannitol, 50 mM
Tris, pH 7.6, and 2 mM EDTA. The yeast were disrupted using
glass beads in five 1-min intervals using a Biospec Products Bead-Beater. The cell extract was diluted with 1 volume of ice-cold Tris/mannitol buffer and centrifuged for 10 min at 1,000 × g at 4 °C to remove intact cells. The supernatant was
saved and recentrifuged at 1,000 × g for 10 min.
Mitochondrial membranes were collected by centrifugation of the
supernatant at 16,000 × g for 20 min. Membranes were
washed twice with 150 mM NaCl and recentrifuged. Final
membrane pellets were resuspended in 50 mM Tris, 1 mM MgSO4, pH 7.6, containing 50% glycerol and
stored at 20 °C. Protein concentrations were determined by the
method of Lowry et al. (50) as modified by Markwell et
al. (51).
Ubiquinol-cytochrome c oxidoreductase activity was assayed
at pH 7.0 and 23 °C using the substrate analog
2,3-dimethoxy-5-methyl-6-n-nonyl-1,4-benzoquinol (52). The
nonenzymatic rate of cytochrome c reduction was determined by the addition of quinol to the assay buffer and allowing the reaction
to proceed for approximately 5 s. The enzymatic rate was then
initiated by adding the membrane fraction to the assay buffer. Rates
were obtained in duplicate and varied less than 10%. The amounts of
proteins used in the enzyme assays are given in the legend of Fig.
2.
Immunoblot analysis on the mitochondrial membranes prepared as
described above was performed with polyclonal antibodies raised against
purified cytochrome bc1 complex as described
previously (29). The amounts of protein used in the immunoblot analysis are given in the legends of Fig. 4.
The mutant CW30/SM45
(abbreviated SM45; Table I) exhibits a
substrate-conditional phenotype commonly observed in strains with
respiratory defective mitochondria. It grows on glucose but cannot grow
on non-fermentable carbon sources such as glycerol, ethanol, or
lactate, the metabolism of which needs functional mitochondria (Fig.
1). The phenotype of this mutant was complemented by
crossing with a rho0 tester strain
(KL14-4A/60), indicating that the respiratory defect is caused by a
nuclear recessive mutation and not by a deficiency of mitochondrial
DNA.
The mutant SM45 is devoid of dithionite-reducible cytochrome
b while the amounts of cytochromes a and
a3 are not markedly modified, indicating a
specific defect at the level of the cytochrome bc1 complex (Fig. 2). A
complementing DNA fragment was selected by transformation of the mutant
SM45 with a plasmid library of yeast nuclear DNA. The restriction
enzyme digestion pattern of the fragment suggested the presence of the
COR2 gene (17), and this was confirmed by complementation of
a strain carrying a null allele in core protein 2 (strain
HR2::40K0). Finally, sequence determination of
the chromosomal COR2 locus in SM45 showed the deletion of 1 base pair at codon 165 in comparison with the wild-type COR2
gene. As a result of this mutation, the last 204 residues of wild-type
core protein 2 are replaced by 32 novel residues as indicated in Fig.
3. This mutation will be referred to as
cor2-45.
Assembly of the Cytochrome bc1 Complex Is Blocked at an Early Step by the Nuclear Mutation cor2-45
In order to better understand how the cor2-45 mutation affects assembly of the cytochrome bc1 complex, we used antibodies that recognize core proteins 1 and 2 (29) to test for the presence of these subunits in the mutant. As shown in Fig. 4, these antibodies also recognize a third protein that migrates ahead of core protein 2 and is distinct therefrom. The identity of this protein is not known. It is unlikely to be a subunit of the MPP protease, analogous to the protease subunit in the potato cytochrome bc1 complex (30), since the yeast MPP subunits have apparent molecular masses of 48 and 51 kDa, and the unknown bc1 protein migrates with an apparent molecular mass of less than 40 kDa. The unknown protein is not derived from the COR2 gene since it is not affected by the loss of core 2 protein (Fig. 4, lane 2), and it is present when the COR2 gene is deleted (Fig. 4, lane 5). Although it is possible that this is a spurious impurity, this seems unlikely since a protein of identical size is present in invariant amounts in highly purified preparations of yeast bc1 complex (see Fig. 11 in Ref. 1). Further experimentation is in progress to establish the identity and possible function of this bc1-associated protein.
As expected, no immunological response corresponding to core protein 2 was detected in the mutant carrying the cor2-45 mutation (Fig. 4, lane 2). We did not observe any signal that could correspond to the mutated protein. A possible explanation is that the antibodies react with the C-terminal half of core protein 2, which is lacking in the mutant, or that the mutated protein is more prone to proteolytic degradation.
As shown previously (18) (see also Figs. 1 and 2), a mutant carrying a null allele in the core protein 2 gene grows slowly on non-fermentable substrates and exhibits a low level of cytochrome c reductase activity. Surprisingly, the growth on non-fermentable substrates as well as the cytochrome c reductase activity are virtually abolished by the cor2-45 mutation (Figs. 1 and 2). Moreover, only traces of core protein 1 are present in cells carrying the cor2-45 mutation (Fig. 4, lane 2), whereas core protein 1 is at a nearly normal level in the core 2 deletion strain (Fig. 4, lane 5).
From these results, it is clear that the cor2-45 frameshift mutation has more severe effects than a deletion allele in the COR2 gene. As shown in Fig. 3, the cor2-45 mutation presumably leads to the synthesis of a truncated protein with a net charge of +15 versus +1 for the wild-type protein. This drastic charge and size modification could be responsible for the toxic effects of cor2-45, for instance, by allowing potentially harmful protein-protein interactions between the modified core protein 2 and other subunits of the cytochrome bc1 complex. This led us, as described in the next section, to search for extragenic suppressor mutations, in order to see if the effects of the cor2-45 mutation can be alleviated by a second mutation in another subunit of the complex.
A Mutation of the Mitochondrial DNA Replacing Serine 223 by Proline in Cytochrome b Restores Assembly and Activity of the Cytochrome bc1 Complex in the Nuclear Mutant Deficient for Core Protein 2Only 1 revertant (SM45/CB2) out of 22 isolates was due
to a mutation in mtDNA. The mutation was mapped by
rho deletion mapping to the mitochondrial
cytochrome b gene (Table II) and sequenced.
The suppressor mutation leads to replacement of the serine codon 223 (TCA) by a proline codon (CCA).
The revertant SM45/CB2 grows on glycerol medium (Fig. 1), exhibits ubiquinol-cytochrome c reductase activity of at least 60% of that of the wild type (Fig. 2, right panel), and spectrally detectable cytochrome b is partially restored (Fig. 2, left panel). In addition, core protein 1, which is dramatically decreased by the cor2-45 mutation, is present at a nearly normal level in the revertant (Fig. 4, lane 3).
We then tested whether the S223P mutation in cytochrome b can also alleviate the effects seen with a null allele in COR2, which encodes core protein 2. To this end, we constructed by cytoduction a yeast strain having the nucleus of the COR2 deletion strain HR2::40K0 and the mtDNA of the revertant SM45/CB2 (Table I). Analysis of this strain (CHR2::40K0) clearly showed that the null allele in COR2 becomes less deleterious in the presence of cytochrome b containing the S223P mutation, as indicated by a markedly faster growth rate on glycerol medium (Fig. 1), a substantially higher content of spectrally detectable cytochrome b (Fig. 2), and a three- to four-fold increase in the ubiquinol-cytochrome c reductase activity (Fig. 2).
As a control, we tested whether the S223P mutation in cytochrome b gives a phenotype when associated with the wild-type core protein 2. To this end, we constructed a strain having the nucleus of the wild-type strain CW30 and the mtDNA of the revertant SM45/CB2 (Table I). This strain (CCW30) exhibits normal cytochrome b content and grows normally on glycerol medium (Fig. 2), indicating that the S223P mutation in cytochrome b by itself has no deleterious effects on the cytochrome bc1 complex.
In the studies reported here, we first describe a frameshift mutation (cor2-45) in the nuclear COR2 gene by which the last 204 residues of core protein 2 are replaced by 32 novel residues and show that this mutation severely impairs assembly of the cytochrome bc1 complex. We then show that assembly of an active bc1 complex is restored by a single base pair transition in mitochondrial DNA, which leads to replacement of serine 223 by proline in cytochrome b. This is the first known example of a homoplasmic missense point mutation of the mitochondrial DNA acting as a functional suppressor of a mutation located in a nuclear gene.
Replacement of serine 223 by proline in cytochrome b not only corrects the cytochrome bc1 complex deficiency resulting from the original frameshift mutation, but it also obviates the need for the core 2 protein since it alleviates the respiratory deficiency resulting from its complete deletion. It should be noted that the frameshift mutation strain is more responsive to the suppression by the cytochrome b mutation than is the cor2 deletion strain (60% and 41%, respectively, in terms of activity relative to the wild type are recovered by the S223P replacement). It is possible that the remaining part of core protein 2 in cor2-45 (about half of the wild-type protein) helps the assembly of or stabilize the bc1 complex, which could explain the observed differences in terms of activity and growth rates of the corresponding strains. These unexpected results illustrate unequivocally that the core 2 subunit of the mitochondrial cytochrome bc1 complex is not essential for the electron transfer and energy transducing functions of this respiratory enzyme, which is consistent with the finding that cytochrome bc1 complexes from bacteria do not contain such proteins (3, 4).
Core proteins 1 and 2 are the two largest subunits, contributing about
half of the protein mass of the bc1 complex.
They are proximal to each other (31), lack any obvious
membrane-anchoring domains (17, 18), and extend from the phospholipid
bilayer into the matrix phase (32). Cytochrome b is a highly
hydrophobic protein with eight -helical segments spanning the inner
mitochondrial membrane, as deduced from hydropathy calculations (33,
34), mutant studies (35-37), and phoA fusion experiments
(38). In the eight-helix cytochrome b models, serine 223 is
located in a matrix-exposed loop that connects transmembrane helices D
and E, as shown in Fig. 5. Thus, the suppressor mutation
affects a subdomain of cytochrome b that may be in contact
with the core proteins.
In yeast, a null mutation in core protein 1 does not significantly affect the steady-state concentration of core 2 (39); similarly, a null mutation in core protein 2 does not markedly change that of core protein 1 (Fig. 4) (18, 39). However, a deficiency in either core protein 1 or 2 results in a drastic decrease in immunodetectable cytochrome b content, whereas mutants lacking cytochrome b have normal core protein contents. Since mRNA levels and expression of cytochrome b in pulse-label experiments are not affected by a core protein deficiency, it is thought that the decrease in the steady-state level of cytochrome b results from an increased susceptibility of cytochrome b to proteolytic degradation when the core proteins are absent (39).
We have shown that a modified core protein 2 can elicit the loss of core protein 1. The C-terminal extension created by the frameshift cor2-45 mutation, which contains many basic residues (Fig. 3), could block the proper folding of core 1, thus making this subunit sensitive to proteolytic degradation. If the core proteins bind to cytochrome b, as suggested by the loss of the cytochrome in core protein-deficient yeast strains, the restoration of core protein 1 by the revertant could simply be due to a conformational change in cytochrome b that strengthens its interaction with core protein 1 and thus protects against any deleterious interaction with the mutated core protein 2.
More important, however, is that a single mutation, S223P, in one of
its matrix-exposed connecting loops makes the cytochrome b
resistant to a core protein 2 deficiency. In N. crassa and
plant mitochondria, the core proteins of the cytochrome
bc1 complex are proteolytically active (6, 7).
In yeast, the core proteins located in the cytochrome
bc1 complex appear to be proteolytically inactive homologues of -MPP and
-MPP (6, 12), which are localized
in the matrix (14-16).
From this relationship and the peripheral location of the core proteins at the matrix side of the bc1 complex, we speculate that the proteolytically inactive core proteins bind to the matrix-exposed loop of cytochrome b and thus protect it from proteolysis by a mitochondrial protease. The S223P mutation may render the matrix-exposed loop of the cytochrome intrinsically resistant to mitochondrial protease, thus obviating the requirement for the core proteins. Notably, the cytochromes b in N. crassa and plant mitochondria, in which the core proteins have been shown to be proteolytically active, and likewise those of most eukaryotic mitochondria except yeast, contain a proline at the position equivalent to serine 223 of yeast (35). Furthermore, a single serine to proline mutation proximal to the MPP cleavage site in the presequence of the iron-sulfur protein of the yeast cytochrome bc1 complex blocks proteolysis by MPP.3
Given the complex evolutionary relationships between the core proteins and MPP and the genetic data presented in this study, it will be particularly interesting to see if, in yeast, the core proteins function to protect the cytochrome b against mitochondrial proteases. Further experiments to test this hypothesis are in progress.
We are grateful to E. Petrochillo for stimulating and helpful discussions. The mutant CW30/45 was isolated by S. Robineau who kindly gave it to us for characterization.