From the Department of Biological Sciences, Columbia University, New York, New York, 10027
Received for publication, December 6, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cytochrome c oxidase (COX), the
terminal enzyme of the mitochondrial respiratory chain, catalyzes the
transfer of electrons from reduced cytochrome c to
molecular oxygen. COX assembly requires the coming together of nuclear-
and mitochondrial-encoded subunits and the assistance of a large number
of nuclear gene products acting at different stages of maturation of
the enzyme. In Saccharomyces cerevisiae, expression of
cytochrome c, encoded by CYC1 and
CYC7, is required not only for electron transfer but also
for COX assembly through a still unknown mechanism. We have attempted
to distinguish between a functional and structural requirement of
cytochrome c in COX assembly. A
cyc1/cyc7 double null mutant strain was
transformed with the cyc1-166 mutant gene (Schweingruber,
M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but
catalytically inactive iso-1-cytochrome c. The COX content
of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized
spectrally, functionally, and immunochemically. The results of these
studies demonstrate that cytochrome c plays a structural
rather than functional role in assembly of cytochrome c
oxidase. In addition to its requirement for COX assembly, cytochrome
c also affects turnover of the enzyme. Mutants containing
wild type apocytochrome c in mitochondria lack COX,
suggesting that only the folded and mature protein is able to promote
COX assembly.
Saccharomyces cerevisiae contains two genes for
cytochrome c. Iso-1-cytochrome c, encoded by
CYC1 (1), accounts for ~95% of the total cytochrome
c in mitochondria (2). The homologous and less abundant
iso-2-cytochrome c is encoded by CYC7 (3). Even
though iso-2-cytochrome c represents only 5% of the total cytochrome c, it is sufficient to support respiration and
growth, albeit at a somewhat reduced rate on non-fermentable carbon
sources such as glycerol and ethanol (4). Mutations in both isoforms lead to a respiratory defect (3). In addition to the absence of
cytochrome c, the double mutant is also deficient in
cytochrome oxidase (COX)1
(5). The same phenotype is observed in cyc3 mutants that are blocked in the covalent attachment of heme to apocytochrome
c (6). The biochemical defect of cytochrome c
mutants has all the hallmarks of COX assembly mutants. Mitochondria
lack the absorption bands corresponding to cytochrome
aa3, and the steady-state concentrations of the
mitochondrially encoded subunits 1, 2, and 3 of COX are reduced,
presumably as a result of increased turnover of unassembled subunits
(5).
The function of cytochrome c in COX assembly is not
understood. Oxidized cytochrome c can accept electrons from
cytochrome c1 of the bc1
complex and cytochrome b2 of lactate
dehydrogenase. The principle function of reduced cytochrome
c is to donate electrons to cytochrome oxidase. Conceivably,
cytochrome c could also promote an oxidation or reduction
event essential for COX assembly. The obvious possibility that
cytochrome c may be involved in heme A biosynthesis has been
excluded (7). Alternatively, cytochrome c could be required
in a structural capacity. For example, its interaction with a COX
intermediate may be necessary for some step in the assembly pathway.
In the present study we have tried to distinguish between a functional
and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant was transformed
with a cyc1 mutant gene that was previously reported to
express stable but catalytically inactive cytochrome c (8).
The COX content of the cyc1/cyc7 mutant
containing the non-functional form of iso-1-cytochrome c has
been characterized spectrally, functionally, and immunochemically. The
results of these analyses are consistent and point to a structural rather than functional role of cytochrome c in COX assembly.
Yeast Strains and Media--
The genotypes and sources of
the strains of S. cerevisiae used in this study are listed
in Table I. Yeast strains were
routinely grown in 2% galactose, 1% yeast extract, and 2% peptone
(YPGal). The compositions of solid media have been described elsewhere (12).
Preparation of Yeast Mitochondria--
Wild type and mutant
yeast were grown to stationary phase in YPGal. Unless otherwise
indicated, mitochondria were prepared by the procedure of Faye et
al. (13), except that zymolyase 20,000 (ICN Biomedicals, Inc.)
instead of Glusulase was used at 24 °C to convert cells to spheroplasts.
Construction of a cyc1 and cyc7 Null
Alleles--
CYC1 was cloned as a
XmaI-HindIII fragment into pUC18. The resultant
plasmid pCYC1/ST1 was used to delete the gene with the bi-directional
primers 5'-GGCGGTACCTATTAATTTAGTGTGTGTATTG and 5'-GGCGGTACCAACAGGCCCCTTTTCCTTTGTC. The PCR-amplified product containing 5'- and 3'-flanking sequences but lacking the entire CYC1 coding sequence was digested with KpnI and
ligated to a 1-kb KpnI fragment containing the yeast
URA3 gene. This plasmid (pCYC1/ST3) was used as a source of
a linear 1.5-kb EcoRI-HindIII fragment with the
To delete CYC7, the gene was first PCR-amplified from yeast
nuclear DNA with primers 5'-GGCGGATCCGAAGGGTCTGCAGTCCCCCGCC and 5'-GGCGGATCCCTGTAAGCGGAAGCGCCTCCAG. The 800-bp fragment containing CYC7 and flanking sequences was digested with
BamH1 and cloned in YEp352B (this plasmid is identical to
YEP352 (14) except that the multiple cloning sequence is replaced by a
single BamH1 site), yielding pCYC7/ST2. CYC7 was
deleted from CYC7/ST2 with the bi-directional primers
5'-GGCGAATTCGTTTTGTTTATGATGTAATGTAGTT and
5'-GGCGAATTCGGCTATGTCGTCGGAGGAG. The linear product containing 5'- and
3'-flanking sequences but lacking CYC7 was digested with EcoR1 and ligated to a 1.7-kb EcoR1 fragment
containing the yeast TRP1 gene. The resultant plasmid
pCYC7/ST4 was digested with BamH1 to obtain a linear 2-kb
fragment with the
The respiratory competent haploid yeast W303-1B was transformed with
the linear 1.5-kb EcoR1-HindIII fragment
containing the cyc1 null allele. A uracil prototrophic
transformant was verified by PCR to be deleted for CYC1.
This mutant, designated W303 Construction of the cyc1 Mutant Gene--
The
cyc1-166 mutation (8) was introduced into CYC1
by PCR amplification of the region between the XmaI and
internal KpnI site of pCYC1/ST1 with primers
5'-GGACCCGGGAGCAAGATCAAGATG and 5'-CTTGGTACCAGGAATATATTTCTTTGGGTTAGTCAAGTACTCTGACATGTTATTTTCGTCCGACAACACG. The fragment with the mutation was digested with a combination of
XmaI and KpnI and was substituted for the
corresponding fragment in pCYC1/ST1, yielding pCYC1/W65S. The mutant
gene was recovered as an XmaI-HindIII fragment
and was transferred to YIp351 and YEp351 (14), yielding pCYC1/ST11 and
pCYC1/ST12, respectively. The mutation was confirmed by sequencing of
the insert in pCYC1/ST11. The mutant gene was integrated at the
leu2 locus of W303-1B after linearization of pCYC1/ST11 at
the ClaI site of the LEU2 marker in the plasmid.
Cytochrome Oxidase Assays--
COX was assayed either
spectrophotometrically by following the oxidation of ferro-cytochrome
c at 550 nm (15) or polarographically by measuring oxygen
utilization with ascorbate plus
N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) as the substrate. In the latter assay the reaction contained 10 mM potassium phosphate, pH 7.5, 5 mM ascorbic
acid, 0.1 mM TMPD, and 50-100 µg of mitochondrial
protein. Both assays were carried out at 24 °C.
Miscellaneous Procedures--
Standard procedures were used for
the preparation and ligation of DNA fragments and for transformation
and recovery of plasmid DNA from Escherichia coli (16).
Yeast strains were transformed by the method of Schiestl and Gietz
(17). Mitochondrial protein synthesis was assayed in vivo in
the presence of cycloheximide as described previously (11). Proteins
were separated by PAGE in the buffer system of Laemmli (18). Cytochrome
c was detected on Western blots using a rabbit polyclonal
antibody raised against SDS-denatured yeast cytochrome c
purchased from Sigma. Antibody-antigen complexes were visualized by a
secondary reaction with the Super Signal detection kit (Pierce).
Alternatively Western blots were first treated with antibody against
cytochrome c followed by incubation with
125I-protein and quantitation of the signals with a Storm
PhosphorImager (Molecular Dynamics). Protein concentrations were
determined by the method of Lowry et al. (19).
Phenotype of the cyc1 and the cyc1/7 Null Mutants--
Deletion of
CYC1 reduces the rate but does not abolish growth of yeast
on rich glycerol/ethanol medium (Ref. 4, Fig.
1A). In contrast, deletion of
both CYC1 and CYC7 completely blocks growth on
the non-fermentable carbon sources (Fig. 1A). Wild type yeast has been shown to contain 95% iso-1-cytochrome c
encoded by the CYC1 gene and 5% iso-2-cytochrome
c encoded by CYC7 (2). Western blot analysis of
mitochondrial iso-2-cytochrome c expressed in the
cyc1 null mutant is 12% of the total cytochrome
c detected in the parental wild type strain (Fig.
1B). This value is 2-3 times higher than the 5%
iso-2-cytochrome c reported previously. This could be
because of a difference in the strains or carbon sources used in two
studies. It is also possible that the absence of CYC1
results in an increased expression of iso-2-cytochrome c.
The spectral properties of the single and double null mutants are
consistent with earlier studies showing that cyc1 and
cyc7 mutants are able to synthesize COX, whereas
cyc1/cyc7 double mutants are totally deficient in the
cytochrome aa3 components of this respiratory
complex (Ref. 4, Fig. 2). The spectral
properties of the mutants correlate with the results of enzymatic
assays. Spectrophotometric and polarographic assays indicate a
reduction of COX activity in the single cyc1 mutant and a
complete absence of activity in the double mutant (Table
II).
Most COX mutants display a decreased incorporation of radioactive
precursors into Cox1p but not Cox2p or Cox3p when mitochondrial translation is measured in vivo in the presence of
cycloheximide to inhibit cytoplasmic protein
synthesis.2 This
characteristic is shared by the cyc1/cyc7
double mutant (Fig. 3). The in
vivo assays also show that the absence of cytochrome c
does not significantly affect the stability of Cox2p and Cox3p during a
90-min chase period. The double mutant also displays greatly reduced
steady-state levels of the mitochondrially translated Cox1p and Cox2p
as reported previously (Ref. 4 and Fig.
4). Although there is some reduction in
Cox3p and some of the imported subunits (Cox4p and Cox5p), these
constituents appear to be more stable (Fig. 4).
Phenotype of a cyc1/cyc7 Double Mutant Expressing
Iso-1-cytochrome c with a W65S Mutation--
Several mutant alleles of
cyc1 were previously shown to express catalytically inactive
forms of iso-1-cytochrome c (4). One such allele
(cyc1-166), coding for a W65S amino acid substitution, produces a protein that is stable at 24 °C but is unable to mediate electron transfer from the bc1 complex to COX
(8). These properties of iso-1-cytochrome c with the W65S
mutation made it possible to examine if COX assembly depends on a redox
active protein.
A gene with the cyc1-166 mutation was made by PCR
amplification of the appropriate coding region with the mutation in one of the primers. The mutant gene was integrated into the chromosomal DNA
of the cyc1/cyc7 double mutant by targeted insertion at the leu2 locus (W303
The cyc1-166 mutant was reported to contain spectrally
detectable cytochrome c but at lower concentrations than
wild type yeast (8). This was also true of the cyc1/cyc7
null strain transformed with the cyc1-166 gene in the
integrative or episomal plasmid (Fig. 2A). The spectra of
mitochondria from both strains indicated only a partial restoration of
cytochrome c (compare the double mutant with the ST11, ST12
transformants in Fig. 2A). The concentrations of cytochrome
c in mitochondria of the transformant with the integrated
mutant gene and of the wild type strain were determined from spectra of
extracts obtained by sonic disruption of mitochondria in the presence
of salt. Under these conditions most of the cytochrome c is
rendered soluble, whereas other cytochromes associated with the
respiratory chain complexes remain in the membrane fraction (Fig.
2B). The concentrations of cytochrome c in wild
type and the mutant mitochondria were estimated to be 0.32 and 0.08 nmol/mg of protein, respectively. The mutant iso-1-cytochrome c represents only 25% of cytochrome c in wild
type yeast. Because the amount of iso-2-cytochrome c in the
mutant detected immunologically is 75% of wild type (Fig.
1B), only 30% of the W65S protein contains heme.
The status of COX in the cyc1/cyc7 null strain harboring the
cyc1-166 allele was examined in several ways. Spectra of
mitochondrial cytochromes indicated the presence of cytochromes
aa3 when the mutant protein was expressed either
from the chromosomally integrated or plasmid-borne gene (Fig. 2). The
presence of the integrated or episomal copy of the cyc1-166
allele restored cytochrome aa3 to more than 50%
of the level seen in wild type mitochondria (Fig. 2A). The
ability of iso-1-cytochrome c with the W65S mutation to
rescue the COX deficiency of the cyc1/cyc7 strain was
confirmed by enzyme assays and by Western analysis of COX subunits
proteins, which indicated that the steady-state levels of the
mitochondrially encoded Cox1p, Cox2p, and Cox3p were restored to nearly
wild type levels (compare Fig. 4 and see Fig. 6C).
Two different assays were used to measure COX activity. The first
relied on the reduction of endogenous cytochrome c in
mitochondria by ascorbate in the presence of TMPD. Using this assay the
cyc1 null mutant had 40% of wild type COX, whereas no
activity was detected in the cyc1/cyc7 double mutant (Table
II). Predictably, the double mutant transformed with the
cyc1-166 gene was also completely inactive in catalyzing
ascorbic acid oxidation by oxygen (Table II). When the assays were
repeated in the presence of exogenous cytochrome c, the
specific activity of COX in the cyc1 mutant was raised to
70% that of wild type, whereas the
cyc1/cyc7 strain with the integrated
cyc-166 gene was comparable with that of wild type (Table
II). Similar results were obtained when the COX activity was assayed
spectrophotometrically by measuring oxidation of substrate amounts of
reduced cytochrome c (Table II). Surprisingly, the COX
activity measured by both assays was lower in the transformant expressing the W65S protein from a multicopy plasmid. This was not true
of the NADH-cytochrome c reductase activities, which were
nearly the same in all the strains (Table II). The spectrum of
mitochondrial cytochromes also showed a lower concentration of
cytochromes aa3 in the high copy transformant
than in the strain with the integrated gene (Fig. 2).
The lower COX activity in the multicopy transformant could be explained
by a kinetic block because of limited accessibility or exchange of the
wild type substrate cytochrome c with the W65S mutant
protein. This was tested by depletion of the iso-1-cytochrome c from mitochondria before the assay. After sonic
irradiation of wild type mitochondria in the presence of 1 M KCl, COX activity was reduced to 15% of the starting
values (Table III). The specific activity
returned to normal levels when the depleted mitochondria were assayed
polarographically or spectrophotometrically in the presence of added
cytochrome c. Under these conditions, however, the specific
activity of mitochondria from the high copy transformant measured in
the presence of cytochrome c was even lower after depletion
(Table III). At present, therefore, the reason for the observed
difference COX between the single and multicopy transformants is not
clear.
Only a Fraction of the W65S Mutant Iso-1-cytochrome c Is in a
Protease-protected Compartment of Mitochondria--
The difference in
the amount of spectrally (25% of wild type) and immunochemically (75%
of wild type) detectable iso-1-cytochrome c in the
cyc1/cyc7 null strain transformed with the
cyc1-166 gene (Figs. 1 and 2) indicates that only 30% of
the protein associated with mitochondria contains heme. The
intra-mitochondrial location of the wild type and of the W65S mutant
proteins was compared by testing their sensitivity to proteinase K in
mitochondria and mitoplasts. Most of the iso-1-cytochrome c
in the strain with the chromosomally integrated cyc1-166
allele was found to be susceptible to digestion by the protease in
intact mitochondria (Fig. 5, upper panel). A small fraction corresponding to 10%, however, was in a
proteinase K-protected compartment. Even though all the protein sedimented with mitoplasts, it was completely sensitive to proteinase K. In contrast, cytochrome c in wild type mitochondria is
digested by the protease only when they are converted to mitoplasts
(Fig. 5, lower panel). Sco1p, an inner membrane protein
previously shown to face the intermembrane space (22), cytochrome
b2, a soluble intermembrane marker, and
The small fraction of W65S protein resistant to proteinase K in
mitochondria probably corresponds to mature protein located in the
intermembrane space, whereas the more abundant fraction digested by the
protease is probably mostly mutant apoprotein. Because transport of
cytochrome c to the intermembrane space of mitochondria has
been shown to be coupled to heme addition (23), the protease
sensitivity of most of the W65S protein suggests, notwithstanding the
fact that the tryptophan at residue 65 is not covalently linked to
heme, its replacement by a serine must reduce the efficiency of heme
attachment to the apoprotein. Mutations in the cysteine ligands of heme
have also been shown to impede apocytochrome c import into
mitochondria in vitro (23). In these studies most of the
mutant apocytochrome c was also found to cosediment with
mitochondria even though it was not protected against the protease
(23).
Role of Cytochrome c in Stability of COX--
The absence of COX
in cyc1/cyc7 mutants has been interpreted to
indicate that cytochrome c plays a role in assembly of this respiratory complex (5). Alternatively, cytochrome c could protect COX against proteolysis. The partial instability of the W65S
protein at 37 °C (8) made it possible to examine the effect of
cytochrome c on the half-life of COX. The wild type parental strain and the cyc1/cyc7 mutant with the
chromosomally integrated cy1c-166 gene were grown to early
stationary phase at 24 °C in rich galactose medium. The cultures
were treated with cycloheximide to prevent synthesis of new proteins
and were further incubated at 37 °C for different times.
Approximately 80% of the protein was degraded as early as 1 h
after the temperature switch. Longer times at 37 °C led to
progressively greater destruction, with only 4% of the starting
protein remaining after a 14-h incubation. Wild type cytochrome
c was also reduced at the higher temperature, although the
extent, even after overnight incubation, was much less (Fig.
6A).
Incubation of the mutant cells at 37 °C caused greater than 90%
loss of COX activity after 14 h of incubation (Fig.
6A). The decrease in the specific activity was accompanied
by a partial reduction in the cytochromes aa3
absorption bands (Fig. 6B). The loss of enzymatic activity,
however, preceded the reduction in the cytochrome
aa3 bands. For example, even though less than
25% of the starting activity remained after 4 h at 37 °C, the
cytochromes aa3 peaks were only marginally
reduced (Fig. 6, A and B). Western analyses of
COX subunits also indicated very partial losses of Cox1p, Cox2p, Cox3p,
and Cox5p during the first 4 h at 37 °C (Fig. 6C).
The most significant change was a large decrease of Cox1p in the mutant
after the overnight incubation. In the wild type strain COX was highly
stable at 37 °C, with more than 90% of enzyme and activity after
14 h of incubation at the high temperature (Fig. 6A).
Western analysis also failed to reveal any significant reductions in
the COX subunits after different times at 37 °C. The NADH-cytochrome
c reductase activities decreased to approximately the same
extent in the wild type and the mutant. At present it is not clear if
this is caused by a destabilization of the dehydrogenase, the
bc1 complex, or the coenzyme Q pool at 37 °C.
The spectrum of mitochondria from mutant cells grown
exponentially at 37 °C exhibited an almost complete absence of
cytochromes aa3, similar to the
cyc1,7 double mutant (Fig. 6D).
The marked decrease of COX activity in mutant cells exposed to 37 °C
for 4 h even though cytochromes aa3 and
steady-state concentrations of COX subunits are unaffected during this
period suggests some more subtle changes in the quaternary structure or
some other aspect of the enzyme. This is also seen in the wild type
strain but to a lesser degree. These results suggest that the main role
of cytochrome c is in assembly but that it also contributes
toward the stability of the enzyme. The latter role is especially
evident after prolonged incubation at 37 °C, which leads to turnover
of Cox1p, more extensive loss of cytochromes aa3, and a virtually complete absence of enzyme
activity (Fig. 6). The degradation of COX in mutant cells undergoing
cytochrome c degradation may be similar to the loss of COX
induced by mitochondrial cytochrome c release in wild type
yeast committed to programmed cell death (24).
Apocytochrome c Does Not Promote COX Assembly--
Apocytochrome
c is imported into the intermembrane space of mitochondria,
where it is matured by covalent attachment of protoheme (23, 25). This
reaction, catalyzed by the heme lyase product of CYC3, fixes
the mature heme protein in the intermembrane space. Apocytochrome
c exits from the intermembrane space when heme attachment is
blocked as a result of mutations in the lyase. Hence cyc3
mutants like cyc1/cyc7 mutants are
COX-deficient (Ref. 5, Fig.
7A).
To test if apocytochrome c is able to promote COX assembly,
a cyc3 null mutant was transformed with CYC1 and
CYC7 on high copy plasmids (ST13 and ST5, respectively. Both
yeast transformants accumulate some apocytochrome c (Fig.
7B). The concentration of apocytochrome c in
mitochondria of the cyc3 mutant transformed with the
respective genes was lower than in wild type and was further reduced
after treatment with proteinase K. The mitochondrial concentrations of
iso-1 or iso-2-apocytochrome c in the proteinase K-protected
compartment is comparable with the amount of iso-2-cytochrome c present in the cyc1 mutant, which is able to
express at least 70% of the normal amount of COX (Fig.
8). Neither of the two cyc3 transformants, however, contained COX either by spectral (Fig. 7A) or enzymatic criteria (not shown). Additionally, Western
analysis of COX subunits indicated that despite the presence of
apocytochrome c in mitochondria of the two transformants,
there was no increase in the steady-state concentrations of Cox2p and
Cox3p. These results indicate that apocytochrome c is unable
to promote COX assembly.
The present study shows that assembly of COX depends on the presence of
cytochrome c in mitochondria even when the latter is unable
to function in electron transport. The requirement for cytochrome
c, therefore, is not related to either reduction or oxidation of some group in a subunit or assembly intermediate of COX.
We estimate that the molar concentration of cytochrome c in
mitochondria of wild type yeast is approximately the same as that of
COX. Assembly of COX, therefore, does not depend on stoichiometric
concentration of cytochrome c. Iso-2-cytochrome c, whose concentration in a cyc1 mutant is only
12% of the total amount of cytochrome c in wild type yeast,
is able to support the expression of 70% of normal amounts of COX.
This lack of requirement for stoichiometry is also supported by the
results obtained with the W65S mutant. Because the apoprotein cannot
substitute for the mature cytochrome, the function of cytochrome
c probably depends on a properly folded protein. This is
consistent with a structural role in COX assembly. In addition to its
requirement for assembly, the results of obtained with the W65S mutant
exposed to 37 °C indicate that cytochrome c also affects
turnover of COX.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Genotypes and sources of S. cerevisiae strains
cyc1::URA3 allele.
cyc7::TRP1 allele.
CYC1, was transformed with the linear
fragment containing the cyc7 null allele. Several uracil-
and tryptophan-independent clones obtained from the transformation were
confirmed by PCR to have the CYC7 deletion. One of the
double mutants, W303
CYC1,7, was used for further studies.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (57K):
[in a new window]
Fig. 1.
Growth properties of mutant and
transformants. A, serial dilutions of the indicated
strains were plated on rich glucose (YPD) medium and
incubated at 30 °C for 1 day. The same dilutions were also plated on
rich glycerol, ethanol (YPEG) medium, and incubated at 24 and 30 °C for 2 days. W303-1B (W303), the respiratory
competent parental strain; W303 CYC1 (
CYC1), a
cyc1 null mutant; W303
CYC1,7 (
CYC1,7), a
cyc1/cyc7 double mutant; W303
CYC1,7
(
CYC1,7/ST7), the double mutant with the wild type
CYC1 gene integrated in chromosomal DNA; W303
CYC1,7
(
CYC1,7/ST11), the double mutant with the
cyc1-166 mutant gene integrated in chromosomal DNA;
W303
CYC1,7 (
CYC1,7/ST12), the double mutant
transformed with the cyc1-166 mutant gene on a high copy
plasmid. B, mitochondria (20 µg of protein) were separated
by SDS-PAGE on a 12% polyacrylamide gel and transferred to
nitrocellulose. The Western blot was probed with an antibody against
yeast cytochrome c, and the antibody-antigen complexes were
visualized with the SuperSignal chemiluminescent substrate kit
(Pierce). The sources of mitochondria are the same as in panel
A. The two additional strains are W303
CYC1,7/ST7 and
W303
CYC1,7/ST13, the cyc1/cyc7 double mutant with the
wild type CYC1 gene integrated at the leu2 locus
and on a multicopy plasmid, respectively. The absorbance unit
(AU) values are the relative absorbance units obtained from
a duplicate Western in which the bands were visualized by a secondary
reaction with 125I-protein A and exposed in a
PhosphorImager (Molecular Dynamics).
View larger version (16K):
[in a new window]
Fig. 2.
Spectra of mitochondrial cytochromes.
A, mitochondria were prepared from the following
strains grown at 24 °C on rich galactose medium (YPGal).
W303 CYC1 (
CYC1), the cyc1 null mutant;
W303
CYC1,7 (
CYC1,7), the cyc1,
cyc7 double mutant; W303
CYC1,7
(
CYC1,7/ST11), the double mutant with the
cyc-166 gene integrated in chromosomal DNA; W303
CYC1,7
(
CYC1,7/ST12), the double mutant transformed with the
cyc1-166 gene on a high copy plasmid. The respiratory
competent parental W303-1B (W303) strain was grown at
30 °C. Mitochondrial cytochromes were extracted with potassium
deoxycholate at a final concentration of 5 mg of protein/ml as
described previously (20). Difference spectra of the reduced (sodium
dithionite) versus oxidized (potassium ferricyanide)
extracts were recorded at room temperature. The
absorption bands
corresponding to cytochromes a and a3
have maxima at 603 nm (a), of cytochrome b at 560 nm (b), and of cytochrome c and
c1 at 550 nm (c). B,
mitochondria obtained from the wild type strains W303-1B
(W303) and the mutant W303
CYC1,7/ST11
(
CYC1,7/ST11) were sonically irradiated
in the presence of 1 M KCl at a protein concentration of
13.2 mg/ml. The suspensions were centrifuged at 230,000 × gav for 15 min. The supernatants were collected,
and difference spectra of the reduced versus oxidized
extract were recorded.
Respiratory and enzymatic activities in cyc mutants and transformants
View larger version (56K):
[in a new window]
Fig. 3.
In vivo synthesis of COX subunits
in mutants and transformants. Synthesis of mitochondrial
translation products was assayed in the following strains.
W303-1B (W303), the respiratory competent parental strain;
W303 CYC1 (
CYC1), the cyc1 null
mutant; W303
CYC1,7 (
CYC1,7), the
cyc1, cyc7 double mutant: W303
CYC1,7
(
CYC1,7/ST7), the double mutant with the wild type
CYC1 gene integrated in chromosomal DNA; W303
CYC1,7
(
CYC1,7/ST11), the double mutant with the
cyc1-166 mutant gene integrated in chromosomal DNA.
Incorporation of [35S]methionine into the mitochondrial
translation products was allowed to proceed for 20 min at 30 °C as
described previously (11). Excess 80 mM cold methionine and
4 µg/ml puromycin were added (zero time), and samples were taken
after 30 and 90 min of chase. Equivalent amounts of total cellular
proteins were separated by SDS-PAGE on a 17.5% polyacrylamide gel,
transferred to a nitrocellulose membrane, and exposed to x-ray film.
The mitochondrially translated ribosomal protein Var1p, subunits 1 (Cox1p), subunit 2 (Cox2p), and 3 (Cox3p) of COX, cytochrome b (Cyt. b),
and subunit 6 (Atp6p) and subunit 8 and 9 (Atp8/9) of the oligomycin-sensitive ATPase are identified
in the margin.
View larger version (55K):
[in a new window]
Fig. 4.
Steady-state levels of COX subunits in
mutants and transformants. Mitochondria were prepared from the
following strains grown at 24 °C on rich galactose medium (YPGal).
W303-1B (W303), the wild type parental strain; W303 CYC1
(
CYC1), the cyc1 null mutant; W303
CYC1,7
(
CYC1,7), the cyc1, cyc7 double
mutant; W303
CYC1,7/ST7 (
CYC1,7/ST7),
the double mutant with a chromosomally integrated copy of
CYC1; W303
SCO1 (
SCO1), a sco1
null mutant; W303
COX15 (
COX15), a cox15
null mutant; W303
MSS51 (
MSS51), a mss51
null mutant. Total mitochondrial proteins (20 µg for detection of
cytochrome c and 40 µg for detection of COX subunits) were
separated by SDS-PAGE electrophoresis on a 12% polyacrylamide gel. The
proteins were transferred to nitrocellulose and reacted with antibodies
to cytochrome c (Cyt. c) and COX subunits 1 (Cox1p), 2 (Cox2p), 3 (Cox3p), 4 (Cox4p), and 5 (Cox5p). The antibody-antigen
complexes were visualized with the Super Signal chemiluminescent kit
(Pierce).
CYC1,7/ST11). The mutant was also
transformed with the gene on a high copy plasmid (W303
CYC1,7/ST12).
Even though mitochondria from both transformants had immunochemically detectable cytochrome c (Fig. 1B), neither the
multicopy nor the integrated mutant gene was able to rescue the growth
defect of the W303
CYC1,7 strain on glycerol at either 24 or 30 °C
(Fig. 1A). Western blot analysis of mitochondria from the
transformant with the integrated mutant gene indicated that the level
of cytochrome c is ~75% that seen in wild type (Fig. 1).
The mitochondrial concentration of cytochrome c was somewhat
lower in the transformant with the gene on a high copy plasmid (60% of
wild type). The failure of the W65S mutant protein to support growth on
non-fermentable carbon sources is in agreement with the previously
noted deleterious effect of the mutation on the catalytic activity of
iso-1-cytochrome c (8).
Effect of cytochrome c depletion on the NADH-cytochrome c and COX
activities of cyc mutants
-ketoglutarate dehydrogenase, a soluble matrix protein, showed the
expected properties in the mitochondria and mitoplasts (Fig.
3B). These results indicate that some 90% of the mutant
protein is associated with mitochondria in a manner that makes it
accessible to proteinase K. Whether it is bound to the outer membrane
or is only partially inserted into the intermembrane space has not been
determined.
View larger version (34K):
[in a new window]
Fig. 5.
Cytochrome c accessibility
to proteinase K. Mitochondria were prepared by the method of Glick
and Pon (21) from wild type (W303) and the
cyc1-166 mutant W303 CYC1,7/ST11
(
CYC1,7/ST11) grown in YPGal to early stationary phase at
24 °C. The mitochondria (Mt) and mitoplasts
(Mp) were incubated in the presence of 100 µg/ml
proteinase K (Prot. K) for 60 min on ice. The reaction was
stopped by the addition of phenylmethylsulfonyl fluoride to a final
concentration of 2 mM, and the mitochondria and mitoplasts
were recovered by centrifugation at 100,000 × gav. The pellets were suspended in 0.6 M sorbitol, 20 mM Hepes, pH 7.5, and proteins
were precipitated by the addition of 0.1 volume of 50% trichloroacetic
acid and heated for 10 min at 65 °C. Mitochondrial and mitoplast
proteins from wild type and the transformant (40 µg) were separated
by SDS-PAGE on a 12% polyacrylamide gel, transferred to
nitrocellulose, and probed with antibody against Sco1p,
-ketoglutarate dehydrogenase (
KGD), cytochrome
b2 (Cyt b2), and
cytochrome c (Cyt. c). Proteins were visualized
with the Super Signal chemiluminescent kit (Pierce).
View larger version (37K):
[in a new window]
Fig. 6.
Cytochrome c and COX
stability in wild type and mutant cells at 37 °C. A,
the wild type W303-1B (W303) and the cyc1-166
mutant W303 CYC1,7/ST11 (
CYC1,7/ST11) were grown to
early stationary phase at 24 °C in YPGal. The cultures were adjusted
to 2 × 10
5 M cycloheximide, and
1/4 of each culture was harvested (zero time). Equal volumes of
the remaining 3/4 of the cultures were collected after for 1-, 4-, and 14-h incubation at 37 °C. Mitochondria were prepared and
assayed for COX (dashed bars) and NADH cytochrome
c reductase (open bars). The relative amount of
cytochrome c (solid bars) in mitochondria was
determined from Western blots as in Fig. 1B. Mitochondrial
cytochrome spectra were obtained as in Fig. 2, except that the protein
concentration was 4 mg/ml during the extraction. C, the
steady-state levels of COX subunits 1 (Cox1p), 2 (Cox2p), 5 (Cox5p), and cytochrome c
were analyzed as in Fig. 4. The COX subunits were visualized with the
SuperSignal substrate kit (Pierce). Cytochrome c was
visualized by a secondary reaction with 125I-protein. The
values of cytochrome c shown in the bar graph in
A were obtained from this gel after quantification in a
PhosphorImager (Molecular Dynamics). D, The
cyc1-166 mutant W303
CYC1,7/ST11 was grown in YPGal at 24 and 37 °C to early stationary phase. Mitochondria were prepared and
extracted at a protein concentration of 5 mg/ml. The spectra were
recorded as in B.
View larger version (21K):
[in a new window]
Fig. 7.
Phenotype of cyc3 mutants
and transformants. Mitochondria were prepared from the
cyc3 null mutant aW303 CYC3 (
CYC3), from
aW303
CYC3/ST5 (
CYC3/ST5), the mutant transformed with
CYC7 on a high copy plasmid, and aW303
CYC3/ST13
(
CYC3/ST13), the mutant transformed with CYC1
on a high copy plasmid. A, mitochondrial cytochromes were
extracted, and spectra were recorded as described in the legend to Fig.
2. B, total mitochondrial proteins (20 µg for detection of
cytochrome c and 40 µg for detection of COX subunits) were
separated by SDS-PAGE on a 12% polyacrylamide gel. After transfer to
nitrocellulose, the Western blot was probed with antibodies to COX
subunits 2 (Cox2p), 3 (Cox3p), and 5 (Cox5p), and to cytochrome c (Cyt. c).
The antibody-antigen complexes were visualized as described in the
legend to Fig. 1.
View larger version (33K):
[in a new window]
Fig. 8.
Apocytochrome c in
mitochondria of cyc3 mutants transformed with
CYC1 and CYC7 on high copy
plasmids. Mitochondria were prepared by the method of Glick and
Pon (21) from the cyc1-166 mutant W303 CYC1,7/ST11
(
CYC1,7/ST11) grown in YPGal to early
stationary phase at 24 °C. Mitochondria were also prepared from the
cyc1 null mutant W303
CYC1 (
CYC1) and
aW303
CYC3/ST5 (
CYC3/ST5) and
aW303
CYC3/ST13 (
CYC3/ST13), the
cyc3 null mutants transformed with CYC7 and
CYC1, respectively, on high copy plasmids. The mitochondria
were incubated in the presence of 100 µg/ml proteinase K for 60 min
on ice. The digestions were stopped with phenylmethylsulfonyl fluoride
and further treated as described in the legend to Fig. 5. Proteinase K
(PK)-treated and untreated samples (50 µg protein) were
separated by SDS-PAGE, transferred to nitrocellulose, and treated with
antiserum to cytochrome c followed by a secondary reaction
with 125I-protein A. The relative amounts of cytochrome
c were quantitated in a PhosphorImager (Molecular Dynamics).
AU, arbitrary absorbance units.
![]() |
Note Added in Proof |
---|
It has been brought to our attention that in the article by Komar-Panicucci et al. (Komar-Panicucci, S., Sherman, F., and McLendon, G. (1996) Biochemistry 35, 4878-4885), mutations affecting the redox potential of yeast iso-1-cytochrome c and resulting in its lower reactivity with the ubiquinol-cytochrome c reductase complex were found to lead to increased expression of cytochrome oxidase.
![]() |
FOOTNOTES |
---|
* This research was supported by National Institutes of Health Grant GM 50187 and by a post-doctoral fellowship MDACU01991001 from the Muscular Dystrophy Association (to A. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, Columbia University, New York, NY 10027. Tel.: 212-854-2920; Fax: 212-865-8246; E-mail: spud@cubpet2.bio.columbia.edu.
Published, JBC Papers in Press, January 8, 2003, DOI 10.1074/jbc.M212427200
2 A. Barrientos and A. Tzagoloff, unpublished information.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: COX, cytochrome c oxidase; TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine; YPGal, 2% galactose, 1% yeast extract, and 2% peptone; kb, kilobase.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Sherman, F., Stewart, J. W., Margoliash, E., Parker, J., and Campbell, W. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 1498-1504[Medline] [Order article via Infotrieve] |
2. | Sherman, F., Taber, H., and Campbell, W. (1965) J. Mol. Biol. 13, 21-39[Medline] [Order article via Infotrieve] |
3. | Downie, J. A., Stewart, J. W., Brockman, N., Schweingruber, A. M., and Sherman, F. (1977) J. Mol. Biol. 113, 369-384[Medline] [Order article via Infotrieve] |
4. |
Sherman, F.,
Stewart, J. W.,
Jackson, M.,
Gilmore, R. A.,
and Parker, J. H.
(1974)
Genetics
77,
255-284 |
5. |
Pearce, D. A.,
and Sherman, F.
(1995)
J. Biol. Chem.
270,
20879-20882 |
6. | Dumont, M. E., Ernst, J. F., Hampsey, D. M., and Sherman, F. (1987) EMBO J. 6, 235-241[Abstract] |
7. | Barros, M. H., and Tzagoloff, A. (2002) FEBS Lett. 516, 119-123[CrossRef][Medline] [Order article via Infotrieve] |
8. | Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143[Abstract] |
9. |
Glerum, D. M.,
Shtanko, A.,
and Tzagoloff, A.
(1996)
J. Biol. Chem.
271,
20531-20535 |
10. |
Glerum, D. M.,
Shtanko, A.,
and Tzagoloff, A.
(1996)
J. Biol. Chem.
271,
14504-14509 |
11. |
Barrientos, A.,
Korr, D.,
and Tzagoloff, A.
(2002)
EMBO J.
21,
43-52 |
12. | Myers, A. M., Pape, L. K., and Tzagoloff, A. (1985) EMBO J. 4, 2087-2092[Abstract] |
13. | Faye, G., Kujawa, C., and Fukuhara, H. (1974) J. Mol. Biol. 88, 185-203[Medline] [Order article via Infotrieve] |
14. | Hill, J. E., Myers, A. M., Koerner, T. J., and Tzagoloff, A. (1986) Yeast 2, 163-167[Medline] [Order article via Infotrieve] |
15. | Wharton, D. C., and Tzagoloff, A. (1967) 10, 245-250 |
16. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
17. | Schiestl, R. H., and Gietz, R. D. (1989) Curr. Genet. 16, 339-346[Medline] [Order article via Infotrieve] |
18. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
19. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
20. | Tzagoloff, A., Akai, A., and Needleman, R. B. (1975) J. Biol. Chem. 250, 8228-8235[Abstract] |
21. | Glick, B. S., and Pon, L. A. (1995) Methods Enzymol. 260, 213-223[Medline] [Order article via Infotrieve] |
22. | Beers, J., Glerum, D. M., and Tzagoloff, A. (1997) J. Biol. Chem. 372, 33191-33196[CrossRef] |
23. |
Dumont, M. E.,
Ernst, J. F.,
and Sherman, F.
(1988)
J. Biol. Chem.
263,
15928-15937 |
24. |
Ludovico, P.,
Rodrigues, F.,
Almeida, A.,
Silva, M. T.,
Barrientos, A.,
and Corte-Real, M.
(2002)
Mol. Biol. Cell
13,
2598-2606 |
25. |
Nicholson, D. W.,
Hergersberg, C.,
and Neupert, W.
(1988)
J. Biol. Chem.
263,
19034-19042 |