From the Department of Biochemistry, West Virginia University
School of Medicine, Morgantown, West Virginia 26506
Previous experiments using deletion mutants of
the iron-sulfur protein had indicated that amino acid residues 138-153
might be involved in the assembly of this protein into the cytochrome bc1 complex. To determine which specific
residues might be involved in the assembly process, charged amino acids
located in the
1-
4 loop of the iron-sulfur protein were mutated
to uncharged residues and tryptophan 152 to phenylalanine. The mutant
genes were used to transform yeast cells (JPJ1) lacking the iron-sulfur
protein gene. Mutants R146I and W152F had almost undetectable growth in medium containing glycerol/ethanol, whereas mutants D143A, K148I, and
D149A grew more slowly than the wild type. Activity of the cytochrome
bc1 complex was decreased 50, 90, 67, 89, and
90% in mutants D143A, R146I, K148I, D149A, and W152F, respectively,
but unchanged in mutants D139A, Q141I, D145L, and V147S. In all of these mutants except W152F, the cytochrome c1
content, determined by immunoblotting, was comparable with that of
wild-type cells. However, immunoblotting revealed that the content of
the iron-sulfur protein was decreased proportionately in the five
mutants with lowered enzymatic activity and growth suggesting that
these amino acids are critical for maintaining the stability of the
iron-sulfur protein. The efficiency of assembly in vitro
compared with the wild type determined by selective immunoprecipitation
was unchanged in the mutants with the exception of R146I, D149A, and
W152F where decreases of 80, 60, and 60%, respectively, were observed
suggesting that these amino acids are critical for the proper assembly
of the iron-sulfur protein into the bc1
complex.
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INTRODUCTION |
The cytochrome bc1
complex1 is an integral
multiprotein complex of the inner mitochondrial membrane which
catalyzes the transfer of electrons from ubiquinol to cytochrome
c coupled to the translocation of protons across the
membrane (1, 2). The bc1 complex of yeast
mitochondria consists of 10 subunits, of which 3 have prosthetic groups
that serve as redox centers: cytochromes b and
c1 and the Rieske iron-sulfur protein. The
iron-sulfur protein is an important component of the catalytic reaction
center P of the bc1 complex where it is involved
in the transfer of electrons to cytochrome c1
from ubiquinol (2). Consistent with its catalytic function, the
iron-sulfur protein is anchored on the outer surface of the inner
mitochondrial membrane where it protrudes into the intermembrane space
(3, 4). The recent resolution of the crystal structure of the
cytochrome bc1 complex of beef heart
mitochondria has revealed that the complex exists as a dimer with 13 membrane-spanning helices (5).
With the exception of cytochrome b, the sole mitochondrial
gene product of the bc1 complex, all the
subunits of this complex are synthesized on free cytoplasmic ribosomes
and in a subsequent step imported into mitochondria where they are
assembled into a functional complex in the membrane (6). The
iron-sulfur protein of Saccharomyces cerevisiae is
synthesized as a precursor protein with a molecular mass of 29 kDa and
possesses a 30-amino acid leader sequence at the amino terminus of the
protein. The precursor form of the iron- sulfur protein is processed
in vivo into the mature form through an intermediate form in
two distinct processing events observed both in vitro and
in vivo (7, 8).
The mechanism of assembly of the subunits of the cytochrome
bc1 complex and, in particular, that of the
iron-sulfur protein has been the subject of several studies (9-11).
These investigations have led to the suggestion that the iron-sulfur
protein may be one of the last proteins to become associated with a
postulated "core" membrane-bound complex during mitochondrial
biogenesis (11, 12). In previous studies in our laboratory, the
assembly of the iron-sulfur protein into the bc1
complex was investigated in vitro by using selective
immunoprecipitation with antiserum against either the iron-sulfur
protein or the intact bc1 complex after import
of radiolabeled precursor into mitochondria lacking the iron-sulfur
protein (9). More recently, the import and assembly of 8 deletion
mutants of the iron-sulfur protein into the bc1
complex was studied using this technique (13, 14). The results obtained
in these studies indicated that the amino acids located in the
extramembranous regions of the iron-sulfur protein might be involved in
assembly with other subunits of the bc1 complex.
By contrast, the amino acid residues in the membranous region of the
protein are apparently not required for the efficient assembly of the
iron-sulfur protein into the bc1 complex (14). Further examination of one of these extramembranous regions containing amino acid residues 138-153 revealed the presence of 6 charged amino
acids located in a loop connecting the
1 helix and the
4 sheet of
a soluble fragment of the iron-sulfur protein from beef heart
mitochondria (15). To determine the possible role of these charged
amino acids in this region of the yeast iron-sulfur protein, which we
assume has a similar if not identical structure, we performed
site-directed mutagenesis of the charged amino acids present in this
region. The effect of these mutations on the activity and the assembly
of the iron-sulfur protein into the bc1 complex has been examined.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis and Transformation of Yeast
Cells--
Site-directed mutagenesis was performed using the
Stratagene Quick ChangeTM site-directed mutagenesis kit.
The codons for the selected amino acids in the predominantly charged
1-
4 loop of the iron-sulfur protein iron-sulfur protein were
altered to encode the desired amino acids according to the
manufacturer's instructions. Mutations were performed using the
RIP gene inserted in the single copy plasmid, pRG415;
however, in some instances the mutations were performed in the high
copy vector pSP64. When this plasmid was used, the mutant
rip genes were subsequently subcloned into the pRG415 vector
that was used to transform yeast cells. The mutant DNA, thus
constructed, was analyzed by restriction enzyme analysis and by
sequencing the mutated gene using the Cy5TM AutoCycle (or
AutoRead) DNA sequencing kit for use with the Alf express automated
sequencer (Amersham Pharmacia Biotech). This sequencing kit is based on
the dideoxy chain termination method (16, 17). DNA containing the
wild-type RIP gene or the mutant rip genes was
used to transform yeast cells (JPJ1), in which the RIP gene
had been deleted, by the lithium acetate method (18) as modified (19).
Mutant yeast cells were selected by their ability to grow on a medium
lacking uracil.
To test for respiratory competence, the transformed JPJI colonies from
the uracil minus plates were streaked on plates containing 1% yeast
extract, 2% peptone, 3% glycerol, and 4% ethanol, pH 5.0, in 1.5%
agar. The plates were incubated at both permissive (30 °C) and
non-permissive (37 °C) temperatures. Subsequently, the transformed
yeast cells were grown in the same liquid medium at 30 °C, and the
rate of growth was monitored by measuring the absorbance at 650 nm over
several hours.
Growth of Yeast Cells and Preparation of Mitochondria--
JPJI,
the yeast strain lacking the RIP gene, was grown aerobically
at 30 °C in a semi-synthetic medium as described previously (8). The
transformed cells were grown in a medium containing 1% yeast extract,
2% peptone, and 2% galactose prior to mitochondrial isolation for
enzymatic studies. In order to prepare mitochondria, yeast cells were
grown to early logarithmic phase (A650 = 0.9-1.2), harvested by centrifugation at 1500 × g for
5 min at room temperature, and washed once with distilled water prior
to mitochondrial isolation. For enzymatic and spectral analyses, yeast
cells were broken using the modified glass bead method (2); however,
for import studies mitochondria was prepared from yeast digested with
Zymolyase as described previously (14).
Enzymatic Assays--
The activity of the cytochrome
bc1 complex was determined by measuring the
reduction of 40 µM horse heart ferricytochrome c at 550-540 nm using the ubiquinol analog decylbenzoquinol
(DBH2) as electron donor. The assay was performed at
25 °C in 50 mM Tris, pH 7.4, 1 mM EDTA, 250 mM sucrose, and 2 mM KCN. The non-enzymatic rate of cytochrome c reduction was determined by adding 33 µM DBH2 and allowing the reaction to proceed
for 5 s after which the enzymatic reaction was initiated by
addition of 0.1 mg of mitochondrial protein. The inhibition of
enzymatic activity by antimycin A and myxothiazol was determined for
each mutant.
Optical Absorption Spectroscopy--
Optical spectra were
recorded using the dual wavelength mode of the Aminco DW-2A
spectrophotometer coupled to a recorder with reference beam set at 539 nm. Mitochondrial membranes were suspended at 2 mg/ml in 25 mM
potassium phosphate buffer, pH 7.4, 1 mM EDTA, and
1% dodecyl maltoside, oxidized by ferricyanide, and then reduced by
adding a few grains of sodium dithionite. The spectra of the oxidized
cytochromes was subtracted from that of the reduced cytochromes to
obtain the reduced versus oxidized difference spectra. The concentration of cytochromes was determined by using the following absorption coefficients and wavelength pairs for the reduced minus oxidized proteins; 20.9 mM
1 cm
1
at 553-539 for cytochrome c-c1 and
25.6 mM
1 cm
1 at 562-575 for
cytochrome b (20).
Import of the Wild-type Iron-Sulfur Protein into Mitochondria in
Vitro--
The wild-type and mutant rip genes, inserted in
the expression vector pSP64, were transcribed and translated in
vitro in the presence of [35S]methionine using the
TNT-coupled reticulocyte lysate system from Promega. The
expressed radiolabeled proteins were imported into mitochondria and
immunoprecipitated with antibodies against the iron-sulfur protein and
the intact cytochrome bc1 complex as described
previously (9, 14). The mitochondria were then reisolated and the
proteins separated by SDS-PAGE. The gels were scanned and
quantitated using a PhosphorImager (Molecular Dynamics) as
described previously (14).
Immunoblotting--
Immunoblotting was performed using the ECL
Western blotting system from Amersham Pharmacia Biotech. Mitochondria
were isolated from yeast cells and the proteins separated by SDS-PAGE
prior to transfer onto polyvinylidene difluoride or nitrocellulose
membranes. Primary antibodies raised against the iron-sulfur protein
were used to locate the proteins immobilized on the membrane. These antibodies were subsequently detected by goat anti-rabbit secondary antibodies labeled with horseradish peroxidase that catalyzes the
oxidation of luminol, thereby emitting small but sustained quantities
of light. The chemiluminescence was then specifically enhanced allowing
an image to be recorded on a photosensitive film (Amersham ECL
bulletin).
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RESULTS |
Growth Characteristics of Yeast Containing Mutant Iron-Sulfur
Proteins--
Previous studies in our laboratory involving deletion
mutants of the iron-sulfur protein had indicated that amino acid
residues 138-153 might be involved in the interaction of this protein
with other subunits during the assembly of the cytochrome
bc1 complex (13, 14). The crystal structure of a
water-soluble fragment of the iron-sulfur protein from beef heart
mitochondria had revealed that these residues are located in the
predominantly charged
1-
4 loop on the surface of the protein
(15). In order to determine which of these residues might be
specifically involved in the proposed interactions of the iron-sulfur
protein with other proteins, site-directed mutagenesis of the following
amino acids was performed as described under "Experimental
Procedures." The acidic amino acids, Asp-139, -143, and -149, were
mutated to alanine (D139A, D143A, D149A), and Asp-145 was mutated to
leucine (D145L), whereas the basic amino acids, Arg-146 and Lys-148,
were mutated to isoleucine (R146I, K148I). As a control, two uncharged
amino acid residues (Gln-141 and Val-147) were mutated to isoleucine
and serine, respectively (Q141I, V147S). In addition, Trp-152 was
mutated to phenylalanine (W152F), as a previous study had reported that
the W152R mutation resulted in a loss of enzymatic activity (21). The
mutations were confirmed by DNA sequencing.
The wild-type RIP gene and each of the mutant rip
genes were used to transform the JPJ1 strain of yeast cells, and growth was monitored on the non-fermentable carbon source glycerol/ethanol. After the initial screening on plates, the rate of growth of the transformed yeast cells was determined in liquid medium with the same
carbon sources at 30 °C. JPJ1 cells transformed with the wild-type
RIP gene, indicated as wild type in Table
I, grew with a doubling time of 2 h.
Two of the mutants, R146I and W152F, had an almost undetectable growth
rate calculated as doubling times of >12 and >10 h, respectively,
whereas mutants D143A, K148I, and D149A grew more slowly than the wild
type with doubling times of 3.5, 4, and 5 h, respectively. Growth
of the remaining mutants, D139A, Q141I, D145L, and V147S, was identical
to that of the wild-type cells.
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Table I
Growth characteristics, enzymatic activities, and cytochrome b and
c1 content in JPJ1 yeast cells transformed with the wild-type
RIP and mutant rip genes
Site-directed mutants were constructed and used to transform JPJ1 yeast
cells as described under "Experimental Procedures." Growth of the
transformed cells on the non-fermentable carbon source glycerol/ethanol
was monitored at 30 °C by measuring changes in optical density at
650 nm. To determine enzymatic activity and cytochrome content,
wild-type and mutant cells were grown in a medium containing galactose.
Mitochondrial membranes were prepared and enzymatic activity and
cytochrome content determined as described under "Experimental
Procedures."
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Ubiquinol-Cytochrome c Oxidoreductase--
The enzymatic activity
of the cytochrome bc1 complex was determined as
DBH2 cytochrome c reductase to determine whether
a lowered activity of the complex correlated with the decreased growth
rates observed for some mutant cells. For these experiments, mitochondria were isolated from the transformed mutant yeast cells that
had been grown in a medium containing galactose as carbon source. To
confirm the enzymatic nature of the observed cytochrome c
reductase activity, the inhibitory effects of the specific inhibitors of the cytochrome bc1 complex, antimycin A and
myxothiazol, were determined. The cytochrome c reductase
activity of the two mutants with very low rates of growth, R146I and
W152F, was less than 10% of that observed with mitochondria from the
wild-type cells (Table I). In addition, the enzymatic activity of the
mutants D143A, K148I, and D149A was reduced approximately 50, 67, and 89%, respectively, compared with the wild type. The remaining mutants
D139A, Q141I, D145L, and V147S retained enzymatic activity at wild-type
levels. In general, these results demonstrate a good correlation
between the observed reduction in the growth rate of these mutants in
the glycerol/ethanol medium and the enzymatic activity of the
cytochrome bc1 complex.
Spectral Analysis of Mitochondria from Mutants--
Our next
approach was to investigate whether changes in the spectral properties
or content of cytochromes b and c-c1
had occurred as a result of the mutations introduced into the
iron-sulfur protein. Spectral analysis of the wild-type mitochondria,
JPJ1 cells transformed with the wild-type RIP, is presented
for comparison with several of the iron-sulfur protein mutants (Fig.
1). The concentration of cytochromes
b and c-c1 in the wild-type
mitochondria was determined to be 0.088 and 0.095 nmol/mg of protein,
respectively (Table I). Examination of the spectra of the mutants
revealed the presence of cytochromes c-c1 at the
same level as that of the wild-type cells with the exception of W152F
in which the cytochrome c-c1 content was
diminished by 30%. By contrast, spectral analysis of mitochondria
obtained from several of the mutants revealed significant changes in
the spectra of cytochrome b leading to calculated decreases
in the amount of cytochrome b present in the mitochondria
(Fig. 1). For example, the iron-sulfur mutants D139A, Q141I, and D149A
had 16-17% less cytochrome b heme than the wild type
whereas the cytochrome b heme content was diminished by 32%
in R146I and 41% in W152F. These values can be compared with the 53%
decrease in cytochrome b heme observed in mitochondria isolated from JPJ1, the strain lacking the iron-sulfur protein. These
reductions in the cytochrome b levels may reflect damage to
the environment of the b hemes due either to the absence of the iron-sulfur protein or to a change in its conformation as a result
of the specific amino acid mutation (8).

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Fig. 1.
Spectral analysis of the cytochrome content
of mitochondrial membranes of the iron-sulfur protein-deficient mutant,
JPJ1, and the wild-type iron-sulfur protein. Mitochondrial
membranes from yeast expressing mutant and wild-type iron-sulfur
protein genes (and grown in media containing galactose) were prepared.
Difference spectra of dithionite-reduced minus ferricyanide-oxidized
samples were recorded at room temperature.
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The decreased levels of cytochrome b observed in several of
these mutants rather than the actual mutation in the iron-sulfur protein may be responsible for the observed lowered enzymatic activity
of the bc1 complex in these mutants. To make
this determination, the turnover numbers of the
bc1 complex in the various mutants were
calculated as enzymatic activity (nmol of cytochrome c
reduced mg
1 of mitochondrial protein
min
1)/the content of cytochrome b (nmol mg
1
of mitochondrial protein) (Fig. 2). A
similar pattern in the loss of enzymatic activity determined as
turnover numbers was observed for the mutants in which the enzymatic
activity was lowered. For example, the decrease in the turnover numbers
was as follows: 50% for D143A, 90% for R146I, 60% for K148I, and
90% for D149A and W152F. The turnover numbers for the remaining
mutants were not affected. These results indicate that the loss of
enzymatic activity observed in these five mutants resulted from the
mutations introduced into the iron-sulfur protein and not from a
decreased level of cytochrome b.

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Fig. 2.
Turnover numbers of JPJ1 and JPJ1 expressing
wild-type and mutant iron-sulfur proteins. Turnover numbers were
determined by dividing the specific enzymatic activity in nmol of
cytochrome c reduced per mg of mitochondrial protein by the
concentration of cytochrome b in nmol per mg of
mitochondrial protein. The turnover numbers are expressed as a percent
of the wild type.
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Western Blot Analysis--
The observation that site-directed
mutagenesis of several charged amino acids plus Trp-152 in the region
containing residues 139-152 of the iron-sulfur protein resulted in a
reduction in the enzymatic activity of the cytochrome
bc1 complex prompted an investigation of the
level of expression of the iron-sulfur protein in these mutants. The
presence of the iron-sulfur protein in mitochondria isolated from these
mutants was determined by Western blotting with an antibody against the
iron-sulfur protein. As a control, the expression of cytochrome
c1 in these mitochondria was compared on the
same gel. Immunoblot analysis revealed the presence of the iron-sulfur
protein in all of the mutants (Fig. 3,
A and C); however, the levels of expression of
the iron-sulfur protein were considerably lower in the mutants D143A,
R146I, K148I, and D149A when compared with the wild-type levels. By
contrast, the levels of cytochrome c1 determined
by immunoblotting with the antibody against cytochrome
c1 were unaffected in the mutants (Fig. 3,
B and D). These results indicate that mutating
two acidic amino acids, Asp-143 and Asp-149, two basic amino acids,
Arg-146 and Lys-148, and the aromatic residue, Trp-152, results in
varying decreases in the levels of the iron-sulfur protein observed
in vivo, perhaps resulting from a decreased expression of
the protein or from an increased instability of the protein. For
example, removal of the charges in this region of the iron-sulfur
protein or changing the tryptophan moiety through mutagenesis may
result in the formation of an unstable protein that is degraded soon after translation.

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Fig. 3.
Immunoblots of mitochondrial membranes from
the yeast cells expressing wild-type RIP and mutant
rip genes. Mitochondrial proteins were separated on
SDS-PAGE and blotted with the antisera against the iron-sulfur protein
(A and C) and against cytochrome
c1 (B and D).
i-ISP is intermediate form and m-ISP is mature
form of the iron-sulfur protein.
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Import and Assembly in Vitro--
The decreased amounts of the
iron-sulfur protein observed in several of the mutants prompted us to
investigate whether the import into mitochondria of these mutant
proteins and their subsequent assembly into the
bc1 complex were affected by the mutations. The
import and assembly of the iron-sulfur protein were determined in
vitro after transcription and translation of these rip
genes inserted into the high copy pSP64 plasmid. After coupled
transcription/translation in vitro, the radiolabeled
precursors of the mutant and the wild-type iron-sulfur protein were all
imported efficiently into the mitochondria isolated from yeast strain
JPJ1, where they underwent the identical two-step cleavage process
(Fig. 4). These results indicate that the
mutations introduced into the iron-sulfur protein had no effect on the
import and processing of the respective precursor proteins.

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Fig. 4.
Import of wild-type and mutant iron-sulfur
proteins in vitro into isolated yeast mitochondria.
Mutant and wild-type iron-sulfur protein genes were expressed in
vitro by coupled transcription/translation in the presence of
[35S]methionine to produce a labeled precursor. The
labeled precursor was incubated with isolated yeast mitochondria
isolated from JPJ1 in the import buffer. After a 30-min incubation, the
mitochondria were reisolated by centrifugation and washed as described
under "Experimental Procedures" before SDS-PAGE on a 12.5%
acrylamide gel and autoradiography. Odd numbered lanes are
lysates (lys) of the unimported precursor whereas even
numbered lanes are the imported (imp) proteins.
p, precursor; m, mature form of the iron-sulfur
protein.
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The assembly of the wild-type and mutant iron-sulfur proteins into the
cytochrome bc1 complex was investigated in
vitro using selective immunoprecipitation with specific
antibodies. The procedure is based on the principle that radiolabeled
precursor and mature forms of the iron-sulfur protein can be
immunoprecipitated with antiserum against the wild-type iron-sulfur
protein; however, the precursor form of the iron-sulfur protein cannot
be immunoprecipitated by the antiserum raised against the intact
complex III (13, 14). Moreover, this antibody does not recognize the
iron-sulfur proteins in immunoblots of mitochondrial membranes.
Consequently, the immunoprecipitation of radiolabeled iron-sulfur
protein with the antiserum against complex III after import reflects
the assembly of the iron-sulfur protein with other subunits of the
bc1 complex recognized by the complex III
antiserum. The efficiency of assembly of the wild-type and mutant
iron-sulfur proteins was determined by dividing the radioactivity
immunoprecipitated by the antiserum against complex III by that
immunoprecipitated by the antiserum against the iron-sulfur protein,
assumed to represent the total amount of the protein imported into
mitochondria (Fig. 5). Slight variations
in the efficiency of assembly of the various mutants were observed
(Fig. 6); however, the most significant
effects were noted with the following mutants: an 80% decrease in
R146I and a 60% decrease in both D149A and W152F. These results
suggest that certain amino acids, especially Arg-146, Asp-149, and
Trp-152, present in the
1-
4 loop of the iron-sulfur protein, may
be critical for the efficient assembly of this protein into the
bc1 complex.

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Fig. 5.
Assembly in vitro of the
wild-type and mutant iron-sulfur protein after import into the
mitochondria of JPJ1 cells. Isolated mitochondria (200 µg) were
incubated in import buffer with the labeled precursor iron-sulfur
protein. The mitochondria were reisolated from the import mixture and
solubilized with 1% Triton X-100 prior to incubation with the antisera
against the intact bc1 complex (CIII)
and against the iron-sulfur protein (ISP) as indicated. The
immunoprecipitates were subject to SDS-PAGE and then analyzed by
autoradiography and PhosphorImager quantitation in arbitrary units
(Abs).
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Fig. 6.
Efficiency of assembly of the mutant
iron-sulfur proteins into the cytochrome bc1
complex of mitochondria from JPJ1. The relative
radioactivities of the mature forms of the mutant and wild-type
iron-sulfur proteins assembled into the bc1
complex were quantified by PhosphorImager analysis. The percentages
indicate the value of normalized immunoprecipitates divided by that of
the wild type.
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DISCUSSION |
In the current study, we have investigated the role of charged
amino acids located in the
1-
4 loop of the iron-sulfur protein on
the activity and assembly of the yeast cytochrome
bc1 complex. These parameters were examined in
mutants in which six charged amino acids in this region were changed to
uncharged residues, and the tryptophan at position 152 at the base of
the
4 sheet was changed to a phenylalanine. Four of these mutants,
D143A, R146I, K148I, and D149A, as well as the W152F mutant grew more slowly than the wild type, whereas growth of two of the mutants, D139A
and D145L, was unaffected. A corresponding decrease in the enzymatic
activity of the bc1 complex was observed in all
of these mutants suggesting that the slower rate of growth reflected a lowered activity of the bc1 complex. For
example, the two mutants with a barely detectable growth rate, R146I
and W152F, had enzymatic activities less than 10% that of the wild
type (Table I). Spectral analysis of cytochrome b and
c-c1 in the mutant mitochondria and the
subsequent determination of turnover numbers indicated that the
reduction in the activity of the bc1 complex did
not result from a decrease in the levels of the two cytochromes.
Indeed, Western blot analysis of mitochondrial proteins from these
mutants revealed that the loss of enzymatic activity of the
bc1 complex was correlated with a decreased
level of expression of the iron-sulfur protein in these mutants. For
example, three of the slowest growing mutants, R146I, D149A, and W152F,
with corresponding very low enzymatic activity had the lowest levels of
the iron-sulfur protein; however, those mutants with moderately reduced
growth rates and enzymatic activities, D143A and K148I, had a
corresponding greater level of immunodetectable iron-sulfur
protein.
The lowered levels of the iron-sulfur protein in these mutants indicate
that four of the charged amino acids in the
1-
4 loop of the
protein plus the tryptophan at residue 152 are necessary for expression
of the iron-sulfur protein in vivo. These charged residues
and Trp-152 may be crucial in maintaining the overall stability of the
protein such that when these amino acids are mutated, the resulting
iron-sulfur protein may become unstable. Such an unstable protein might
be degraded soon after translation in the cytosol or during its import
into mitochondria. Alternately, the mutated protein might not assemble
efficiently into the bc1 complex resulting in
its subsequent degradation in the mitochondria. To distinguish among
these possibilities, the effect of these mutations on the import and
assembly of the mutated iron-sulfur proteins into the cytochrome
bc1 complex was investigated in
vitro. None of these mutations of the iron-sulfur protein had any
effect on the import of the mutated proteins into mitochondria in
vitro or on the subsequent two-step processing of the precursor to
the mature form in the mitochondrial matrix. By contrast, differences in the efficiency of assembly in vitro of the five mutations
that affect growth and activity were observed. Three of the mutants, R146I, D149A, and W152F, were assembled into the
bc1 complex with very low efficiency when
compared with the wild type. It should also be noted that these three
mutations resulted in the most severe effects on growth and enzymatic
activity of the cytochrome bc1 complex. We
suggest that Arg-146, Asp-149, and Trp-152 may be involved in
establishing the conformation of the iron-sulfur protein necessary for
its efficient assembly with the other proteins in the
bc1 complex. The charged amino acids may form
hydrogen bonds or salt bridges with other charged amino acids to
maintain the
1-
4 loop in its extended conformation, whereas the
location of Trp-152 at the base of the
4 sheet may be critical for
the overall conformation of the protein. Recent reports have indicated that mutations of the iron-sulfur protein of Rhodobacter
capsulatus also affect the conformation of the iron-sulfur protein
and consequently its stability (22).
By contrast, two of the mutants with lower enzymatic activity and
decreased levels of the iron-sulfur protein in vivo, D143A and K148I, assembled into the bc1 complex
in vitro almost as efficiently as the wild-type protein. It
should be noted that the effects of these mutations on the enzymatic
activity of the bc1 complex and the
mitochondrial content of the iron-sulfur protein in vivo were moderate in comparison with the mutations in Arg-146, Asp-149, and
Trp-152. We suggest that the charged amino acids Asp-143 and Lys-148
may not affect the conformation of the protein and its assembly into
the bc1 complex; however, these charged amino
acids may be necessary for maintaining the stability of the iron-sulfur protein. Consequently, mutating them to uncharged amino acids may have
led to a partial degradation of the mutated iron-sulfur protein during
or soon after its translation on cytosolic ribosomes.
In conclusion, we suggest that several charged amino acids and Trp-152
located in the
1-
4 loop of the iron-sulfur protein of the yeast
cytochrome bc1 complex are required to maintain
the stability of the protein in vivo. As a consequence of
this instability, lowered levels of the iron-sulfur protein were
observed in vivo resulting in a decreased activity of the
bc1 complex and slower growth of the cells.
Currently, we are extending these investigations to mutations of
charged amino acids located in the
1 helix of the iron-sulfur
protein to obtain further evidence in support of this suggestion.