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INTRODUCTION |
The Rieske iron-sulfur protein is an essential subunit of
mitochondrial cytochrome bc1 complexes (1). Like
the majority of mitochondrial proteins it is encoded by a nuclear gene,
translated on cytosolic ribosomes, and then targeted to the
mitochondria by an amino-terminal presequence (2, 3). During
import and assembly of the iron-sulfur protein into Saccharomyces
cerevisiae mitochondria (4-6), a 30-amino acid amino-terminal
targeting sequence is removed in two steps. A matrix processing
peptidase (MPP)1 first
removes a 22-amino acid peptide from the presequence of the precursor
iron-sulfur protein (p-ISP) to form intermediate iron-sulfur protein
(i-ISP). Because MPP is a matrix protein (7-10), this cleavage step
presumably takes place in the mitochondrial matrix. A mitochondrial
intermediate peptidase (MIP) then removes an octapeptide from i-ISP to
generate mature length iron-sulfur protein (m-ISP). It is not clear,
however, whether this last processing step takes place before or after
the protein is inserted into the bc1 complex. It
is also not known at what step during proteolytic processing the 2 Fe:2
S cluster is inserted into the apoprotein.
Although previous experiments indicated that i-ISP can be found in the
bc1 complex in vivo (11-13),
in vitro studies suggested that only m-ISP can be assembled
into the complex (5). In the present study we employed yeast strains
that express iron-sulfur proteins in which the recognition sites for
MPP or MIP in the presequence have been destroyed by site-directed
mutagenesis. Mitochondrial membranes isolated from these strains
contain only minor amounts of mature length iron-sulfur protein, but
their cytochrome c reductase activities are comparable with
membranes from wild type yeast, indicating that the iron-sulfur cluster is inserted into the protein before MIP processes i-ISP to m-ISP. Immunoprecipitation of bc1 complex after import
of iron-sulfur protein into mitochondria in vitro
demonstrates that intermediate iron-sulfur protein is assembled into
the bc1 complex.
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EXPERIMENTAL PROCEDURES |
Materials--
Reagents for in vitro transcription
and translation of proteins were from Promega. The in vitro
translation product was labeled using Tran35S-label
(methionine) from ICN. EDTA was from Fisher, and
o-phenanthroline was from Sigma. Automated sequencing was
performed using the Dye Terminator Sequencing kit from Applied
Biosystems Inc. The yeast strains and plasmids used are shown in Table
I.
Isolation of Mitochondria--
S. cerevisiae strain
W303-1A was grown in 2% yeast extract, 2% peptone, 2% dextrose at
30 °C to an optical density at 600 nm of 2-4. Mitochondria were
isolated from spheroplasts and frozen as described previously (14).
In Vitro Transcription and Translation--
In vitro
transcription and translation using the TnT®-coupled reticulocyte
lysate system were performed according to supplier recommendations.
Before use in the import experiment, polyribosomes were removed by
centrifugation at 230,000 × g for 20 min.
Import of Iron-Sulfur Protein into Mitochondria in
Vitro--
The in vitro import mixture contained 4-12%
(v/v) translated iron-sulfur protein precursor in rabbit reticulocyte
lysate and an additional 10-18% (v/v) of rabbit reticulocyte lysate.
The mixture also contained 154 mM sucrose, 49 mM KCl, 7 mM MOPS-KOH, pH 7.2, 2.1% bovine
serum albumin, 1.4 mM MgCl2, 1 mM
ATP, 4 mM NADH, and 20-50 µg of mitochondrial protein in
a total volume of 0.1 ml. Import was performed as described previously
(15).
Immunoprecipitation--
The immunoprecipitation was performed
as described (5) with minor modifications. After import of the labeled
iron-sulfur protein into mitochondria in vitro, the washed
mitochondria from 0.2 ml of import mixture were solubilized with 3%
SDS, 1% Triton X-100, or 0.8 mg of dodecyl maltoside/mg of protein in
100 µl of a buffer containing 0.1 M Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 5 mM
diisopropylfluorophosphate. The SDS-dissociated sample was vigorously
shaken at room temperature for 30 min, whereas the samples treated with
Triton X-100 or dodecyl maltoside were shaken at 4 °C for 30 min.
Subsequently all the samples were diluted 10× with the above buffer
containing 1% Triton X-100, and in some experiments 10 mM
EDTA and 4 mM o-phenanthroline were also included.
For immunoprecipitation the samples were then incubated with the
indicated antiserum for 16 h at 4 °C. After centrifugation at
16,000 × g for 3 min 100 µl of agarose bound protein
G (Boehringer Mannheim) was added to the supernatants, and the mixture
was incubated with shaking for 1 h at room temperature. The
agarose beads were collected by centrifugation and then washed two
times for 20 min at 4 °C with 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 0.2% Triton X-100
and two times in the same buffer without Triton X-100. The washed
agarose-bound precipitates were resuspended in 50 µl of 2×
concentrated SDS-PAGE sample buffer and incubated at 95 °C for 5 min. The beads were removed by centrifugation at 16,000 × g for 1 min, and the supernatants were analyzed by SDS-PAGE and fluorography of the dried gels.
Amino-terminal Sequencing--
Purified
bc1 complex from strain JN16 was subjected to
SDS-PAGE and then transferred to Pro BlottTM membranes using 10 mM CAPS, pH 11, 10% methanol as transfer buffer. After
drying between blotting paper the bands of interest were excised and
subjected to NH2-terminal sequencing in an Applied
Biosystems Protein Sequencer (model 476A).
Western Analysis of Mitochondrial Membranes and bc1
Complexes--
Mitochondrial membranes (15) or purified
bc1 complexes (16) were resolved on 15%
SDS-PAGE gels (17) and either stained with Coomassie blue or blotted to
nitrocellulose membranes. Iron-sulfur protein and cytochrome
c1 were detected by Western blotting (18) using
monoclonal antibodies to the iron-sulfur protein or cytochrome c1. The intensities of the signals were
quantified using an Apple Color One Scanner and the NIH Image software.
Cytochrome c Reductase Activity
Measurements--
Ubiquinol-cytochrome c oxidoreductase
activities of mitochondrial membranes and purified
bc1 complexes were assayed in 50 mM
potassium phosphate, pH 7.0, 250 mM sucrose, 0.2 mM EDTA, 1 mM NaN3, 0.1% (w/v)
bovine serum albumin at 23 °C using 80 µM 2,3-dimethoxy-5-methyl-6n-decyl-1,4-benzoquinol as substrate and 50 µM cytochrome c. Reduction of cytochrome
c was monitored in an Aminco DW-2aTM spectrophotometer at
550 versus 539 nm in dual wavelength mode. Data were
collected and analyzed using an Online Instrument Systems Inc. computer
interface and software. Turnover numbers of the
bc1 complex in membranes and of the purified
enzymes were calculated on the basis of the concentration of cytochrome b, which was determined from optical spectra of the
dithionite reduced minus ferricyanide oxidized samples (19). For each
yeast strain membranes and purified enzymes from three individual
isolations were assayed in triplicate. Activities are expressed as a
percentage of the turnover number of membranes or
bc1 complex from yeast strain JN24, carrying
wild type RIP1 on a low copy expression vector. The turnover numbers of
the enzyme from JN24 ranged between 200 and 220 s
1, as
reported previously for the enzyme from wild type strains (20).
Site-directed Mutagenesis of the Iron-Sulfur Protein
Gene--
Site-directed mutagenesis was performed using the
CLONTECH Transformer Mutagenesis kit. The plasmid
pGem3-RIP, carrying the RIP1 gene for the iron-sulfur protein, was used
as the template for construction of pJN49. For construction of pJN38
and pJN63 the HindIII-PstI fragment of
YEP351-RIP1, encoding iron-sulfur protein and the promoter region, was
subcloned into pGem3, and the resulting vector was used as template for
site-directed mutagenesis. The mutagenized fragments were then excised
with HindIII and PstI and subcloned into the
expression vector pFL39:RIP1, a CEN, TRP vector carrying the gene
encoding iron-sulfur protein on a HindIII-SacI fragment. The construction of pFL39:RIP-S183C has been previously described (21).
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RESULTS |
Intermediate Length Iron-Sulfur Protein Is Assembled into the S. cerevisiae bc1 Complex When the MPP or MIP Processing Site
in the Presequence Is Destroyed--
In Neurospora crassa
and S. cerevisiae, p-ISP is translocated into the matrix,
where it is processed to i-ISP by MPP (4, 22). The remaining
octapeptide is subsequently removed by MIP to generate m-ISP. However,
it has not been established where this last processing step takes
place. We previously reported that i-ISP can be found in isolated
bc1 complex (11-13), suggesting that i-ISP is
assembled into the bc1 complex and that
processing of i-ISP to m-ISP occurs in the bc1
complex. On the other hand, the results of other studies had suggested
that only mature length iron-sulfur protein can be assembled into the
bc1 complex (5).
To test the hypothesis that i-ISP is assembled into the
bc1 complex we constructed yeast expression
plasmids that generate iron-sulfur proteins in which the MPP or the MIP
recognition sites have been destroyed. We have shown previously that
changing Lys-20 and Arg-21 to glycines in the yeast iron-sulfur protein
presequence results in complete inhibition of processing by MPP and
accumulation of precursor iron-sulfur protein in vitro (Fig.
1 and Refs. 23 and 24).

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Fig. 1.
Amino acid sequences of the mutagenized
presequences used in this study. Shown are the presequence of the
Rieske iron-sulfur protein and the amino acid replacements that were
made by site-directed mutagenesis of the cloned gene. Amino acids that
were introduced by site-directed mutagenesis are underlined.
The MPP and MIP processing sites are marked by solid arrows ( ). The
arrow in parentheses indicates that MIP cuts at
this site with very low activity. It has not been determined which
proteases are responsible for the processing steps in JN16, in which
the MPP site is blocked. The first two processing sites indicated by
arrows have been determined by sequencing of the
intermediate length proteins in purified bc1
complex isolated from this strain.
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The MIP recognition site is defined by a large hydrophobic residue in
position
8 and a small hydroxylated residue or glycine in position
5 from the final MIP cleavage site (25), equivalent to residues
Ile-23 and Ser-26 in the yeast iron-sulfur protein presequence. To
destroy this site we first changed Ile-23 to glycine. This mutation
resulted in accumulation of i-ISP after import in vitro;
however, a significant amount of m-ISP was still formed (results not
shown). To further improve the inhibition of processing by MIP we then
also changed Ser-26 into phenylalanine. As shown in Fig.
2, this mutated iron-sulfur protein
(pJN49) is processed at an extremely low rate by MIP, and only trace
amounts of m-ISP are formed.

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Fig. 2.
Import into mitochondria of iron-sulfur
protein containing altered presequence. Iron-sulfur protein in
which the MIP site is blocked was imported into mitochondria in
vitro for the indicated times. After import half of each sample
was treated with proteinase K.
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To determine whether insertion of the iron-sulfur cluster is required
before MIP can perform the final processing step, we also included an
iron-sulfur protein in which Ser-183 had been changed to cysteine
(pFL39:RIP-S183C). We previously showed that this mutation results in
the loss of the iron-sulfur cluster but has no effect on the stability
of the protein (21). To determine the effect of these mutations on
processing of the iron-sulfur protein presequence, we isolated
mitochondrial membranes from yeast strains expressing wild type and
mutated iron-sulfur proteins and performed Western blots on them. As
shown in Fig. 3A, the main
species that is formed when the MIP site is blocked is i-ISP, as was
expected. In this strain very little m-ISP is present in the
mitochondrial membranes, and the ratio of i-ISP to m-ISP is the inverse
of that in mitochondrial membranes from the wild type strain.

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Fig. 3.
Immunoblot analysis and polyacrylamide gel
electrophoresis of mutant iron-sulfur proteins in mitochondrial
membranes and isolated bc1 complexes.
Mitochondrial membranes containing 2 pmol of bc1
complex (A) or 2 pmol of purified bc1
complexes (B) isolated from yeast strains expressing wild
type and mutated iron-sulfur proteins (see Fig. 1) were separated by
SDS-PAGE and blotted to nitrocellulose, and the blots were probed with
antibodies against iron-sulfur protein and cytochrome
c1. In C samples containing 110 pmol
of cytochrome bc1 complexes purified from yeast
strains expressing wild type or mutated iron-sulfur proteins were
separated on a 15% SDS-PAGE gel and stained with Coomassie blue to
visualize the protein bands. The arrows point to the two
intermediates that were subjected to amino-terminal sequencing. The
asterisk marks an impurity that migrates slightly ahead of
m-ISP.
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In membranes from JN16, where the MPP site has been destroyed,
alternative processing of the presequence occurs, such that several
intermediates of different lengths can be detected. We determined two
of the additional processing sites by NH2-terminal sequencing of the intermediates, indicated by arrows in
Figs. 1 and 3C. One cleavage occurs after Arg-5 in the
presequence, and another one occurs after Met-15 with additional
cleavage sites in between. The basic residues in the presequence are
not part of the recognition motif for those cleavage sites, because
changing each basic residue in the presequence individually did not
alter the processing pattern compared with that in JN16 (results not shown). The iron-sulfur protein that possesses the S183C mutation is
processed like the wild type protein, suggesting that insertion of the
iron-sulfur cluster is not an obligatory prerequisite for processing
(Fig. 3A). However, the relative amount of m-ISP in the
S183C form of the iron-sulfur protein is decreased in comparison to
that from the wild type strain (Fig. 3C), suggesting that
processing from i-ISP to m-ISP is retarded if the cluster is not inserted.
To determine whether the intermediate length forms of the iron-sulfur
protein are assembled into the bc1 complex, we
isolated bc1 complexes from the mutant and wild
type strains and performed Western blots on them (Fig. 3B).
From the Western blots of the mitochondrial membranes in Fig.
3A and of the purified bc1 complexes in Fig. 3B, it is clear that basically all of the
intermediate length species that are found in the membranes are
assembled into the bc1 complex. In a parallel
experiment we stained an SDS-PAGE gel of these
bc1 complexes with Coomassie blue (Fig.
3C). These findings confirm that i-ISP is assembled into the
bc1 complex.
Intermediate Length Iron-Sulfur Protein Is Functionally Active in
the bc1 Complex of S. cerevisiae--
To test whether the
intermediate length species in the bc1 complexes
of JN16 and JN20 contain iron-sulfur cluster, we determined the
cytochrome c reductase activities of membranes and
bc1 complexes isolated from these strains. We
also determined the relative amounts of i-ISP and m-ISP from the
intensity of the bands on the Western blots.
From the results in Fig. 4, it is clear
that basically all of the i-ISP in the strain in which the MIP site is
blocked (JN20) and at least a major portion of the intermediates in the
strain in which MPP is blocked (JN16) are functionally active and
therefore must contain the iron-sulfur cluster. The densitometry
measurements (Fig. 4A) indicate that m-ISP accounts only for
about 5-15% of the total iron-sulfur protein when the MIP or MPP
processing site is blocked, whereas mitochondrial membranes isolated
from these strains exhibit bc1 complex activity
comparable with those from the wild type strain (Fig.
4B).

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Fig. 4.
Effect of iron-sulfur proteins containing
mutated presequences on cytochrome c reductase
activities of the cytochrome bc1
complex. In A immunoblots of mitochondrial membranes
and purified bc1 complexes were scanned and
quantified by densitometry. In B cytochrome c
reductase activities of mitochondrial membranes and isolated cytochrome
bc1 complexes were measured. The activities are
expressed as percentages of the activity measured with membranes or
bc1 complex isolated from the strain expressing
the wild type iron-sulfur protein.
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These results show that i-ISP is assembled and functionally active in
the bc1 complex of S. cerevisiae.
However, it is noteworthy that the bc1 complex
isolated from JN20, in which the MIP site is blocked, has only about
50% activity when compared with that from the wild type strain. A
similar drop in turnover number in the isolated
bc1 complex in comparison to the mitochondrial
membranes is observed in the case of JN16. It therefore seems that
activity is lost during purification of the complex in which i-ISP is
not processed to m-ISP.
Intermediate Length Iron-Sulfur Protein Can Be Assembled into the
bc1 Complex of S. cerevisiae in Vitro--
To determine
whether the intermediate length forms of the iron-sulfur protein also
interact with other subunits of the bc1 complex
in vitro, we imported wild type iron-sulfur protein and iron-sulfur proteins in which the MIP or MPP recognition sites were
destroyed into mitochondria in vitro. After import the
membranes were extracted with different detergents, and the extracts
were precipitated with antibodies against the iron-sulfur protein or antibodies that recognize the bc1 complex but
not the iron-sulfur protein. As shown in Fig.
5A, if membranes are extracted
with either Triton X-100 or dodecyl maltoside, which do not denature the bc1 complex, i-ISP is precipitated by the
antibodies against the complex. If the membranes are extracted with
SDS, which denatures the bc1 complex, antibodies
raised against the iron-sulfur protein precipitate the iron-sulfur
protein, but antibodies raised against the bc1
complex do not. These results clearly show that i-ISP can also be
assembled into the bc1 complex in
vitro.

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Fig. 5.
Immunoprecipitation of iron-sulfur protein
and cytochrome bc1 complex after import of
mutated iron-sulfur proteins into mitochondria in
vitro. Iron-sulfur proteins were imported into
mitochondria in vitro as described under "Experimental
Procedures." After the import reaction mitochondria were solubilized
with the indicated detergents and precipitated with the indicated
antibodies. In A the immunoprecipitations were performed in
the absence of metal chelators, and in B metal chelators
were included to inhibit protease processing during the precipitation.
The precipitated proteins were then subjected to SDS-PAGE, and the
dried gels were analyzed by fluorography. TX, Triton X-100;
DM, dodecyl maltoside.
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During the 16-h incubation for immunoprecipitation there appears to be
some proteolytic processing of i-ISP and m-ISP (Fig. 5A).
When we included high concentrations of EDTA and
o-phenanthroline during the overnight incubation step (but
not during import), this processing was strongly inhibited (Fig.
5B). In the precipitations in Fig. 5A the amounts
of iron-sulfur protein precipitated by the antibodies to the
bc1 complex are comparable with that
precipitated by antibodies to the iron-sulfur protein, provided that
the membranes were extracted with nondenaturing detergents. If
o-phenanthroline is included during the incubation and
precipitation, the amounts of iron-sulfur protein precipitated by the
antibodies to the bc1 complex are significantly
decreased, relative to what is precipitated by antibodies specific to
the iron-sulfur protein (Fig. 5B). This suggests that
o-phenanthroline destabilizes the binding of iron-sulfur protein to the rest of the bc1 complex, probably
as a result of disruption of the iron-sulfur cluster as described by
Boumans and co-workers (26, 27).
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DISCUSSION |
We have investigated the order of processing and assembly steps
during import of the Rieske iron-sulfur protein into S. cerevisiae mitochondria. In N. crassa p-ISP is
translocated into the matrix where the first part of the presequence is
removed by MPP, and then i-ISP is retranslocated across the inner
mitochondrial membrane and assembled into the
bc1 complex (22). At some point during this step the second part of the presequence is removed by MIP, and the
iron-sulfur cluster is inserted. It had been controversial whether
i-ISP was first incorporated into the bc1
complex and then cleaved (11-13) or whether only m-ISP could be
assembled into the bc1 complex (5).
Isaya and co-workers (28) showed that approximately 40% of the total
amount of MIP in the mitochondria fractionates with the mitochondrial
membranes and that the majority of the i-ISP also fractionates with the
mitochondrial membranes in a MIP-deficient strain. We therefore changed
Ile-23 and Ser-26 in the presequence to glycine and phenylalanine,
respectively, so that the in vitro processing of i-ISP to
m-ISP was inhibited and i-ISP accumulated in mitochondrial membranes
in vivo. Although the membranes only contained 5-15% of
the amount of m-ISP of wild type membranes, they exhibited 100%
cytochrome c reductase activity. This result clearly shows
that not only is i-ISP assembled into the bc1
complex but also that it contains the iron-sulfur cluster. We cannot
conclude from our results, however, whether the iron-sulfur cluster is inserted into i-ISP while it is still in the matrix or after it has
been translocated to the outer side of the inner mitochondrial membrane. The last step of the assembly process then seems to be the
processing of i-ISP containing the 2 Fe:2 S cluster to m-ISP by
MIP.
In bovine mitochondria the cleaved presequence of the iron-sulfur
protein is retained as a subunit in the bc1
complex (29). In the crystal structure of the bovine
bc1 complex, the amino terminus of m-ISP and the
cleaved presequence are located on the matrix side of the complex (30,
31). It thus seems likely that the bovine iron-sulfur protein is
processed after it is assembled into the complex (29).
A variation of this mechanism could be applicable to the S. cerevisiae iron-sulfur protein. No subunit analogous to the bovine iron-sulfur protein presequence has been identified so far in S. cerevisiae. However, in contrast to the bovine iron-sulfur protein, the S. cerevisiae presequence is processed in two
steps. In unpublished experiments we have found that the 22-amino acid peptide that is removed by MPP is not present in the
bc1 complex or in mitochondrial membranes.
However, the octapeptide that is cleaved from i-ISP by MIP may stay in
the complex as a yet unidentified subunit. This might explain why
mitochondrial membranes isolated from JN20, in which the blocked MIP
site prevents formation of the octapeptide, exhibit 100%
bc1 complex activity, whereas isolated bc1 complex is only 50% active. The absence of
the cleaved octapeptide might impair structural integrity in a manner
that indirectly affects catalytic activity but does not cause loss of
iron-sulfur protein from the complex.
In the purified bc1 complexes from the strains
in which the MIP and MPP sites were blocked, we noticed reduced levels
of subunit 10 (Fig. 3C). The amount of subunit 10 was also
decreased, but to a lesser extent, in the bc1
complex from the S183C mutant. In the crystal structure of the bovine
enzyme, the homologous subunit interacts with the iron-sulfur protein
and is peripherally located (30, 31). Taken together these results
suggest that subunit 10 is added after i-ISP is processed to m-ISP.
To determine whether the iron-sulfur cluster must be inserted before
i-ISP is processed to m-ISP, we included a mutant in which the
iron-sulfur cluster is not properly inserted but the apoprotein is
stably assembled into the complex (21). The ratio of m-ISP to i-ISP in
isolated bc1 complex or mitochondrial membranes from the S183C mutant as judged by Coomassie blue staining is diminished compared with the ratio in the membranes from wild type
yeast. Therefore we conclude that although the iron-sulfur cluster
normally is inserted into i-ISP, the processing from i-ISP to m-ISP
does not require cluster insertion, but either the processing is slowed
in the absence of cluster insertion or m-ISP is less stable without the cluster.
In previous experiments, Fu and Beattie (5) imported iron-sulfur
protein into S. cerevisiae mitochondria in vitro
and precipitated the radioactively labeled protein with antibodies
specific to iron-sulfur protein or with antibodies that had been raised
against bc1 complex but did not recognize
iron-sulfur protein. After in vitro import of iron-sulfur
protein and extraction of the mitochondria with detergent, both
antibodies precipitated m-ISP. When MIP processing of i-ISP to m-ISP
was inhibited by addition of EDTA and o-phenanthroline, the
antibodies against iron-sulfur protein precipitated i-ISP, whereas the
antibodies raised against bc1 complex did not.
From this it was concluded that m-ISP but not i-ISP could be assembled into the bc1 complex.
We performed similar experiments, but instead of blocking the
processing of the presequence with metal chelators we imported iron-sulfur proteins with modified presequences in which the MPP or MIP
recognition sites had been destroyed. Our data show that i-ISP is
assembled with other components of the bc1
complex in these mutant strains and that even in mitochondrial membrane
extracts from wild type yeast a significant amount of i-ISP can be
precipitated with antibodies against bc1 complex.
Boumans and co-workers (26, 27) showed that o-phenanthroline
binds to solubilized bc1 complex and disrupts
the iron-sulfur cluster. This can explain our finding that
coimmunoprecipitation of iron-sulfur protein with the
bc1 complex in the presence of o-phenanthroline is much less efficient, if after
o-phenanthroline has disrupted the iron-sulfur cluster the
protein is less stably associated with the complex. In addition, we
have shown previously that high concentrations of EDTA and
o-phenanthroline not only block processing of the
iron-sulfur protein but also inhibit import of the precursor protein
into the mitochondrial matrix (24). Either of these
o-phenanthroline effects might explain why Fu and Beattie
(5) were unable to detect i-ISP associated with the
bc1 complex after they inhibited MIP with
o-phenanthroline.