Intermediate Length Rieske Iron-Sulfur Protein Is Present and Functionally Active in the Cytochrome bc1 Complex of Saccharomyces cerevisiae*

Jürgen H. Nett and Bernard L. TrumpowerDagger

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To investigate the relationship between post-translational processing of the Rieske iron-sulfur protein of Saccharomyces cerevisiae and its assembly into the mitochondrial cytochrome bc1 complex we used iron-sulfur proteins in which the presequences had been changed by site-directed mutagenesis of the cloned iron-sulfur protein gene, so that the recognition sites for the matrix processing peptidase or the mitochondrial intermediate peptidase (MIP) had been destroyed. When yeast strain JPJ1, in which the gene for the iron-sulfur protein is deleted, was transformed with these constructs on a single copy expression vector, mitochondrial membranes and bc1 complexes isolated from these strains accumulated intermediate length iron-sulfur proteins in vivo. The cytochrome bc1 complex activities of these membranes and bc1 complexes indicate that intermediate iron-sulfur protein (i-ISP) has full activity when compared with that of mature sized iron-sulfur protein (m-ISP). Therefore the iron-sulfur cluster must have been inserted before processing of i-ISP to m-ISP by MIP. When iron-sulfur protein is imported into mitochondria in vitro, i-ISP interacts with components of the bc1 complex before it is processed to m-ISP. These results establish that the iron-sulfur cluster is inserted into the apoprotein before MIP cleaves off the second part of the presequence and that this second processing step takes place after i-ISP has been assembled into the bc1 complex.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table I
Yeast strains and plasmids

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (down-arrow ). 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.

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.

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.

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.

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.

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).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

We thank Dr. Diana Beattie for the gift of antibodies against bc1 complex. We also thank Dr. Chris Snyder for bc1 complex isolated from yeast strain CHS14 and many helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 20379.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, Dept. of Biochemistry, 7200 Vail, Hanover, NH 03755. Tel.: 603-650-1621; Fax: 603-650-1389.

    ABBREVIATIONS

The abbreviations used are: MPP, matrix processing peptidase; MIP, mitochondrial intermediate peptidase; p-ISP, precursor iron-sulfur protein; i-ISP, intermediate iron-sulfur protein; m-ISP, mature iron-sulfur protein; MOPS, 3-(N-morpholino) propanesulfonic acid; CAPS, cyclohexylaminopropane sulfonic acid; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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