From the Department of Biochemistry, Dartmouth
Medical School, Hanover, New Hampshire 03755 and the
§ Universitätsklinikum Frankfurt, ZBC, Institut
für Biochemie I, D-60590 Frankfurt, Germany
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ABSTRACT |
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The iron-sulfur proteins of the cytochrome
bc1 complexes of Schizosaccharomyces
pombe and Saccharomyces cerevisiae contain the three
amino acid motif
RX()(F/L/I)XX(T/S/G)XXXX(
)
that is typical for proteins that are cleaved sequentially in two steps by matrix processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). Despite the presence of this recognition sequence the
S. pombe iron-sulfur protein is processed only once during import into mitochondria, whereas the S. cerevisiae protein
is processed in two steps. Import of S. pombe iron-sulfur
protein in which the putative MIP or MPP recognition sites are
eliminated by site-directed mutagenesis and import of iron-sulfur
protein into mitochondria from yeast mutants that lack MIP activity
indicate that one step processing of the S. pombe
iron-sulfur protein is independent of those sites and of MIP activity.
Sequencing of the mature protein obtained after import in
vitro and of the endogenous iron-sulfur protein isolated from
mitochondrial membranes by preparative 2D-electrophoresis shows that
MPP recognizes a second site in the presequence and processing occurs
between residues 43 and 44.
If proline-20 of the S. pombe presequence is changed into a serine, a second cleavage step is induced. Conversely, if serine-24 of the S. cerevisiae presequence is changed to a proline, the first cleavage step that is normally catalyzed by MPP is blocked, causing precursor iron-sulfur protein to accumulate. Together these results indicate that a single amino acid change in the presequence is responsible for one-step processing in S. pombe versus two-step processing in S. cerevisiae.
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INTRODUCTION |
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The Rieske iron-sulfur protein of the mitochondrial cytochrome bc1 complex (1), like the majority of mitochondrial proteins, is encoded in the nucleus, translated on cytosolic ribosomes, and then targeted to the mitochondria by an amino-terminal presequence (2-4). During or after translocation of the precursor ISP1 into the mitochondria the presequence is cleaved by specific proteases.
The iron-sulfur protein of bovine heart mitochondria is processed in one-step by MPP (5), whereas the iron-sulfur proteins of Neurospora crassa (2) and Saccharomyces cerevisiae (6) are processed in two sequential steps, in which MPP removes the first part of the presequence from precursor ISP to form intermediate iron-sulfur protein, after which MIP removes an octapeptide to generate mature iron-sulfur protein (m-ISP). Why the presequence of the iron-sulfur protein is removed in two steps in some species and one step in others, and what properties of the presequence or the processing machinery determine two-step or one-step processing are not known.
Although no consensus sequences recognized by the two proteases have
been found, precursors that are cleaved in two steps are characterized
by a three amino acid motif
RX()(F/L/I)XX(T/S/G)XXXX(
)2
at the carboxyl terminus of their presequences (7, 8, 10). Recently the
gene for the iron-sulfur protein of Schizosaccharomyces pombe has been cloned (9). Comparison of the presequences of the
S. pombe, S. cerevisiae and N. crassa
iron-sulfur proteins indicates that all three proteins have the three
amino acid motif characteristic for two-step processing. However, upon
in vitro import of the S. pombe iron-sulfur
protein, it is processed to m-ISP in only one step. In the present
study we have investigated the basis for the difference between
one-step processing in S. pombe and two-step processing in
S. cerevisiae.
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EXPERIMENTAL PROCEDURES |
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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 and lysing enzymes from Trichoderma harzianum were from Sigma. Zymolyase was from ICN. Automated sequencing was performed using the Dye Terminator Sequencing Kit from Applied Biosystems Inc.
Isolation of Mitochondria-- S. cerevisiae strains W303-1A and Y6040 (11) were grown in YPD (1% 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 (12).
S. pombe mitochondria were isolated essentially as described previously (13, 14). Haploid colonies of strain 972 (mating type h-) were grown in 4 liters of BactoTM YM broth (DIFCO) to late exponential phase. Cells were harvested at 2,000 × g for 5 min, washed twice in distilled water and incubated at 0.5 g/ml in 0.5 MImport of Iron-Sulfur Protein into Mitochondria in Vitro-- The in vitro import mixture contained 4-9% (v/v) translated iron-sulfur protein precursor in rabbit reticulocyte lysate and an additional 13-18% (v/v) of rabbit reticulocyte lysate. The import 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 20-40 µg of mitochondrial protein in a total volume of 0.1 ml. When S. pombe mitochondria were used or when import was performed in the presence of o-phenanthroline, the mixture was supplemented with an additional 1 M sorbitol. Prior to the addition of radioactive precursor the import mixture was kept on ice for 5 min to energize the mitochondria and to allow o-phenanthroline to penetrate the mitochondrial membranes. To obtain deenergized mitochondria, a sample was incubated for 5 min on ice in the presence of 20 µM antimycin, 20 µg/ml valinomycin, and 20 µg/ml carbonyl cyanide p-trifluoromethoxyphenylhydrazone before addition of precursor. To all other samples, precursor was added and import was performed for 20 min or the indicated times at 30 °C while the deenergized mitochondria were kept on ice. Import was stopped by placing the samples on ice and adding antimycin, valinomycin, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the concentrations indicated above. Proteinase K treatment was used to assess the extent of import (15).
Isolation of Mitochondrial Membranes--
A 40-liter culture of
S. pombe was grown to late exponential phase, the cells were
sedimented by centrifugation, washed once with distilled water, and
once with disruption buffer (0.1 M Tris-HCl, pH 8.0, 0.25 M sorbitol, 5 mM MgCl2, 0.15 M potassium acetate, 1 mM dithiothreitol). The
pellet was resuspended in a total volume of 30 ml of disruption buffer
and frozen by slowly pouring as a thin stream into liquid nitrogen. The
frozen cells were blended in liquid nitrogen for a total of 5 min at 1 min intervals in a stainless steel Waring blender. Additional liquid
nitrogen was periodically added to prevent thawing of the cells. The
lysed cell powder was thawed under warm water, and diisopropyl
fluorophosphate was added to a final concentration of 1 mM.
The cell debris was removed by centrifugation at 3000 × g for 10 min, and the resulting supernatant was centrifuged
at 20,000 × g for 30 min to sediment the mitochondrial
membranes. The membranes were washed twice in B1 (50 mM
Tris acetate, pH 8.0, 0.4 M mannitol, 2 mM
EDTA, 1 mM diisopropyl fluorophosphate), once in B2 (50 mM Tris acetate, pH 8.0, 0.15 M potassium
acetate, 2 mM EDTA) and stored in B2 + 50% glycerol at
20 °C.
In Vitro Transcription and Translation-- In vitro transcription using Phage SP6 RNA polymerase and in vitro translation using rabbit reticulocyte lysate were performed according to supplier recommendations. In later experiments the TnT® coupled reticulocyte lysate system was used. Before use in the import experiment, polyribosomes were removed by centrifugation at 160,000 × g for 40 min.
Gel Electrophoresis-- After in vitro import samples were analyzed on 15% SDS-PAGE gels, and protein bands were visualized by autoradiography (16). Blue native-PAGE and 2D-electrophoresis of proteins from mitochondrial membranes of S. pombe was performed as described previously (17). Cytochrome bc1 complex was solubilized from mitochondrial membranes using a Triton X-100/protein ratio of 1.5. Linear 3.5-13% acrylamide gradient gels were used for Blue native-PAGE, and 13% acrylamide gels for Tricine/SDS-PAGE in the second dimension (18). The band of bc1 complex, which was visible during Blue native-PAGE was excised from a preparative gel and cut into 4 pieces. A stack of these 4 pieces was processed by Tricine/SDS-PAGE in a second dimension and electroblotted onto Immobilon P membranes (19). A 473A protein sequencer (Applied Biosystems) was used for amino-terminal sequencing of electroblotted proteins.
Subcloning and Site-directed Mutagenesis of Iron-Sulfur Protein Genes-- The S. pombe iron-sulfur protein gene was amplified by polymerase chain reaction using pFL61-SpISP (9) as template. The sense primer was 5'-TAATAAAAGCTTAGTAATTTAGACCGAATATTTTC-3' (introducing a HindIII site at the 5'-end) and the antisense primer was 5'-TAATAAGAATTGTATTTATCCGATGATAATTTTG-3'. The amplified product was purified and subcloned into the TA cloningTM Vector, introducing an EcoRI site at the 3'-end. The HindIII-EcoRI fragment was isolated and cloned into a HindIII-EcoRI-digested pGEM-3 plasmid, creating pJN36. Site-directed mutagenesis was performed using the CLONTECH Transformer mutagenesis kit. The mutations that were introduced into the S. pombe and S. cerevisiae genes were verified by sequencing the relevant coding regions and are compiled in Fig. 1, along with the names of the corresponding plasmids.
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Radiosequencing-- After in vitro transcription and translation, the wild-type S. pombe iron-sulfur protein was imported into S. pombe mitochondria using 22% (v/v) translated precursor in 0.3 ml of in vitro import mixture. Following import the mitochondria were treated with proteinase K and phenylmethylsulfonyl fluoride as described (15), and proteins were separated by SDS-PAGE using 50 µl of sample for each of six lanes. They were then transferred to Pro BlottTM membranes using 10 mM CAPS, pH 11, 10% methanol as transfer buffer. After drying between blotting paper the bands corresponding to m-ISP were excised from six lanes and subjected to NH2-terminal sequencing in an Applied Biosystems Protein Sequencer (model 476A).
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RESULTS |
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S. pombe Iron-Sulfur Protein Is Processed in One Step When Imported
into S. pombe or S. cerevisiae Mitochondria--
Mitochondrial
precursor proteins that are cleaved in two sequential steps by MPP and
MIP share a highly conserved three amino acid motif
RX()(F/L/I)XX(T/S/G)XXXX(
) at
the carboxyl terminus of their leader sequences (7, 8, 10). The residue
in position
11 from the final cleavage site also seems to be
important for recognition of the precursor by MPP (20) and is often a
basic amino acid. When we compared the deduced amino acid sequence of the iron-sulfur protein of S. pombe with the sequences for
the homologous proteins of S. cerevisiae and N. crassa which are both processed in two steps by MPP and MIP, we
found that the S. pombe protein also contained the three
amino acid motif typical of sequential cleavage by MPP and MIP (Fig.
2). The S. pombe protein also
contains a basic residue (Arg-16) at position
11 from the predicted
final cleavage site between Ser-26 and Ser-27.
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Processing of the S. pombe Iron-Sulfur Protein Still Occurs in a Single Step When MIP Activity Is Inhibited by EDTA and o-Phenanthroline-- MPP and MIP are metal-dependent proteases, and their activity can be blocked by addition of metal chelators EDTA and o-phenanthroline. In S. cerevisiae mitochondria MIP activity is blocked completely by 10 mM EDTA and 2.5 mM o-phenanthroline (6), whereas the concentrations sufficient to block MPP activity also abolish import of the precursor protein itself (21). To detect a possible intermediate, we inhibited the S. pombe processing peptidase(s) with increasing concentrations of metal chelators and imported S. pombe iron-sulfur protein into the mitochondria. Whereas it was confirmed that increasing concentrations of the chelators inhibit conversion of intermediate ISP to m-ISP when S. cerevisiae iron-sulfur protein was imported into S. cerevisiae mitochondria (Fig. 4A), no intermediate length protein was observable when the same concentrations of chelators were used to inhibit the peptidase activities during import of the S. pombe protein into the S. pombe mitochondria (Fig. 4B).
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Mutations That Destroy the Putative MPP and MIP Sites Do Not Affect
Processing of S. pombe Iron-Sulfur Protein--
To clarify whether the
putative MPP and MIP recognition sites are essential for processing of
the S. pombe iron-sulfur protein, we destroyed each of these
sites by site-directed mutagenesis. As a control we first mutagenized
the S. cerevisiae iron-sulfur protein gene to destroy the
MPP and MIP sites in that protein. We have shown previously that
changing Lys-20 and Arg-21 to glycines, which are at positions 11 and
10 from the final cleavage site, results in complete inhibition of
processing in vitro of S. cerevisiae iron-sulfur
protein by MPP (15).
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Mutation of a Proline to Serine in the S. Pombe Iron-Sulfur Protein Presequence Results in Two-step Processing, Whereas Mutation of a Serine to Proline in the S. Cerevisiae Iron-Sulfur Protein Presequence Blocks the First Step of Two-Step Processing-- In searching for an explanation as to why the potential MPP site in the S. pombe iron-sulfur protein was not recognized in either S. pombe or S. cerevisiae mitochondria, we noted the presence of a proline in position 20 of the presequence, which is at position +2 relative to the predicted MPP site (Fig. 2). In the S. cerevisiae iron-sulfur protein, a serine residue (Ser-24) is found at the equivalent position. Since replacement of a serine with a proline would have a marked effect on local secondary structure of the presequence proximal to the MPP cleavage site, we suspected that this sequence difference might account for the difference in processing of the two presequences.
When we changed Pro-20 in the S. pombe iron-sulfur protein presequence to serine (creating pJN43), an additional intermediate length protein could be detected upon in vitro import into either S. pombe or S. cerevisiae mitochondria as shown in Fig. 6, A and B, respectively. The two processing steps that result seem to be independent, since m-ISP can also be formed in a single step. Although this does not demonstrate that this intermediate length protein is converted to m-ISP, there are numerous precedents that MPP will cleave very short presequences, and on this basis we infer that this is a bona fide intermediate in two-step processing. This does not preclude formation of m-ISP by an independent pathway in a single step as shown in Fig. 7.
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Tyrosine-44 Is the Amino Terminus of the Mature S. pombe Iron-Sulfur Protein-- After having established that the predicted protease recognition sites are not necessary for processing of the S. pombe iron-sulfur protein we determined the actual processing site by radiosequencing of the mature protein after import and processing of [35S]methionine-labeled protein in vitro. As shown in Fig. 8A the methionine at position 50 is released in sequencing cycle seven, thus establishing Tyr-44 as the amino-terminal residue of the mature S. pombe iron-sulfur protein. To rule out that the observed radioactivity was due to release of methionines 32 and 33 in cycles seven and eight, we also performed radiosequencing on the mature form of an S. pombe iron-sulfur protein in which Met-32 and -33 had been changed to alanines by site-directed mutagenesis of the encoding DNA (pJN93). The same pattern for release of radioactivity as for the wild-type protein was observed, confirming the earlier findings (results not shown).
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Processing of S. pombe Iron-Sulfur Protein Is Probably Performed by
MPP and Is Independent of MIP Activity--
To determine which
protease is responsible for the single-step cleavage of the S. pombe protein we changed the amino acids in positions 2 and
3
from the processing site to glycines by site-directed mutagenesis of
the encoding DNA (creating pJN92; see Fig. 1). The arginine residue
that is found in position
2 is typical for proteins that are
processed by MPP, and we and others have shown that glycines at
position
2 and
3 inhibit processing by MPP almost completely
(21-23). When we imported the altered S. pombe protein
encoded by pJN92 into S. pombe mitochondria in
vitro, inhibition of processing could be observed when compared with the wild-type protein (Fig. 8B). We therefore conclude
that MPP is most likely the protease that cleaves the S. pombe iron-sulfur protein in a single step.
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DISCUSSION |
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The iron-sulfur proteins of the bc1
complexes of S. cerevisiae (6) and N. crassa (2)
are processed sequentially by MPP and MIP upon import into
mitochondria, whereas the iron-sulfur protein of beef heart
mitochondria is cleaved once by MPP only (5). All three proteins
contain the three amino acid motif RX()(F/L/I)XX(T/S/G)XXXX(
) that
is thought to direct two-step processing, but the bovine protein also
has an additional MPP site downstream, which is used as the cleavage
site. The properties of this iron-sulfur protein that direct two-step
processing in one species and one-step processing in another, and the
reason why this protein would be processed in two steps in some species and in one step in others are not understood. The deduced amino acid
sequence for the iron-sulfur protein of S. pombe (9) shows the three amino acid motif typical of processing by MPP and MIP, and
therefore it had been proposed that this protein was cleaved twice
(5).
The aim of our studies was to investigate the processing characteristics of the S. pombe iron-sulfur protein and to compare that processing to the processing of the homologous proteins in S. cerevisiae and bovine heart mitochondria. When the S. cerevisiae iron-sulfur protein is imported in vitro, intermediate and mature forms of the protein can easily be distinguished. Although the S. pombe iron-sulfur protein has a presequence very similar to the S. cerevisiae protein, our results clearly show that the S. pombe iron-sulfur protein is only processed in one step when imported into either S. pombe or S. cerevisiae mitochondria. To investigate whether S. pombe mitochondria contain MIP activity, we also imported S. cerevisiae iron-sulfur protein as a control. The protein was processed in two steps, albeit at a very low rate, suggesting that the S. pombe MIP either has a very low activity or an altered specificity. Another possible explanation is that an alternative protease cleaves at the MIP site unspecifically.
When we inhibited the processing proteases of S. pombe or
S. cerevisiae mitochondria by increasing concentrations of
metal chelators, no intermediate could be detected in either case,
again suggesting only one step processing. In another approach to
accumulate any possible intermediate, we destroyed the putative
recognition sites for MPP and MIP by site-directed mutagenesis. We had
shown before that changing Lys-20 and Arg-21 into glycines in the
S. cerevisiae iron-sulfur protein (see Fig. 2) abolished
in vitro processing by MPP completely, leading to
accumulation of precursor (15). Here we have shown that the MIP site of
the S. cerevisiae iron-sulfur protein can be destroyed by
changing the large hydrophobic residue, Ile-23, at position 8 from
the final cleavage site into glycine, and changing the small residue,
Ser-26, at position
5 into phenylalanine. Those amino acid changes do
not seem to block processing of the S. cerevisiae protein by
MPP, since intermediate length iron-sulfur protein accumulates when
imported into S. cerevisiae mitochondria in
vitro. When we made the same changes in the S. pombe
iron-sulfur protein and imported the S. pombe proteins into S. pombe or S. cerevisiae mitochondria,
processing was not affected at all. This confirmed that processing of
the S. pombe iron-sulfur protein occurs in one step and is
independent of those sites.
In an attempt to determine the factors that prevent MPP and MIP from
processing at the predicted sites, we changed Pro-20 in the S. pombe iron-sulfur protein, which is residue 7 from the predicted
MIP cleavage site, into a serine and also changed Ser-24 in the
S. cerevisiae iron-sulfur protein into a proline. Whereas
the first mutation resulted in the formation of an additional processing step, the second one inhibited the processing of the altered
protein almost completely. In a study that tried to identify new
natural substrates for MIP, Isaya and co-workers (10) found that serine
seems to be the preferred amino acid at position
7 from the MIP
cleavage site. It is thought that the presequences of mitochondrial
precursor proteins act by forming amphipatic helices (24), and it has
been shown that helical elements in the presequence can be recognized
by MPP (25). Proline on the other hand is a helix-breaking residue.
When in position
7 of the proposed MPP/MIP recognition site proline
seems to be a key residue, preventing the first and possibly also the
second processing step from occurring. We also substituted the prolines
in the MPP/MIP consensus motif ( ... RPLVASVSLNVPA ... ) in
the bovine iron-sulfur protein (see Ref. 5) with leucine and serine,
respectively, without any significant effect on processing upon import
into S. pombe or S. cerevisiae mitochondria
(results not shown). Ogishima et al. (26) have shown
recently that a proline in the presequence of malate dehydrogenase is
important for processing by MPP. This suggests that the function of the
proline is dependent on its position within the consensus motif.
Because our results indicated that the S. pombe iron-sulfur protein was not processed at the predicted cleavage sites, we determined the amino terminus of the mature protein by radiosequencing of the in vitro imported protein and thus established that processing occurred before Tyr-44. We confirmed this result by also microsequencing mature S. pombe iron-sulfur protein that had been generated in vivo.
To determine whether MPP cleaves at the Tyr-44 site, we mutagenized the
arginine and isoleucine residues that are found in positions 2 and
3 into glycines. It has been suggested that MPP from rat liver,
N. crassa, or S. cerevisiae mitochondria can only
cleave efficiently when an arginine is found in position
2. It has
also been shown that glycine at this position almost completely
prevents processing (21-23). The processing of the altered S. pombe protein was strongly inhibited, as would have been expected for MPP dependent cleavage. It therefore seems that processing of the
S. pombe iron-sulfur protein is more closely related to that
of the protein from bovine heart mitochondria than to processing of the
iron-sulfur protein from S. cerevisiae. To our knowledge there has been no conclusive evidence for the functional relevance of
two-step processing to date, and it is possible that during evolution
the second processing step has become obsolete and has been substituted
by one-step processing.
Acknowledgments-- We thank Dr. Grazia Isaya for providing the MIP deletion strain Y6040.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 20379 and by a fellowship from the Deutsche Forschungsgemeinschaft (to J. N.).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 Biochemistry, Dartmouth Medical School, 7200 Vail, Hanover, NH 03755. Tel.: 603-650-1621; Fax: 603-650-1389.
1 The abbreviations used are: ISP, iron-sulfur protein; MPP, matrix-processing peptidase; MIP, mitochondrial intermediate peptidase; m-ISP, mature iron-sulfur protein; MOPS, 3-(N-morpholino)propanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine.
2 Arrows indicate the peptidase cleavage sites.
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REFERENCES |
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