Activation of a Matrix Processing Peptidase from the Crystalline Cytochrome bc1 Complex of Bovine Heart Mitochondria*

Kaiping DengDagger , Li ZhangDagger , Anatoly M. KachurinDagger , Linda YuDagger , Di Xia§, Hoeon Kim§, Johann Deisenhofer§, and Chang-An YuDagger

From the Dagger  Oklahoma State University, Stillwater, Oklahoma 74078-0454 and the § Howard Hughes Medical Institute and University of Texas, Southwestern Medical Center, Dallas, Texas 75235

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

No mitochondrial processing peptidase (MPP) activity is detected in crystalline bovine heart mitochondrial cytochrome bc1 complex, which possesses full electron transfer activity. However, when the complex is treated with increasing concentrations of Triton X-100 at 37 °C, the electron transfer activity decreases, whereas peptidase activity increases. Maximum MPP activity is obtained when the electron transfer activity in the complex is completely inactivated with 1.5 mM of Triton X-100. This result supports our suggestion that the lack of MPP activity in the mammalian cytochrome bc1 complex is because of binding of an inhibitor polypeptide to the active site of MPP located at the interface of core subunits I and II. This suggestion is based on the three-dimensional structural information for the bc1 complex and the sequence homology between subunits of MPP and the core subunits of the beef complex. Triton X-100, at concentrations that disrupt the structural integrity of the bc1 complex as indicated by the loss of its electron transfer activity, weakens the binding of inhibitor polypeptide to the active site of MPP in core subunits, thus activating MPP. The Triton X-100-activated MPP is pH-, buffer system-, ionic strength-, and temperature-dependent. Maximum activity is observed with an assay mixture containing 15 mM Tris-HCl buffer at neutral pH (6.5-8.5) and at 37 °C. Activated MPP is completely inhibited by metal ion chelators such as EDTA and o-phenanthroline and partially inhibited by myxothiazol (58%), ferricyanide (28%), and dithiothreitol (81%). The metal ion chelator-inhibited activity can be partially restored by the addition of divalent cations such as Zn2+ (68%), Mg2+ (44%), Mn2+ (54%), Co2+ (62%), and Fe2+ (92%), indicating that metal ion is required for MPP activity. The cleavage site specificity of activated MPP depends more on the length of amino acid sequence from the mature protein portion and less on the presequence portion, when a synthetic peptide composed of NH2-terminal residues of a mature protein and the COOH-terminal residues of its presequence is used as a substrate.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Bovine heart mitochondrial ubiquinol-cytochrome c reductase (1, 2), the cytochrome bc1 complex, which catalyzes electron transfer from ubiquinol to cytochrome c, has recently been crystallized (3-6) and its structure determined at 2.9-Å resolution (7). This complex contains four redox prosthetic groups: cytochromes b566 and b562, cytochrome c1, and a high potential iron-sulfur cluster (2Fe-2S Rieske center). The crystalline complex is composed of 11 protein subunits; the three (subunits III, IV, and V), which house b-type cytochromes, cytochrome c1, and the iron-sulfur cluster, respectively, are the major subunits, whereas the eight (subunits I, II, VI-XI) containing no redox prosthetic groups are the supernumerary subunits (8). Biochemical and biophysical studies of the three major subunits have been extensive and a wealth information has been obtained. However, the functions of the supernumerary subunits are largely unknown. The delay in functional assessment of supernumerary subunits is mainly because of the unavailability of a reconstitutively active cytochrome bc1 complex depleted of a given supernumerary subunit.

By using gene deletion and complementation approaches, the functions of supernumerary subunits in the yeast bc1 complex have been suggested. Subunit I (core I) (9) and subunit II (core II) (10) are essential for maintaining proper conformation of apocytochrome b for the addition of heme; subunit VI (11, 12) is involved in manipulating dimer/monomer transition; subunits VII and VIII (13) are essential for assembly of the bc1 complex; and subunit IX (14) interacts with the iron-sulfur protein and cytochromes b and c1.

Recently it was reported that the plant mitochondrial cytochrome bc1 complex from potato tuber (15-17) and spinach leaves (18, 19) possesses a mitochondrial processing peptidase (MPP)1 activity in addition to the electron transfer activity. The MPP activity is associated with the core subunits of the plant complex based on sequence homology (15, 16) and immunological similarity (15, 18, 20) between subunits of non-plant MPPs and core subunits of the plant bc1 complex.

MPP belongs to the pitrilysin family of zinc metalloproteases with an inverted zinc binding motif, whose members include insulin degrading enzymes from mammals and protease III from bacteria (21). MPP has been purified from Neurospora crassa (22), Saccharomyces cerevisiae (23), and rat liver (24) and shown to contain two nonidentical subunits, alpha -MPP and beta -MPP, that cooperate in processing. Both subunits are matrix proteins except for beta -MPP from N. crassa, which is partially attached to the mitochondrial inner membrane. Most mitochondrial precursor proteins are cleaved to the mature form in a single step by MPP. MPPs of different species share common amino acid motifs, including the zinc binding HXXEH(X)72-76E motif in the beta -subunit and the HXXEK(X)72-76E or HXXD/ER(X)72-76E in the alpha -subunit (25). Both histidines and the distal glutamate in the HXXEH(X)72-76E motif of the beta -subunit are zinc binding residues and constitute the active site of MPP. The spacing of about 72-76 amino acids between the putative 2nd and 3rd metal binding residues seems to be of importance for enzyme function, as this distance is conserved during evolution from S. cerevisiae to plant and mammalian MPPs.

Sequence analysis reveals that core I protein of the bovine bc1 complex (26)2 has 56% identity with the beta -subunit of rat MPP, 38% with yeast, and 42% with potato, and core II protein (26) is 27% identical to the alpha -subunit of rat MPP, 28% to yeast, and 30% to potato. In addition, a sequence of Y57XXE60H61(X)76E137, similar to the zinc-binding motif, HXXEHX76E, in the beta -subunit of MPP, is present in the core I protein of the bovine complex. The recent three-dimensional structural information on the bc1 complex shows that glutamic acid 137 is close to the other two metal binding residues, tyrosine 57 and histidine 61, in the putative zinc binding motif of the core I protein (no electron density is apparent for zinc atom in the structure at the current level of resolution (7)). Also, lysine 286 and arginine 287 of the core II protein are structurally close to the putative zinc binding motif of the core I protein and may contribute to the active site (see Fig. 1).


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Fig. 1.   The three-dimensional structure of the bc1 complex in the core subunit region containing the putative MPP active site. The amino acid residue numbers are based on the sequence of mature protein.

Despite the sequence homology between the core subunits of the beef bc1 complex and those of MPP and the presence of a zinc binding motif, similar to that of MPP, in the core I protein, no MPP activity is detected in the active beef bc1 complex. In the bc1 crystal, the overall shape of each core protein resembles a bowl with approximate twofold integral symmetry. The NH2-terminal domain of core I is interacting with the COOH-terminal domain of core II and vice versa. Core I and core II enclose a big cavity. Because the two internal approximate 2-fold rotation axes of core I and core II differ in direction by 14.5°, the two bowls come together in the form of a ball, with a crack leading to the internal cavity. The crack is filled with part of the 50-residue sequence of an unassigned polypeptide (see Fig. 1B of Ref. 7). Based on this structural information, we suggest that the lack of MPP activity in bovine bc1 complex is because of the binding of a polypeptide to the active site of MPP in the core subunits upon complex maturation. If this suggestion is correct, one would expect to see MPP activity when this inhibitor polypeptide is released or its binding to the core proteins is weakened. Herein we report the conditions for activation of MPP from the crystalline cytochrome bc1 complex and the properties of activated MPP. Preliminary results on this subject have been reported (28).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Cytochrome c, Type III, Triton X-100, and sodium cholate were purchased from Sigma. Diheptanoyl phosphatidylcholine was a product of Avanti Polar Lipids Inc. 2,3-Dimethoxyl-5-methyl-6-geranyl-1,4-benzoquinone (Q2) and its reduced form (Q2H2) were synthesized as reported previously (29). Asolectin was a product of Associate Concentrate. Other chemicals were of the highest purity commercially available.

Polypeptide Substrates-- Polypeptides that are composed of COOH-terminal regions of presequences and NH2-terminal regions (as indicated by bold letters) of matured subunits of V, IV, and beta -F1 were synthesized by the Recombinant DNA/Protein Resource Core Facility at Oklahoma State University. These synthetic polypeptides are: 1) V P A S V R Y S H T D I K (-7V+6); 2) A S V R Y S H T D I K V P D F S D Y R R P E V L D (-5V+20); 3) R P L V A S V S L N V P A S V R Y S H T D I K V P D F (-17V+10); 4) A V A L H S A V S A S D L E L H P P S Y (-10IV+10); 5) A V A L H S A V S A S D L E L H P P S Y P W S H R G L L S S (-10IV+20); 6) A L Q P A R D Y A A Q A S P S P K A (-8beta +10); and 7) A L Q P A R D Y A A Q A S P S P K A G A T T G R I V A V (-8beta +20).

Enzyme Preparations and Assays-- Purified cytochrome bc1 complex (ubiquinol-cytochrome c reductase) suitable for protein crystallization was prepared as reported previously (2). Crystals of the cytochrome bc1 complex were obtained using diheptanoyl phosphatidylcholine as detergent (2). The crystals were collected and washed with precipitating buffer containing no detergent and redissolved in 50 mM Tris-HCl buffer (tris(hydroxymethyl)aminomethane base titrated to the appropriate pH with hydrochloric acid), pH 8.0, containing 0.2% potassium deoxycholate.

For ubiquinol-cytochrome c reductase activity assay, an appropriate amount of the bc1 complex was added to an assay mixture (1 ml) containing 50 mM potassium/sodium phosphate buffer, pH 7.0, 1 mM EDTA, 100 µM cytochrome c, and 25 µM Q2H2. Activity was determined by measuring the reduction of cytochrome c (the increase in absorbance at 550 nm) in a Shimadzu UV 2101 PC spectrophotometer at 22 °C. A millimolar extinction coefficient of 18.5 was used to calculate activity. Nonenzymatic oxidation of Q2H2, determined under the same conditions in the absence of enzyme, was subtracted.

For MPP activity assay, an appropriate amount of the cytochrome bc1 complex was added to an assay mixture (20 µl) containing 15 mM Tris-HCl, pH 8.0, 1.5 mM Triton X-100, and 40 µg substrate polypeptide and incubated at 37 °C for 24 h. After incubation the mixture was centrifuged at 28,000 × g for 15 min, and the supernatant solution was applied to a C-8 HPLC column. The column was eluted with a linear gradient formed from 72 ml each of 0.1% trifluoroacetic acid and 90% acetonitrile in 0.1% trifluoroacetic acid with a flow rate of 0.8 ml/min. The peptides were monitored at 214 or 280 nm. The MPP activity calculation is based on the amount of product peak produced. The product peptide is indicated by its dependence on the amount of bc1 used in the assay system. A typical HPLC assay profile for MPP activity is shown in Fig. 2. The product peaks were collected, dried, and subjected to partial NH2-terminal sequence analysis to determine the cleavage site of MPP.


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Fig. 2.   MPP activity assay by HPLC. Twenty µl of assay mixture containing 15 mM Tris-HCl buffer, pH 8.0, 1.5 mM Triton X-100, 40 µg of substrate peptide-2, A S V R Y S H T D I K V P D F S D Y R R P E V L D (-5V+20) were added to 0 (chromatogram 1), 40 µg (chromatogram 2), and 80 µg (chromatogram 3) of the bc1 complex. The mixtures were incubated at 37 °C for 24 h and centrifuged at 28,000 × g for 15 min. The supernatant solutions were subjected to HPLC separation using the conditions described under "Experimental Procedures." Peak A is nonproteineous component. Peak B is a product peptide with the sequence ASVRY, and Peak C is a mixture of a product peptide having the sequence S H T D I K V P D F S D Y R R P E V L D and the remaining substrate peptide.

    RESULTS AND DISCUSSION
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Introduction
Procedures
Results & Discussion
References

Detergent Concentration-dependent Inactivation of Ubiquinol-cytochrome c Reductase Activity and Activation of MPP Activity of Crystalline Cytochrome bc1 Complex-- Crystalline cytochrome bc1 complex dissolved in 50 mM Tris-HCl buffer, pH 8.0, containing 0.2% deoxycholate, exhibits full activity in catalyzing antimycin-sensitive electron transfer from Q2H2 to cytochrome c but shows no MPP activity. This is different from the plant mitochondrial cytochrome bc1 complex in which both MPP and electron transfer activities are observed. Based on the three-dimensional structural information for the beef bc1 complex (7), the lack of MPP activity may be because of the binding of an inhibitor peptide to the putative active site of MPP located at the interface of the core subunits during complex maturation. One way to confirm this suggestion is to activate MPP in the beef cytochrome bc1 complex by removal of the binding of inhibitor polypeptide or by weakening its binding with the core subunits in the complex.

Weakening the binding between inhibitor polypeptide and the core subunits can be achieved by disrupting the structural integrity (or loosening the structure) of the complex with detergents or protein denaturing reagents, such as urea or guanidine HCl. Because electron transfer activity requires the structural integrity of the complex, the extent of activity lost indicates the extent of bc1 structure disruption. Fig. 3 shows Triton X-100 concentration-dependent inactivation of the electron transfer activity and activation of MPP of the cytochrome bc1 complex. Electron transfer activity decreases, whereas MPP activity increases with increasing concentrations of Triton X-100. Maximum MPP activity is obtained when Triton X-100 concentration reaches 1.5 mM at which point more than 98% of the ubiquinol-cytochrome c reductase activity is lost. The MPP activity remains unchanged even at a Triton X-100 concentration of 4.5 mM. These results confirm our suggestion that MPP activity in the beef cytochrome bc1 complex is inhibited by the binding of a polypeptide to its active site in the core subunits. Disruption of the structural integrity of the bc1 complex by Triton X-100, as indicated by the loss of its electron transfer activity, is most likely because of modification of the hydrophobic interactions in the complex. Because the core proteins are hydrophilic, they are expected to be less sensitive to the Triton X-100 treatment. Apparently the conditions that weaken the binding between the inhibitor peptide and MPP do not affect the enzymatic activity of MPP.


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Fig. 3.   Effect of Triton X-100 concentration on ubiquinol-cytochrome c reductase and MPP activity of the cytochrome bc1 complex. Twenty-µl aliquots containing 15 mM Tris-HCl buffer, pH 8.0, 40 µg of the cytochrome bc1 complex, and 40 µg of substrate peptide, V P A S V R Y S H T D I K (-7V+6), were treated with the indicated concentrations of Triton X-100, incubated at 37 °C for 24 h, and assayed for ubiquinol-cytochrome c reductase activity (QCR) (open circle ) and MPP activity (×). The 100% ubiquinol-cytochrome c reductase activity equals 10 µmol of cytochrome c reduced per min/nmol of cytochrome b, at room temperature. The highest product peak was assumed to represent 100% MPP activity.

Other nonionic detergents, such as Zwittergent, decanoyl-N-methylglucamide and octyl glucoside, also activate MPP in the beef cytochrome bc1 complex. Ionic detergents, such as sodium cholate, deoxycholate, and sodium dodecylsulfate, and chaotropic reagents, such as urea and guanidine HCl, do not activate MPP even though they inactivate the electron transfer activity of the complex. The failure to detect MPP activity in complex treated with ionic detergents or chaotropic reagents may result from their inhibitory effect on MPP rather than from ineffective weakening of the binding of inhibitory polypeptide with core subunits.

Time Course of Product Peptide Generation by Activated MPP-- Fig. 4 shows time course of MPP product generation, inactivation of electron transfer activity, and activation of MPP. Cytochrome bc1 complex in 15 mM Tris-HCl buffer, pH 8.0, containing 1.5 mM Triton X-100 was incubated at 37 °C. Under these conditions the half-life of bc1 complex is less than 1 h. The cleavage of substrate peptide by activated MPP is a slow reaction. Product peptide formation increases as the reaction time increases, but at a decreasing rate, and reaches a maximum after 48 h. When MPP activity was determined after various lengths of incubation (activation) time, the maximal activity was observed with 2 h of activation. Prolonged incubation of the cytochrome bc1 complex under activation conditions causes only a slight decrease in MPP activity. More than 75% of MPP activity remains after 40 h of incubation. Because in the routine assays a reaction time of 12-24 h was used, prior activation of MPP is not necessary.


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Fig. 4.   Time course of MPP product generation, inactivation of electron transfer activity, and activation of MPP activity. Two-hundred µl of the cytochrome bc1 complex (400 µg), in 15 mM Tris-HCl, pH 8.0, containing 1.5 mM Triton X-100, was incubated at 37 °C. At the indicated incubation time 10-µl aliquotes were withdrawn for assay of ubiquinol-cytochrome c reductase (bullet ) activity and 20 µl for MPP (×) activity determination, using 400 µg of substrate peptide (-7V+6) and 12-h reaction time. For product accumulation, the MPP assay mixture was continuously incubated at 37 °C, and at the indicated time intervals 20-µl aliquots of reaction mixture were withdrawn and assayed by HPLC for product peptide generation (open circle ).

Effect of Buffer System, pH, Ionic Strength, and Temperature on Activated MPP Activity-- MPP is buffer system- and ionic strength-dependent. Its activity is higher in Tris-HCl buffer than in phosphate buffer, at pH 8.0. Maximal activity is observed at Tris-HCl buffer concentrations between 15 and 50 mM and gradually decreases at higher Tris-HCl concentrations. In 15 mM Tris-HCl buffer the activity is not affected by addition of KCl up to 100 mM, but decreases at higher salt concentrations. In phosphate buffer, maximal MPP activity is only about 80% of that observed in Tris-HCl buffer. The MPP activity decreases as the concentrations of phosphate buffer increase beyond 15 mM. The requirement of low ionic strength for maximal activity suggests involvement of ionic interaction during catalysis. This is consistent with the observation that MPP activity in spinach leaf bc1 complex is completely inhibited at 1.2 M KCl (30), but not with the observation that MPP in potato bc1 complex is most active at high salt concentrations (17, 31).

The optimal pH for MPP is between pH 6.5 and 8.5. At pH values higher than 8.5 a drastic decrease in activity is observed. At pH values below 6.5 the decrease observed is less drastic. The sharp decrease at pH values above 8.5 suggests involvement of an amino acid residue with a pKa between 8.5 and 9.0 in the catalytic or substrate binding site. This is consistent with the report of involvement of a histidine residue in the active site of MPP (25, 32) but inconsistent with the observation that MPP from plant bc1 complex is fully active at pH 9.0 (30-33).

As expected the MPP activity is temperature-dependent with a maximum at 37 °C. At 42 °C a drastic decrease is observed, indicating that activated MPP is unstable and becomes partially denatured. In contrast, MPP in the plant bc1 complex is active at 50 °C (31).

Effect of Electron Transfer and Proteinase Inhibitors on Activated MPP-- Table I summarizes the effect of commonly used electron transfer inhibitors of the bc1 complex and of proteinase inhibitors on activated MPP. Myxothiazol, a Qo site inhibitor, at 1 mM, inhibits 58% of activated MPP in beef bc1 complex. Antimycin A, a Qi site inhibitor, and hemin, which have been reported to inhibit MPP activity in the plant mitochondrial bc1 complex, show no effect on activated MPP even at a concentration of 1 mM. Activated MPP is not sensitive to the commonly used proteinase inhibitors, such as phenylmethylsulfonyl fluoride, N-ethylmaleimide, and pepstatin. Ferricyanide and dithiothreitol inhibit 28 and 81%, respectively.

                              
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Table I
Effect of electron transfer and proteinase inhibitors on the activated MPP activity

Requirement of Metal Ion for MPP Activity-- Activated MPP is inhibited by metal ion chelators such as EDTA or o-phenanthroline (Fig. 5). Activity decreases as chelator concentration increases. Complete inactivation is observed with o-phenanthroline at 0.5 mM or EDTA at 1.75 mM. A linear concentration-dependent inhibition is observed with o-phenanthroline; inhibition is 60% at 100 µM.


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Fig. 5.   Inhibition of activated MPP by EDTA and o-phenanthroline. 20-µl aliquots of the cytochrome bc1 complex in 15 mM Tris-HCl, pH 8.0, containing 1.5 mM Triton X-100, 40 µg of substrate peptide (-7V+6) and indicated concentrations of EDTA (open circle ) or o-phenanthroline (×) were incubated at 37 °C for 24 h and assayed. 100% MPP activity is represented the product peptide obtained without EDTA or o-phenanthroline.

The EDTA-inhibited MPP activity can be partially restored by addition of divalent cations, such as Fe2+ (92%), Zn2+ (68%), Co2+ (62%), Mn2+ (54%), and Mg2+ (44%) (see Fig. 6), indicating that metal ion is required for activity. This is in line with the report that metal ion is required for MPP activity in the plant mitochondrial bc1 complex and in isolated MPP (19, 24).


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Fig. 6.   Restoration of the EDTA-inhibited MPP activity by divalent cations. One ml of the cytochrome bc1 complex (2.0 mg) in 15 mM Tris-HCl buffer, pH 8.0, containing 2.0 mg of substrate peptide (-7V+6), 1.5 mM Triton X-100, and 0.5 mM EDTA was incubated at 37 °C for 22 h. After incubation, 20-µl aliquots were withdrawn, treated with various concentrations of Fe2+ (open circle ), Zn2+ (×), Mg2+ (triangle ), Mn2+ () and Co2+ (bullet ) ions and the incubation continued at 37 °C for another 22 h before the MPP activity was assayed.

Substrate Specificity of Activated MPP-- Several subunits of the beef cytochrome bc1 complex are nuclear gene-encoded and synthesized in the cytosol with presequences. Probably the function of MPP of core subunits I and II is removal of presequences from these subunits during maturation of the complex. Therefore, we synthesized polypeptides composed of various lengths of COOH-terminal presequences and NH2-terminal sequences of mature subunits to determine the substrate specificity of activated MPP. Table II summarizes the cleavage sites in the seven synthetic peptides tested.

                              
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Table II
Substrate specificity of activated MPP
Twenty µl of the cytochrome bc1 complex (40 µg) in 15 mM Tris-HCl buffer, pH 8.0, containing 1.5 mM Triton X-100 and 40 µg of the indicated synthetic substrate peptides were incubated at 37 °C for 24 h. Product peptides were separated on HPLC using a C-8 column and quantitated by peptide sequencing. Bold and thin arrows indicate the specific and nonspecific cleavage sites, respectively.

When a synthetic peptide composed of seven amino acid residues from the COOH terminus of the presequence and six from the NH2 terminus of mature subunit V (the iron-sulfur protein) (-7V+6) is used as substrate for activated MPP, two product peptides are obtained: one with the NH2-terminal amino acid corresponding to that of mature subunit V and the other with the NH2-terminal amino acid one residue upstream from the NH2 terminus of mature subunit V. When -7V+6 substrate peptide is elongated by increasing the number of residues from the NH2 terminus of mature subunit V to 20 (Table II), only one product peptide with the NH2 terminus corresponding to that of mature subunit V is obtained, indicating that activated MPP cleaves this peptide at a specific site. However, when the -7V+6 substrate peptide is elongated by increasing the number of residues from the COOH terminus of presequence to 17, a product peptide with the NH2-terminal amino acid three residues downstream from the NH2 terminus of mature subunit V is obtained, indicating that MPP cleaves this peptide at a different specific site. These results clearly indicate that length of the amino acid sequence from the NH2 terminus of mature protein determines the cleavage site specificity of activated MPP.

It has been reported that the presequence of subunit V is processed in a single step and retained as subunit IX in the mature complex (34). This is the first instance in which a cleaved targeting presequence has been shown to be retained in the cell. Because subunit IX is absent from the plant mitochondrial cytochrome bc1 complex, it is possible that subunit IX, once it is clipped off from the iron-sulfur protein precursor by MPP of core subunits, binds to the active site of MPP to inhibit it prior to complex maturation. Final proof for this possibility must await definitive assignment of the amino acid sequence of the polypeptide binding to the interface of core subunits.

To further confirm that peptide length from the NH2 terminus of mature protein is a determining factor for site-specificity of activated MPP, four peptides containing different lengths of presequence and mature protein of subunit IV (cytochrome c1) and the beta  subunit of F1 ATPase were synthesized and used as substrates for activated MPP (see Table II). When a peptide containing 10 residues from the COOH-terminal end of presequence and 10 residues from the NH2 terminus of mature subunit IV (-10IV+10) is used, the MPP cleavage site is at 7 amino acid residues downstream from the NH2 terminus of mature subunit IV. When the length of the amino acid sequence from the NH2-terminal end of mature subunit IV is elongated to 20 residues (10IV+20), two product peptides are obtained: one with the correct partial NH2-terminal amino acid sequence of mature subunit IV and the other with the NH2 terminus two residues upstream (Table II). These results suggest that MPP specificity increases with the length of the mature protein sequence.

The recombinant beta -subunit (with its presequence) of F1 ATPase is the most commonly used substrate for assaying MPP activity. When a synthetic peptide containing 10 amino acid residues from the NH2-terminal end of mature beta  subunit of F1 ATPase and 8 amino acid residues from its presequence (-8beta +10) is used as substrate for MPP, no product peptide is obtained. However, when the NH2-terminal end of mature beta  subunit is increased to 20 residues (-8beta +20), one product peptide with the correct NH2 terminus of mature beta  subunit is obtained (Table II). This further suggests that the amino acid sequence of the mature protein is a determining factor for cleavage site specificity of MPP.

The Possible Function of MPP in the Cytochrome bc1 Complex-- Although there is little doubt about the association between MPP activity and core proteins I and II of crystalline bc1 complex, the low turnover number of the activated processing activity merits some discussion. The activated MPP activity of the cytochrome bc1 complex is about one-hundredth that of rat liver mitochondrial intermediate peptidase estimated from the assay conditions reported (35). The low activity may be due partly to the incomplete removal of the inhibitor peptide, competition between substrate peptide and inhibitor peptide for the active site, and the association of core proteins with other subunits, except subunit VIII (36). It is likely that before core proteins I and II are assembled into the mature cytochrome bc1 complex, they may be present as soluble peptidases in the matrix. In this form, they may have better activity toward the precursors of the imported proteins than the activated MPP does toward the substrate peptides used. Thus MPP may play an essential role in the processing of imported proteins despite the observed low activity. Alternatively, the activated MPP of the cytochrome bc1 complex may represent an enzymatic activity which was located within bc1 complexes from all organisms during establishment of endosymbiosis between mitochondria and eukaryotic cells. According to this hypothesis the core proteins are relics of an ancient processing peptidase (27).

    ACKNOWLEDGEMENT

We thank Dr. Roger Koeppe for the critical review of this manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM 30721 and Agricultural Experimental Station Project 1819, Oklahoma State University.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. Tel.: 405-744-6612; Fax: 405-744-7799.

The abbreviations used are: MPP, matrix processing peptidase or mitochondrial processing peptidase; HPLC, high performance liquid chromatography; Q2, 2,3-dimethoxy-5-methy-6-geranyl-1,4-benzoquinoneQ2H2, 2,3-dimethoxy-5-methy-6-geranyl-1,4-benzoquinol.

2 GenBankTM accession number X59692 (revised 12/11/96).

    REFERENCES
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Abstract
Introduction
Procedures
Results & Discussion
References

  1. Hatefi, Y. (1985) Annu. Rev. Biochem. 54, 1015-1069[CrossRef][Medline] [Order article via Infotrieve]
  2. Yu, C. A., Xia, J. Z., Kachurin, A. M., Yu, L., Xia, D., Kim, H., and Deisenhofer, J. (1996) Biochim. Biophys. Acta 1275, 47-53[Medline] [Order article via Infotrieve]
  3. Yue, W. H., Zou, Y. P., Yu, L., and Yu, C. A. (1991) Biochemistry 30, 2303-2306[Medline] [Order article via Infotrieve]
  4. Kubota, T., Kawamoto, M., Fukuyama, K., Shinzawa-Itoh, K., Yoshkawa, S., and Matsubara, H. (1991) J. Mol. Biol. 221, 379-382[Medline] [Order article via Infotrieve]
  5. Berry, E. A., Huang, L.-S., Earnest, T. N., and Jap, B. K. (1992) J. Mol. Biol. 224, 1161-1166[Medline] [Order article via Infotrieve]
  6. Yu, C. A., Xia, D., Deisenhofer, J., and Yu, L. (1994) J. Mol. Biol. 243, 802-805[Medline] [Order article via Infotrieve]
  7. Xia, D., Yu, C. A., Kim, H., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1997) Science 277, 60-66[Abstract/Free Full Text]
  8. Yang, X., and Trumpower, B. L. (1988) J. Biol. Chem. 263, 11962-11970[Abstract/Free Full Text]
  9. Tzagoloff, A., Wu, M., and Crivellone, M. (1986) J. Biol. Chem. 261, 17163-17169[Abstract/Free Full Text]
  10. Oudshoorn, P., Van Steeg, H., Swinkels, B. W., Schoppink, P., and Grivell, L. A. (1987) Eur. J. Biochem. 163, 97-103[Abstract]
  11. Schoppink, P. J., Hemrike, W., and Berden, J. A. (1989) Biochim. Biophys. Acta 974, 192-202[Medline] [Order article via Infotrieve]
  12. Schmitt, M. E., and Trumpower, B. L. (1990) J. Biol. Chem. 265, 17005-17011[Abstract/Free Full Text]
  13. Maarse, A. C., DeHann, J., Schoppink, P. J., Berden, J. A., and Grivell, L. A. (1988) Eur. J. Biochem. 172, 179-184[Abstract]
  14. Phillips, J. D, Schmitt, M. E., Brown, T. A., Beckmann, J. D., and Trumpower, B. L. (1990) J. Biol. Chem. 265, 20813-20821[Abstract/Free Full Text]
  15. Braun, H.-P., Emmermann, M., Kraft, V., and Schmitz, U. K. (1992) EMBO J. 11, 3219-3227[Abstract]
  16. Emmermann, M., Braun, H.-P., Arretz, M., and Schmitz, U. K. (1993) J. Biol. Chem. 268, 18936-18942[Abstract/Free Full Text]
  17. Braun, H.-P., and Schmitz, U. K. (1995) J. Bioenerg. Biomembr. 27, 423-436[Medline] [Order article via Infotrieve]
  18. Eriksson, A. C., Sjoling, S., and Glaser, E. (1994) Biochim. Biophys. Acta 1186, 221-231
  19. Glaser, E., Eriksson, A. C., and Sjoling, S. (1994) FEBS Lett. 346, 83-87[CrossRef][Medline] [Order article via Infotrieve]
  20. Emmermann, M., Braum, H.-P., and Schmitz, U. K. (1993) Biochim. Biophys. Acta 1142, 306-310
  21. Rawlings, N. D., and Barrett, A. J. (1991) Biochem. J. 275, 389-391[Medline] [Order article via Infotrieve]
  22. Hawlitschek, G., Schneider, H., Schmidt, B., Tropschug, M., Hartl, F. U., and Neupert, W. (1988) Cell 53, 795-806[Medline] [Order article via Infotrieve]
  23. Yang, M., Jensen, R. E., Yaffe, M. P., Oppliger, W., and Schatz, G. (1988) EMBO J. 7, 3857-3862[Abstract]
  24. Ou, W., Ito, A., Okazaki, H., and Omura, T. (1988) EMBO J. 8, 2605-2612[Abstract]
  25. Striebel, H.-M., Rysavy, P., Adamea, J., Spizek, J., and Kalouek, F. (1996) Arch. Biochem. Biophys. 235, 211-218[CrossRef]
  26. Gencic, S., Schagger, H., and von Jagow, G. (1991) Eur. J. Biochem. 199, 123-131[Abstract]
  27. Braun, H-P., and Schmitz, U. K. (1995) Trends Biochem. Sci. 20, 171-175[CrossRef][Medline] [Order article via Infotrieve]
  28. Deng, K. P., Xia, D., Kachurin, A. M., Kim, H., Deisenhofer, J., Yu, L., and Yu, C. A. (1996) Biophys. J. 72, 319a
  29. Yu, L., and Yu, C. A. (1982) Biochemistry 21, 4096-4101[Medline] [Order article via Infotrieve]
  30. Eriksson, A. C., Sjoling, S., and Glaser, E. (1996) J. Bioenerg. Biomembr. 28, 283-290
  31. Emmermann, M., and Schmitz, U. K. (1993) Plant Physiol. 103, 615-620[Abstract/Free Full Text]
  32. Kitada, S., Shimokata, K., Niidome, T., Ogishima, T., and Ito, A. (1995) J. Biochem. (Tokyo) 117, 1148-1150[Abstract]
  33. Eriksson, A. C., and Glaser, E. (1992) Biochim. Biophys. Acta 1140, 208-214
  34. Brandt, U., Yu, L., Yu, C. A., and Trumpower, B. L. (1993) J. Biol. Chem. 268, 8387-8390[Abstract/Free Full Text]
  35. Kalousek, F., Isaya, G., and Rosenberg, L. E. (1992) EMBO J. 11, 2803-2809[Abstract]
  36. Kim, H., Xia, D., Yu, C. A., Xia, J. Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J. (1998) Proc. Natl. Acad. Sci. U. S. A., in press


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