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.
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INTRODUCTION |
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,
-MPP and
-MPP, that cooperate in
processing. Both subunits are matrix proteins except for
-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
-subunit and the HXXEK(X)72-76E
or HXXD/ER(X)72-76E in the
-subunit (25). Both histidines and the distal glutamate in the
HXXEH(X)72-76E motif of the
-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
-subunit of rat MPP, 38% with yeast, and 42% with potato, and
core II protein (26) is 27% identical to the
-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
-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.
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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).
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EXPERIMENTAL PROCEDURES |
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
-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
(
8
+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
(
8
+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.
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 |
RESULTS AND DISCUSSION |
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) ( ) 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.
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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 ( ) 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 ( ).
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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.
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 ( ) 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.
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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+ ( ), Zn2+ (×),
Mg2+ ( ), Mn2+ ( ) and Co2+
( ) ions and the incubation continued at 37 °C for another 22 h before the MPP activity was assayed.
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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.
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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
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
-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
subunit of F1
ATPase and 8 amino acid residues from its presequence
(
8
+10) is used as substrate for MPP, no
product peptide is obtained. However, when the NH2-terminal
end of mature
subunit is increased to 20 residues
(
8
+20), one product peptide with the correct NH2 terminus of mature
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).
We thank Dr. Roger Koeppe for the critical
review of this manuscript.