New Insights into the Co-evolution of Cytochrome c
Reductase and the Mitochondrial Processing Peptidase*
Stefanie
Brumme
,
Volker
Kruft§,
Udo K.
Schmitz
, and
Hans-Peter
Braun
¶
From the
Institut für Angewandte Genetik,
Universität Hannover, Herrenhäuser Strasse 2, 30419 Hannover and § Applied Biosystems GmbH, Brunnenweg 13,
64331 Weiterstadt, Germany
 |
ABSTRACT |
The mitochondrial processing peptidase (MPP) is a
heterodimeric enzyme that forms part of the cytochrome c
reductase complex from higher plants. Mitochondria from mammals and
yeast contain two homologous enzymes: (i) an active MPP within the
mitochondrial matrix and (ii) an inactive MPP within the cytochrome
c reductase complex. To elucidate the evolution of MPP, the
cytochrome c reductase complexes from lower plants were
isolated and tested for processing activity. Mitochondria were prepared
from the staghorn fern Platycerium bifurcatum, from the
horsetail Equisetum arvense, and from the colorless algae
Polytomella, and cytochrome c reductase
complexes were purified by a micro-isolation procedure based on
Blue-native polyacrylamide gel electrophoresis and electroelution. This
is the first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail. The cytochrome c
reductase complexes from P. bifurcatum and E. arvense are shown to efficiently process mitochondrial precursor
proteins, whereas the enzyme complex from Polytomella lacks
proteolytic activity. An evolutionary model is suggested that assumes a
correlation between the presence of an active MPP within the cytochrome
c reductase complex and the occurrence of chloroplasts.
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INTRODUCTION |
The mitochondrial cytochrome c reductase (also called
the bc1 complex or complex III) is a
multisubunit enzyme of the respiratory chain that catalyzes reduction
of cytochrome c by oxidation of ubiquinol. Coupled to this
reaction, it translocates protons from the mitochondrial matrix to the
space between the two mitochondrial membranes and thereby contributes
to the chemiosmotic gradient across the inner mitochondrial membrane.
The function of cytochrome c reductase is based on three
subunits, which are directly involved in electron transport:
cytochromes b and c1 and the
"Rieske" iron-sulfur protein (reviewed in Refs. 1 and 2). In
several bacteria, the bc1 complex only contains
these three so-called "respiratory" subunits. However, all
mitochondrial cytochrome c reductase complexes characterized
so far comprise six to eight additional subunits, the functions of
which are not entirely understood. Two of them are large and have
molecular masses around 50 kDa, whereas four to six are small (<15
kDa). The primary structures of the large subunits, which are
traditionally called the "core I" and "core II" proteins, are
known for man, beef, yeast, potato, and Euglena (reviewed in
Ref. 3). Both core proteins are located on the matrix exposed side of
cytochrome c reductase, as revealed by electron microscopy
of membrane crystals from Neurospora crassa (4) and more
recently by x-ray analysis of cytochrome c reductase crystals from beef (5). Mutational analysis of the two core proteins
from yeast revealed involvement of both subunits in assembly of the
multisubunit complex (6-8).
The two core subunits exhibit some sequence similarity. Furthermore
their primary structures resemble the sequences of the two subunits of
the mitochondrial processing peptidase
(MPP1;
- and
-subunits), which is a heterodimeric matrix-localized enzyme that
removes presequences of nuclear encoded mitochondrial proteins upon
their transport into the organelle (reviewed in Refs. 9-11). In fact,
in potato, the two core subunits of cytochrome c reductase
even represent the subunits of MPP and the cytochrome c
reductase complex from this plant is a highly active processing peptidase (12). The same results have been shown for cytochrome c reductase from spinach and wheat (13, 14). An intermediate situation was found in N. crassa; the core I protein has
-MPP activity, whereas the
-subunit of MPP is localized in the
mitochondrial matrix (15, 16). These molecular data can be explained by an evolutionary model, which was suggested recently (3, 17). (i) Early
in evolution, MPP developed starting from a preexisting bacterial
protease and became part of the cytochrome c reductase complex; (ii) later in evolution, the two subunits of MPP became detached from the enzyme complex to allow independent regulation of
protein processing and respiration in some organisms; (iii) the
detachment was realized by gene duplications, because due to the
co-evolution of cytochrome c reductase and MPP, the two subunits of MPP became indispensable for assembly of this respiratory protein complex. From the perspective of this model, the core subunits
are relics of an ancient processing peptidase and the bifunctional
cytochrome c reductase complex in plants represents a
situation that was originally present in the mitochondria from all
organisms.
To test this model on the co-evolution of the cytochrome c
reductase complex and the mitochondrial processing peptidase, we decided to analyze the processing activity of cytochrome c
reductase from lower plants. A micro-isolation procedure is employed
for the purification of cytochrome c reductase from the
staghorn fern Platycerium bifurcatum, from the horsetail
Equisetum arvense, and from the alga Polytomella,
which is based on organelle preparations, Blue-native-polyacrylamide
gel electrophoresis (BN-PAGE), and electroelution. We present data on
the subunit composition and the function of the purified cytochrome
c reductase complexes. Interestingly, the enzyme complexes
from P. bifurcatum and E. arvense exhibit highly
specific processing activity, whereas cytochrome c reductase
from Polytomella is proteolytically inactive. Evolutionary implications of these findings are discussed.
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EXPERIMENTAL PROCEDURES |
Isolation of Mitochondria from Solanum tuberosum, Polytomella
spp., P. bifurcatum, and E. arvense
S. tuberosum (Potato)--
Mitochondria from potato tubers were
isolated as described in Ref. 18. The organelles were suspended in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4,
pH 7.2, at a concentration of 10 mg of mitochondrial protein/ml. For
the purification of the bc1 complex by BN-PAGE,
100 µl of purified mitochondria were used per lane.
Polytomella spp.--
Polytomella ssp. (198.80, E. G. Pringsheim) was obtained from the "SAG-Sammlung von
Algenkulturen" at the University of Göttingen (Göttingen,
Germany) and was grown in 2.5-liter culture flasks without shaking in
the dark using the medium described by Schlösser (19) at
25 °C. Mitochondria were isolated basically as described in Ref. 20.
The resulting crude mitochondria were washed with 0.4 M
mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4, pH 7.2, and purified by
Percoll step gradient centrifugation (14%, 22%, 45% Percoll in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4,
pH 7.2) at 70,000 × g for 45 min. Pure mitochondrial fraction was washed twice; resuspended in 0.4 M mannitol,
0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4, pH 7.2, at a
concentration of 2 mg of mitochondrial protein/ml; and divided into
aliquots of 200 µl.
P. bifurcatum (Staghorn Fern)--
Plants were obtained from the
greenhouse of the "Berggarten" at the University of Hannover. The
young round, barren leaves ("mantle" leaves) were separated from
the plants and cut into small pieces. All subsequent steps of
mitochondria isolation were carried out at 4 °C. Pieces of leaves
were homogenized three times in two volumes of 0.4 M
mannitol, 0.1% BSA, 1 mM EGTA, 15.0 mM
-mercaptoethanol, 0.05 mM PMSF, 25 mM MOPS,
pH 7.8, for 3 s using a Waring blender. The homogenate was
filtered through two layers of muslin. The resulting suspension was
centrifuged at 3000 × g for 5 min. To obtain the
mitochondrial fraction, the supernatant was centrifuged at 18,000 × g for 30 min. The mitochondrial fraction was resuspended
in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4,
pH 7.2; homogenized in a Dounce homogenizer; and purified by Percoll
step gradient centrifugation (14%, 26%, 45% Percoll in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM KH2PO4,
pH 7.2, at 70,000 × g for 45 min). Mitochondria formed
two light brown bands in the 26% phase. They were washed twice in
resuspension buffer, solved at a concentration of 1 mg of mitochondrial
protein/ml, and divided into aliquots of 600 µl.
E. arvense (Horsetail)--
Wild plants were used. Mitochondria
were isolated from the etiolated part of the stems from vegetative
shoots, which are located near to the surface of the soil. All steps
were carried out at 4 °C. The brown, silicified epidermis was peeled
off. Stems were homogenized by grinding in a mortar in 10 volumes of
0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 15.0 mM
-mercaptoethanol, 0.05 mM PMSF, 25 mM MOPS, pH 7.8. Subsequent steps of differential
centrifugation and Percoll step gradient centrifugation were carried
out as described above for isolating mitochondria from P. bifurcatum. Mitochondria formed a light, reddish brown band in the
26% phase of the gradient. The mitochondria were removed, washed
twice, and finally solved in 0.4 M mannitol, 0.1% BSA, 1 mM EGTA, 0.2 mM PMSF, 10 mM
KH2PO4, pH 7.2, at a concentration of ~1.5 mg
of mitochondrial protein/ml. Aliquots of 300 µl were used as starting
material of sample preparation for BN-PAGE.
Purification of Mitochondrial Protein Complexes by BN-PAGE
Blue-native-polyacrylamide gel electrophoresis and sample
preparation for BN-PAGE was carried out as described in Ref. 21. Starting points for sample preparation were the obtained aliquots of
mitochondria from S. tuberosum, Polytomella spp.,
P. bifurcatum, and E. arvense. The mitochondrial
protein complexes from each organism were resolved on a separate gel
consisting of a stacking gel (4% acrylamide) comprising 10 slots and a
separating gel (4.95-12.6% acrylamide), respectively.
Electroelution of Cytochrome c Reductase Complexes
The blue protein bands representing cytochrome c
reductase complexes were cut out after BN-PAGE and electroeluted as
described in Ref. 22. Electroelution was performed with the
electroelutor/concentrator ECU-040 (CBS Scientific Co., Del Mar, CA)
overnight at 150 V and 4 °C with electrode buffer (25 mM
Tricine, 7.5 mM Bis-Tris, 0.1 mM PMSF, pH
7.0).
Tricine-SDS-PAGE for Second Gel Dimension
Entire lanes from BN-PAGE with separated native-mitochondrial
protein complexes were used for the resolution of subunits and identification of complexes by Tricine-SDS-PAGE as described in Ref.
21.
Analysis of Purified Protein Complexes by SDS-PAGE and
Immunoblotting
Tricine-SDS-PAGE was carried out in the PROTEAN II cell from
Bio-Rad (gel dimensions: 20 × 16 × 0.1 cm) according to the
protocol published by Schägger and von Jagow (23). Approximately
1 µg of electroeluted enzyme complex was mixed with equal volumes of 2× loading buffer (10% SDS, 30% glycerol, 100 mM Tris,
4%
-mercaptoethanol, 0.006% bromphenol blue) and loaded into the
slots. The electrophoresis was carried out at 20 °C; it was started
at 30 V for 45 min and continued at 30 mA for 16 h. Proteins were
either silver-stained or blotted onto nitrocellulose membranes
(Schleicher & Schüll, Dassel, Germany) and incubated overnight
with 1000-fold diluted antiserum. Visualization of immunopositive
proteins was performed as described in Ref. 24.
Processing Assays
In vitro processing of radiolabeled precursor
proteins was carried out for 1 h at 28 °C in a final volume of
120 µl, including 88 µl of processing buffer (22 mM
Tris-HCl, 25 mM NaCl, 0.6% Triton X-100, 1 mM
PMSF, 300 µM ZnCl2), 1-4 µl of
radiolabeled precursor protein, and about 2 µg of electroeluted
cytochrome c reductase. The processing reaction was stopped
with an equal volume of 2-fold concentrated loading buffer (10% SDS,
30% glycerol, 100 mM Tris-HCl, 4%
-mercaptoethanol,
0.006% bromphenol blue). Inhibition of processing activity was carried
out by addition of 3 mM EDTA. Processing products were
separated by SDS-PAGE as described by Laemmli (25). The gel was fixed
with 50% acetic acid, incubated with Amplify (Amersham, Little
Chalfont, United Kingdom) and exposed to X-OMATTM films (Eastman Kodak
Corp.).
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RESULTS |
Isolation of Cytochrome c Reductase Complexes from S. tuberosum,
Polytomella spp., P. bifurcatum, and E. arvense--
The isolation of
highly pure mitochondria from plants devoid of chloroplast
contaminations is facilitated by the use of etiolated starting material
like tubers or dark grown seedlings. Hence, most biochemical studies on
plant mitochondria were carried out with organelles from potato tuber
or etiolated seedlings from different higher plants. However, lower
plants lack suitable etiolated tissues for the preparation of
mitochondria. The starting materials for our investigation were mantle
leaves from the staghorn fern P. bifurcatum, which have a
reduced content of chloroplasts, and subterranean parts of stems from
the horsetail E. arvense. Furthermore, the colorless algae
Polytomella spp. was used for isolating mitochondria, which
is related to Chlamydomonas but lacks chloroplasts.
Mitochondria from potato tuber were prepared as control organelles.
Procedures for the isolation of mitochondria from potato and
Polytomella were adapted from preexisting protocols (18,
20), whereas the method for the purification of fern and horsetail
mitochondria was newly established (see "Experimental Procedures").
Owing to limited starting material, the overall amount of organelles
was low for the fern and the horsetail. Therefore, a micro-isolation procedure was used for the purification of respiratory enzyme complexes, which is based on BN-PAGE and electroelution. BN-PAGE is a
powerful tool for the separation of membrane-bound protein complexes
(26); proteins are solubilized under mild conditions using non-ionic
detergents, and Coomassie dyes are employed to introduce charge shifts
on the polypeptides prior to electrophoresis. A two-dimensional
resolution of mitochondrial protein complexes from P. bifurcatum, E. arvense, and Polytomella spp.
is shown in Fig. 1.

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Fig. 1.
Two-dimensional resolution of the
mitochondrial protein complexes from P. bifurcatum
(A), E. arvense (B), and
Polytomella spp. (C) by
Blue-native-polyacrylamide gel electrophoresis and
Tricine-SDS-PAGE. The organelles from all three organisms were
isolated as described under "Experimental Procedures," and the
protein complexes were solubilized by 1.5%
n-dodecylmaltoside. The gels were silver-stained. Schemes of
the gels are given on the right, and the molecular sizes of
standard proteins are given in the middle (in kDa).
CR, cytochrome c reductase; Cyt,
cytochrome; FeS, iron-sulfur protein; SU,
subunit; F0F1,
F0F1-ATP synthase.
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Subunit Compositions of the Purified Cytochrome c Reductase
Complexes--
The cytochrome c reductase complexes were
identified by their subunit compositions and by immunostaining (see
below). Protein bands corresponding to the native cytochrome
c reductase complexes from potato, P. bifurcatum,
E. arvense, and Polytomella spp. were cut out
from one-dimensional Blue-native gels and the enzyme complexes were
electroeluted. Subsequently, the obtained fractions were analyzed by
Tricine-SDS-PAGE and silver staining. Fig.
2 shows the subunits of cytochrome
c reductase complexes from all four organisms after the
electroelution step. The resolved cytochrome c reductase
complexes were blotted onto membrane filters and analyzed by
immunostaining with antibodies directed against the core I protein and
the iron-sulfur protein (Fig. 3). Some
additional further protein bands were identified by direct protein
sequencing (Table I). The combined data
from Figs. 1-3 and Table I allow us to draw the following conclusions
on the subunit compositions of the purified protein complexes.

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Fig. 2.
Tricine-SDS-PAGE of the cytochrome
c reductase complexes from S. tuberosum
(St), P. bifurcatum (Pb), E. arvense (Ea), and Polytomella spp.
(Ps). All protein complexes were isolated by
Blue-native-polyacrylamide gel electrophoresis and electroelution as
described under "Experimental Procedures." The gel was
silver-stained. A scheme of the gel is given on the right,
and the sizes of standard proteins are given in the middle
(in kDa). C1, core I protein; C2, core II
protein; Cb, cytochrome b;
Cc1, cytochrome c1;
FeS, iron-sulfur protein; QCR8, protein
homologous to the yeast qcr8 gene product. The numbers
indicate small subunits according to their calculated molecular mass in
kDa; ? indicates subunits that could not be unambiguously identified.
Bands shown in gray in the scheme are contaminations. The
8-kDa subunit of cytochrome c reductase from potato appears
to be a doublet on this particular gel for unknown reasons.
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Fig. 3.
Identification of the core I and the
iron-sulfur subunit by immunoblotting. The cytochrome c
reductase complexes from S. tuberosum (St),
P. bifurcatum (Pb), E. arvense
(Ea), and Polytomella spp. (Ps) were
resolved by Tricine-SDS-PAGE as shown in Fig. 2 and subsequently
blotted onto nitrocellulose membranes. A, incubation of a
blot with an antiserum directed against the core I protein from
N. crassa; B, incubation of a blot with an
antiserum directed against the iron-sulfur protein from N. crassa. Molecular masses of standard proteins are given on the
left of the blots (in kDa).
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Table I
N-terminal sequences of subunits of the cytochrome c reductase
complexes from P. bifurcatum and E. arvense
Amino acids are given in the one-letter-code; X stands for
amino acids that could not be unambiguously identified.
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(i) Cytochrome c reductase from potato, which is a well
characterized protein complex (2) and which was analyzed as a control enzyme complex, comprises 10 subunits (Fig. 2): the core I protein (apparent molecular mass: 56 kDa), the core II protein (52 kDa), cytochrome b (35 kDa), cytochrome c1
(31 kDa), the FeS protein (24 kDa), a "14-kDa" subunit, and four
subunits below 10 kDa.
(ii) The cytochrome c reductase from P. bifurcatum is contaminated by some subunits of the
F0F1-ATP synthase complex (both enzyme
complexes have a very similar size on the native gel dimension, see
Fig. 1). The core I protein (apparent molecular mass: 53 kDa) was
identified by immunoblotting (Fig. 3A, lane 2).
The core II protein is assumed to migrate just above (Fig. 2,
lane 2), as (a) the band at 53 kDa appeared to be
a doublet on several gels and (b) the upper part of the band
has a brown color on silver-stained gels typical for core II proteins,
whereas the lower part of the band has a red-brown color typical for
core I proteins. The N-terminal sequence of the 30-kDa band (Table I)
exhibits significant sequence identity to the mature N terminus of
cytochrome c1 from potato. The band at 26 kDa
strongly cross-reacts with an antiserum directed against the
iron-sulfur protein from N. crassa (Fig. 3B). The N-terminal sequence of the 26-kDa protein was determined by direct protein sequencing but only exhibits low sequence identity to iron-sulfur proteins from other species. However, this result is not
unexpected due to the low degree of sequence conservation at the N
terminus of iron-sulfur subunits from different organisms. At least
three small proteins (apparent molecular masses 14 kDa, two proteins
<10 kDa) form part of the cytochrome c reductase complex
from P. bifurcatum (Fig. 2, lane 2). For unknown
reasons, the cytochrome b subunit of P. bifurcatum could not be detected on the gel in Fig. 2. However, it
is highly unlikely that cytochrome b is absent, as it forms
the core of the cytochrome c reductase complexes and as the
apparent molecular mass of the dimeric protein complex from P. bifurcatum lies at 500 kDa on native gels (data not shown) in
accordance with the molecular mass of cytochrome c reductase
complexes from other organisms (2).
(iii) The cytochrome c reductase complex from the horsetail
E. arvense could be obtained in highly pure form (Fig. 2)
and comprises 10 subunits: the core I and core II proteins (apparent molecular masses: 53 kDa), cytochrome b (36 kDa), cytochrome
c1 (32 kDa), the FeS protein (26 kDa), the
14-kDa protein (which runs at 15 kDa), and four proteins below 10 kDa.
The core I protein was identified by immunoblotting (Fig.
3A), cytochrome c1 by direct protein
sequencing (Table I), and the FeS protein by protein sequencing and
immunoblotting (Table I, Fig. 3B). The core II protein most
likely has a molecular mass identical to the core I protein, as the
53-kDa band seemed to be a doublet on several gels (data not shown).
The first 20 amino acids of the second smallest subunit of cytochrome
c reductase from E. arvense could be determined
by cyclic Edman degradation. The protein exhibits 65% sequence
identity to the potato 8.2-kDa subunit of the cytochrome c
reductase complex, which is homologous to the yeast qcr8 gene product.
(iv) Likewise, the cytochrome c reductase complex from
Polytomella spp. was isolated in very pure form (Fig. 2) and
could be resolved into nine different subunits: core I protein (53 kDa), core II protein (49 kDa), cytochromes b and
c1 at 32 kDa, FeS protein (24 kDa), 14-kDa
protein, and three or more subunits below 12 kDa. Cytochrome
c reductase from this organism was purified before by
González-Halphen and co-workers (20), and similar values were
reported for the molecular weights of the subunits. The identities of
the subunits were determined by direct protein sequencing and
immunoblotting (20). However, González-Halphen and co-workers
were able to resolve one more small subunit below 12 kDa.
Processing Activities of the Purified Cytochrome c Reductase
Complexes--
To investigate whether the purified cytochrome
c reductase complexes include the activity of the
mitochondrial processing peptidase, an in vitro processing
assay was carried out using two radiolabeled mitochondrial precursor
proteins: the
subunit of the ATP synthase complex and the FeS
protein of cytochrome c reductase (Fig.
4). Both precursor proteins are converted
into their mature form when incubated with cytochrome c
reductase complex from potato, which was shown previously to be an
efficient processing peptidase (12). Potato MPP is a metallopeptidase,
and the processing reaction can be inhibited by the metal chelator EDTA
(Fig. 4, lanes 2). Identical results were obtained with the
isolated cytochrome c reductase complexes from P. bifurcatum and E. arvense. Both enzyme complexes
efficiently process the tested precursor proteins into their mature
forms in the absence of EDTA (Fig. 4, lanes 3-6). In
contrast, cytochrome c reductase from Polytomella
spp. did not exhibit detectable processing activity. Hence, the
cytochrome c reductase complexes from potato, P. bifurcatum, and E. arvense do contain both subunits of
MPP, whereas Polytomella spp. lacks at least one of the MPP
proteins.

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Fig. 4.
Processing activity of isolated cytochrome
c reductase from S. tuberosum (St),
P. bifurcatum (Pb), E. arvense
(Ea), and Polytomella spp.
(Ps). The precursor of the -subunit of the
F1-ATP synthase from tobacco (A) and the
precursor of the iron-sulfur protein from potato (B) were
synthesized in vitro in the presence of
[35S]methionine and treated with isolated cytochrome
c reductase in the presence or absence of EDTA as indicated.
The radiolabeled proteins were subsequently resolved by SDS-PAGE and
visualized by fluorography. C, control; p,
precursor protein; m, mature protein.
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DISCUSSION |
Blue-native-polyacrylamide gel electrophoresis has been shown here
to be a suitable method for the isolation of mitochondrial protein
complexes from P. bifurcatum, E. arvense and
Polytomella. The procedure is especially valuable if the
starting material for organelle preparations is limited.
To our knowledge, this is the first report on a biochemical preparation
of a mitochondrial enzyme from a fern or a horsetail. The cytochrome
c reductase complexes from E. arvense and
Polytomella could be obtained in highly pure form, whereas
the cytochrome c reductase complex from the fern P. bifurcatum was contaminated by subunits of the ATP synthase
complex.
Cytochrome c reductase complexes from three very different
eukaryotes (beef, yeast, and potato) have been studied extensively (2,
27, 28). All three enzyme complexes comprise 10 subunits: 3 respiratory
proteins, 2 large core proteins, and 5 small polypeptides. In beef, the
presequence of the Rieske iron-sulfur protein is retained in the
cytochrome c reductase complex after proteolytic cleavage of
the precursor protein and is considered to be an eleventh subunit (29,
30). Additionally, the cytochrome c reductase complex from
E. arvense comprises 10 subunits, which have molecular weights very similar to the one reported for potato. The same number of
subunits was reported for the cytochrome c reductase complex
from Polytomella (20). Hence, the structure of the
mitochondrial cytochrome c reductase complex, which differs
remarkably from the structure of the corresponding prokaryotic protein
complex, seems to be highly conserved in very different organisms and
most likely evolved early in the development of the eukaryotic
cell.
The electron transfer activities of the cytochrome c
reductase complexes from potato, P. bifurcatum, E. arvense, and Polytomella prepared by
Blue-native-polyacrylamide gel electrophoresis and electroelution were
not measured, but there are several indications that the enzyme
complexes are intact and most likely active. (i) The iron-sulfur
protein, which easily dissociates from cytochrome c
reductase complexes causing low enzymatic activity, forms part of all
four cytochrome c reductase complexes as monitored by direct protein sequencing and immunoblotting; (ii) the apparent molecular masses of the cytochrome c reductase complexes from potato,
P. bifurcatum, and E. arvense complexes during
native gel electrophoresis was in the range of 500 kDa (data not
shown), which is similar to the values reported for the intact dimeric
cytochrome c reductase complexes from yeast and beef (2).
Only the enzyme complex from Polytomella had a smaller size,
which is in line with previous reports (20).
The activity of the mitochondrial processing peptidase was shown to
reside within the mitochondrial matrix in yeast, Neurospora, and rat (31-33). In vitro processing assays with the
isolated cytochrome c reductase complexes from yeast,
Neurospora, and beef revealed no detectable processing
activity of this respiratory enzyme complex (14). In contrast, the MPP
activity forms part of cytochrome c reductase in all plants
investigated so far: potato, spinach, and wheat (12-14). These
experimental findings are in line with the sequence data presently
available for the large subunits of the cytochrome c
reductase complex, the so-called core subunits (Fig.
5). The core I proteins from man, beef,
and yeast exhibit some sequence similarity to
-MPP subunits from
different organisms, but they have an incomplete inverse zinc-binding
motif, which was shown to be essential for the proteolytic activity of
MPP (34, 35) (Fig. 5A). Similarily, the sequences of the
core II subunit from man, beef, and yeast resemble
-MPP sequences from various organisms, but lack a highly conserved stretch of uncharged amino acids, which is present in all
-MPP proteins sequenced so far and which is believed to be a prerequisite for the
function of
-MPP in protein processing (Fig. 5B). In
contrast, the core I protein from potato includes a complete inverse
zinc-binding motif and the core II protein from this organism comprises
the characteristic uncharged "
-MPP domain" (12, 36). The fungus Neurospora represents an intermediate situation; the core I
protein has an inverse zinc-binding motif, but the core II protein
lacks the
-MPP domain.2
Hence, the cytochrome c reductase complex from
Neurospora does not exhibit processing activity (14, 15).
Additionally, the cytochrome c reductase complex from
Euglena, which was not tested for processing activity so
far, seems to represent such an intermediate situation; a zinc-binding
motif is present in the the core I protein but no
-MPP domain in the
core II protein (Fig. 5, A and B).

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Fig. 5.
Alignment of two sequence stretches of MPP
and core proteins from different organisms. A, alignment of
the zinc-binding region. Residues directly involved in metal binding
are in black boxes; other residues conserved in at least six
organisms are in gray boxes. B, alignment of the
-MPP domain. Residues that are identical in at least three of the
four -MPP proteins are in black boxes ( -MPP domain);
other residues conserved in at least six organisms are in gray
boxes. The numbers on the left of the
sequences indicate the position of the first amino acid of the sequence
stretches as deduced from the open reading frame of the corresponding
nucleotide sequences. Accession numbers of the proteins are given on
the right of the sequences.
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Processing activity of cytochrome c reductase complexes from
three other plants: the fern P. bifurcatum, the horsetail
E. arvense, and the colorless alga Polytomella,
was tested. In agreement with the data from other plants, cytochrome
c reductase from the fern and the horsetail comprise
processing activity, whereas the enzyme complex from the colorless alga
Polytomella has no proteolytic activity. These data allow us
to draw some new conclusions on the evolution of the MPP subunits and
the core proteins. To date, experimental or structural data on the
proteolytic activity of the large subunits of cytochrome c
reductase complexes from 12 organisms are known: the vertebrata man,
beef, and rat; the fungi yeast and Neurospora; the plants
potato, spinach, wheat, P. bifurcatum, and E. arvense; and the nongreen algae Polytomella and
Euglena. It is generally accepted that mitochondria arose
from purple bacteria and have a monophyletic origin (37, 38). As MPP is
a heterodimer in all organisms investigated so far, the early
mitochondria most likely contained
- and
-MPP, which probably
both formed part of cytochrome c reductase. This situation
is still valid in all plants. In other organisms, one or two gene
duplications occurred, which were the prerequisite for the development
of a MPP enzyme in the soluble mitochondrial fraction: (i) a
duplication of the
-MPP protein, which gave rise to the core II
protein, and (ii) a duplication of the
-MPP protein, which gave rise
to the core I protein. Both gene duplications occurred in mammals and
yeast, one duplication in Neurospora,
Euglena, and probably Polytomella. Considering
the current knowledge about the phylogeny of eukaryotes, these
duplications must have taken place several times independently. This is
in line with very recent findings that duplication of entire genomes
occurred in the evolution of several higher eukaryotes like vertebrata
or yeast (39, 40). The question is: why did these genome duplications
lead to the evolution of core subunits in some organisms but not in
others? Interestingly, all organisms that comprise proteolytically
inactive core subunits and a soluble matrix-localized MPP are non-green
organisms, which generally or temporarily lack chloroplasts. Possibly,
the presence of chloroplasts, a second organelle involved in the
bioenergetics of the cell, alleviated the evolutionary pressure on
plant mitochondria, so that a soluble MPP was dispensable. Plant
mitochondria have unique functions in the metabolism of the green cell,
which seem not to require an independent regulation of respiration and
protein processing. Investigation of cytochrome c reductase
from further organisms may give further insights into the co-evolution
of this respiratory enzyme complex and the subunits of MPP.
 |
ACKNOWLEDGEMENTS |
The staghorn fern P. bifurcatum
was kindly provided by the Berggarten of the Hannover University. We
thank Elke Buchsteiner for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.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.:
49-511-7622674; Fax: 49-511-7623608; E-mail:
braun{at}mpimg-berlin-dahlem.mpg.de.
1
The abbreviations used are: MPP,
mitochondrial processing peptidase; BN, Blue-native; PAGE,
polyacrylamide gel electrophoresis; Tricine,
N-tris(hydroxymethyl)methylglycine; BSA, bovine serum albumin; PMSF, phenylmethylsulfonyl fluoride; MOPS,
3-(N-morpholino)propanesulfonic acid; Bis-Tris,
bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane.
2
S. Brumme, U. K. Schmitz, and H.-P. Braun,
unpublished data.
 |
REFERENCES |
-
Trumpower, B. L.
(1990)
Microbiol. Rev.
54,
101-129
-
Braun, H. P.,
and Schmitz, U. K.
(1995)
J. Bioenerg. Biomembr.
27,
423-436[Medline]
[Order article via Infotrieve]
-
Braun, H. P.,
and Schmitz, U. K.
(1995)
Trends Biochem. Sci.
20,
171-175[CrossRef][Medline]
[Order article via Infotrieve]
-
Leonard, K.,
Wingfield, P.,
Arad, T.,
and Weiss, H.
(1981)
J. Mol. Biol.
149,
259-274[Medline]
[Order article via Infotrieve]
-
Xia, D.,
Yu, C. A.,
Kim, H.,
Xia, J. Z.,
Kachurin, L. Z.,
Yu, L.,
and Deisenhofer, J.
(1997)
Science
277,
60-66[Abstract/Free Full Text]
-
Oudshoorn, P.,
van Steeg, H.,
Swinkels, B. W.,
Schoppink, P.,
and Grivell, L. A.
(1987)
Eur. J. Biochem.
163,
97-103[Abstract]
-
Crivellone, M. D.,
Wu, M. A.,
and Tzagoloff, A.
(1988)
J. Biol. Chem.
263,
14323-14333[Abstract/Free Full Text]
-
Gatti, D. L.,
and Tzagoloff, A.
(1990)
J. Biol. Chem.
265,
21468-21475[Abstract/Free Full Text]
-
Brunner, M.,
Klaus, C.,
and Neupert, W.
(1994)
in
Signal Peptidases (von Heijne, G., ed), pp. 73-86, Landes Co., Austin, TX
-
Luciano, P.,
and Geli, V.
(1996)
Experientia (Basel)
52,
1077-1082
-
Braun, H. P.,
and Schmitz, U. K.
(1997)
Int. J. Biochem. Cell Biol.
29,
1043-1045[CrossRef][Medline]
[Order article via Infotrieve]
-
Braun, H. P.,
Emmermann, M.,
Kruft, V.,
and Schmitz, U. K.
(1992)
EMBO J.
11,
3219-3227[Abstract]
-
Eriksson, A. C.,
Sjöling, S.,
and Glaser, E
(1994)
Biochim. Biophys. Acta
1186,
221-231
-
Braun, H. P.,
Emmermann, M.,
Kruft, V.,
Bödicker, M.,
and Schmitz, U. K.
(1995)
Planta
195,
396-402[Medline]
[Order article via Infotrieve]
-
Schulte, U.,
Arretz, M.,
Schneider, H.,
Tropschug, M.,
Wachter, E.,
Neupert, W.,
and Weiss, H.
(1989)
Nature
339,
147-149[CrossRef][Medline]
[Order article via Infotrieve]
-
Weiss, H.,
Leonard, K.,
and Neupert, W.
(1990)
Trends Biochem. Sci.
15,
178-180[Medline]
[Order article via Infotrieve]
-
Braun, H. P.,
Jänsch, L.,
and Schmitz, U. K.
(1997)
in
Proteolysis and Protein Turnover (Hopsu-Havu, V. K., Järvinen, M., and Kirschke, H., eds), pp. 290-297, IOS Press, Amsterdam
-
Braun, H. P.,
and Schmitz, U. K.
(1995)
Biochim. Biophys. Acta
1229,
181-186[Medline]
[Order article via Infotrieve]
-
Schlösser, U. G.
(1994)
SAG-Sammlung von Algenkulturen Catalogue of Strains 1994: BOACEJ, Vol. 3, pp. 113-186, University of Göttingen, Göttingen, Germany
-
Gutiérrez-Cirlos, E. B.,
Antaramian, A.,
Vázquez-Acevedo, M.,
Coria, R.,
and González-Halphen, D.
(1994)
J. Biol. Chem.
269,
9147-9154[Abstract/Free Full Text]
-
Jänsch, L.,
Kruft, K.,
Schmitz, U. K.,
and Braun, H.-P.
(1996)
Plant J.
9,
357-368[CrossRef][Medline]
[Order article via Infotrieve]
-
Schägger, H.
(1995)
Methods Enzymol.
260,
190-201[Medline]
[Order article via Infotrieve]
-
Schägger, H.,
and von Jagow, G.
(1987)
Anal. Biochem.
166,
368-379[Medline]
[Order article via Infotrieve]
-
Braun, H. P.,
Emmermann, M.,
Kruft, V.,
and Schmitz, U. K.
(1992)
Mol. Gen. Genet.
231,
217-225[Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Schägger, H.,
and von Jagow, G.
(1991)
Anal. Biochem.
199,
223-231[Medline]
[Order article via Infotrieve]
-
Schägger, H.,
Link, T. A.,
Engel, W. D.,
and von Jagow, G.
(1986)
Methods Enzymol.
126,
181-191[Medline]
[Order article via Infotrieve]
-
Brandt, U.,
Uribe, S.,
Schägger, H.,
and Trumpower, B. L.
(1994)
J. Biol. Chem.
269,
12947-12953[Abstract/Free Full Text]
-
Borchart, U.,
Machleidt, W.,
Schägger, H.,
Link, T. A.,
and von Jagow, G.
(1985)
FEBS Lett.
191,
125-130[CrossRef][Medline]
[Order article via Infotrieve]
-
Brandt, U.,
Yu, L.,
Yu, C.-A.,
and Trumpower, B. L.
(1993)
J. Biol. Chem.
268,
8387-8390[Abstract/Free Full Text]
-
Yang, M.,
Jensen, R. E.,
Yaffe, M. P.,
Oppliger, W.,
and Schatz, G.
(1988)
EMBO J.
7,
3857-3862[Abstract]
-
Hawlitschek, G.,
Schneider, H.,
Schmidt, B.,
Tropschug, M.,
Hartl, F. U.,
and Neupert, W.
(1988)
Cell
53,
795-806[Medline]
[Order article via Infotrieve]
-
Ou, W. J.,
Ito, A.,
Okazaki, H.,
and Omura, T.
(1989)
EMBO J.
8,
2605-2612[Abstract]
-
Kitada, S.,
Shimokata, K.,
Niidome, T.,
Ogishima, T.,
and Ito, A.
(1995)
J. Biochem. (Tokyo)
117,
1148-1150[Abstract]
-
Striebel, H. M.,
Rysavy, P.,
Adamec, J.,
Spizek, J.,
and Kalousek, J.
(1996)
Arch. Biochem. Biophys.
335,
211-218[CrossRef][Medline]
[Order article via Infotrieve]
-
Emmermann, M.,
Braun, H. P.,
Arretz, M.,
and Schmitz, U. K.
(1993)
J. Biol. Chem.
268,
18936-18942[Abstract/Free Full Text]
-
Gray, M. W.
(1993)
Curr. Opin. Genet. Dev.
3,
884-890[Medline]
[Order article via Infotrieve]
-
Lang, B. F.,
Burger, G.,
O'Kelly, C. J.,
Cedergren, R.,
Golding, G. B.,
Lemieux, C.,
Sankoff, D.,
Turmel, M.,
and Gray, M. W.
(1997)
Nature
387,
493-497[CrossRef][Medline]
[Order article via Infotrieve]
-
Spring, J.
(1997)
FEBS Lett.
400,
2-8[CrossRef][Medline]
[Order article via Infotrieve]
-
Wolfe, K. H.,
and Shields, D. C.
(1997)
Nature
387,
708-713[CrossRef][Medline]
[Order article via Infotrieve]
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