From the Institut de Biologie Moléculaire des
Plantes du CNRS, Université Louis Pasteur, 12 rue du
Général Zimmer, F-67084 Strasbourg, France and the
¶ Institut für Mikrobiologie, Eidgenössische
Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich,
Switzerland
Received for publication, September 28, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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The maturation of c-type cytochromes
requires the covalent attachment of the heme cofactor to the
apoprotein. For this process, plant mitochondria follow a pathway
distinct from that of animal or yeast mitochondria, closer to that
found in Electron transport constituents of respiratory chains in
prokaryotes and eukaryotes are composed mainly of proteins containing a
variety of cofactors including metal ions, iron-sulfur clusters, nucleotides, or hemes. During the last 10 years, there has been an
increasing interest in their biogenesis, which involves the transport
of both the apoprotein and prosthetic groups, their folding, and their
subsequent assembly into the corresponding complexes. In the case of
c-type cytochromes, the covalent attachment of heme to the
apoprotein occurs at the site of function, which is a different
subcellular compartment from that in which synthesis of heme or
apocytochromes occurs. These two characteristics add a further
complexity to the post-translational maturation pathway of these
heme-containing proteins.
Genetic analysis of model organisms has led to the identification of
three different systems for the maturation of c-type cytochromes (1, 2) in Gram-negative bacteria (system I), chloroplast
(system II), and fungal mitochondria (system III). Although system I
and system II share common elements, genome sequencing projects have
shown that no homology could be found between systems I and III. In
Escherichia coli (system I) at least eight genes,
ccmA-ccmH1, are essential for the
production of holocytochrome c (3, 4). In contrast, in yeast
mitochondria (system III), only two proteins, cytochrome
c and cytochrome c1 heme lyases were
shown to be the central components of the formation of holocytochromes (5, 6). Orthologues of fungal heme lyases were found in vertebrate and
invertebrate nuclear genomes but not in plant genomes. Unexpectedly,
land plant mitochondrial genomes encode orthologues of bacterial
ccm genes, namely ccmB, ccmC, and
ccmF (7, 8). These genes are also found in the mitochondrial
genome of early diverging protists such as Reclinomonas
americana (9) but not in the mitochondrial or nuclear genomes of
yeast. This finding reveals that at least two distinct routes for
c-type cytochrome assembly were established during
mitochondrial evolution as exemplified by yeast and land plants. Plant
mitochondria have evolved unique strategies for the structure and
expression of their genetic information (10) and have developed
specific biochemical and physiological functions as organelles
belonging to photosynthetic organisms (11). The c-type
cytochrome maturation pathway is an example of the specific features
displayed by plant mitochondria compared with their mammalian or fungal counterparts.
All mitochondria contain two c-type cytochromes: cytochrome
c1 is part of complex III (cytochrome
c reductase), whereas cytochrome c shuttles from
complex III to complex IV (cytochrome c oxidase). Both
apoproteins are synthesized in the cytosol and are imported post-translationally into the mitochondrial inner membrane and intermembrane space.
Heme needs to be translocated from the matrix side of the inner
membrane where ferrochelatase, the last enzyme in its biosynthesis, is
located (12) to the intermembrane space where ligation to apocytochrome
takes place. In plants, In bacteria, CcmE is crucial for cytochrome c maturation.
This periplasmic protein is anchored in the membrane by its N-terminal hydrophobic domain (18). The CcmE protein from E. coli was
shown to be a heme-binding protein (19, 20). The bacterial CcmE protein
is characterized by two stretches of conserved amino acids, of which
one contains a strictly conserved histidine shown to bind heme
covalently (19). This protein is believed to capture heme in the
periplasm in the presence of CcmC (21, 22) and transfer it to
the apocytochrome in the presence of one or more of the CcmF,
CcmG, or CcmH proteins (19). In this paper we present the
characterization of Arabidopsis thaliana CCME, a
plant orthologue of E. coli CcmE. We show that the nuclear
gene AtCCME encodes a mitochondrial protein attached to the
inner membrane and oriented toward the intermembrane space. We have
tested the extent to which the plant mitochondrial CCME could
substitute for its E. coli orthologue in
vivo.
Strains and Plasmids--
AtCCME cDNA clone
(GenBankTM accession number ATU72502) was obtained from F. Grellet (CNRS, Perpignan France). Plasmids expressing AtCCME under an
arabinose-inducible promoter were constructed by
PCR.1 To generate the plasmid
pAT1, the coding region of AtCCME corresponding to
Met79-Ser256 was amplified using O1 and
O2 as 5' and 3' primers respectively. The PCR fragment was
cloned in the BamHI site of pUC19. Clones with the correct
orientation were checked by sequencing, the
NdeI/EcoRI fragment was cut out and cloned into
pISC-2. The His222 to Ala AtCCME mutant was generated by
"overlap extension" PCR technique. Two overlapping complementary
oligonucleotides, O3 and O4, were designed to introduce the mutation.
Two amplifications were performed separately using O1-O3 and O4-O5
primer pairs. The purified PCR products were annealed and amplified
with O1 and O5. The final PCR product was cut by NdeI and
EcoRI and cloned into pISC-2, generating pAT2, as follows:
O1, 5'-CGGGATCCATATGCAGAATCGCCGTTTATGG-3'; O2, 5'-CTCCGGATCCTAAGAAGCCGCAACTTCAGC-3'; O3,
5'-CTCATCAGCCTTAGCCAAAACTTCAGTC-3'; O4,
5'-GGCTAAGGCTGATGAGAAGTATATGCCA-3'; O5,
5'-GAAGAATTCAGGATCCTAAGAAGCCGCAACTTC-3'. Restriction sites are shown in italics. The E. coli
ccmE gene was amplified by PCR and cloned as an
NdeI-EcoRI fragment into pISC-2, resulting in
pEC412, and the cloned DNA was sequenced. Plasmid pEC101, containing
ccmABCD, was cloned by inserting a 1.2-kilobase pair
AflII-SspI fragment containing ccmBCD
into AflII-FspI-digested pEC86 (23), which
provided ccmA. The information concerning strains and
plasmids used is this work is summarized in Table I.
Overexpression of AtCCME and Antibody Production--
A portion
of the AtCCME cDNA (corresponding to
Phe111-Ser256) was amplified by PCR
using the following two primers:
5'-ATCGGATCCTCTTCTACCTAACGCCA-3' and 5'-TAAAAAGAA
TGAATTCTATACCAATC-3'. The PCR product was cut by
BamHI and EcoRI and cloned into the corresponding
sites of pRSET-C (Invitrogene) downstream of a His tag sequence. After induction with 2 mM
isopropyl-1-thio- Western Blot and Immunodetection--
Proteins were separated by
SDS-polyacrylamide gel electrophoresis and transferred onto an
Immobilon-P membrane (Amersham Pharmacia Biotech). Western blots with
AtCCME purified antibodies were performed at a dilution of 1/5000. Goat
anti-rabbit antibodies conjugated to horseradish peroxidase (Amersham
Pharmacia Biotech) were used as secondary antibodies and revealed with
ECL reagents (Amersham Pharmacia Biotech). Antibodies directed against
the spinach large subunit (LSU) of ribulose bisphosphate carboxylase
(a gift from B. Camara, IBMP-CNRS, Strasbourg, France) and
Southern Hybridization--
A. thaliana ecotype
Columbia were grown on soil in a greenhouse for ~4 weeks. Total DNA
was extracted as described (28). 25 µg of DNA were digested with each
restriction endonuclease and analyzed on 0.6% agarose gels. After
Southern transfer to a nylon membrane, hybridization was performed
under standard conditions at 65 °C with a 32P-labeled
AtCCME cDNA probe prepared by random hexamer extension. The membrane was washed in 2× SSC, 0.1% SDS for 30 min at 65 °C and twice in 0.2× SSC, 0.1% SDS for 30 min at 65 °C.
Bacterial Growth Conditions and Cell Fractionation--
E.
coli was grown aerobically in LB medium or anaerobically in
minimal salt medium with 5 mM nitrite as the electron
acceptor (29). For the expression of Bradyrhizobium
japonicum cytochrome c550, E. coli cells were grown to midexponential phase and then induced
with 0.4% arabinose. Whole-cell protein analysis, isolation of
periplasmic and membrane fractions, and heme staining were performed as
described previously (3, 21, 22, 30).
Purification of A. thaliana Mitochondria and
Chloroplast--
Arabidopsis protoplasts were prepared from
3-4-day-old suspension cell cultures as described previously (31). The
washed protoplasts were resuspended in an extraction buffer (400 mM sucrose, 50 mM Tris-HCl, pH 7.5, 3 mM EDTA, 0.1% bovine serum albumin, and 2 mM
dithiothreitol), and disrupted by filtrations through nylon membranes
(32). The broken cells were diluted in a large volume of the extraction
buffer, and differential centrifugations were carried out as described
previously (33). The chloroplast-enriched fraction was loaded onto a
40/80% Percoll step gradient, and intact chloroplast were collected as
described (31). Mitochondria were layered onto a 13.5-21-45% Percoll
step gradient and spun at 75,000 × g for 45 min. The
mitochondria were collected at the 21/45% interface and washed in the
extraction buffer without bovine serum albumin and dithiothreitol.
Mitoplast Preparation and Submitochondrial
Fractionation--
Mitoplasts were prepared as described (34) with
some modifications (35). The mitochondria were resuspended in a
swelling buffer, and the outer membrane rupture was achieved by
Dounce homogenization. Mitoplast and outer membrane fractions
were isolated after centrifugation through a bovine serum albumin-free
discontinuous gradient of 22, 33, and 47% sucrose. Outer membranes
were collected at the 8.6/22% interface, diluted, and recovered by
centrifugation at 38,000 rpm in a Ti-75 rotor. Intact mitoplasts were
collected from the 33/47% interface, washed and resuspended in 20 mM MOPS, pH 7.2, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride at a protein concentration of 3 mg/ml,
and broken by three freeze/thaw cycles followed by sonication (5×
10 s, 300 W, Sonic Vibra Cells). The membrane (P) and soluble (S)
fraction of mitoplasts were separated by a 30-min centrifugation at
100,000 × g in a Beckman TLA-100 rotor. The
soluble proteins were precipitated by 10% trichloroacetic acid. The
membrane fraction was subjected to alkaline treatment (0.1 M Na2CO3, pH 11.5, for 30 min at
4 °C) to extract peripheral proteins (36). A 30-min centrifugation
at 100,000 × g in a Beckman TLA-100 rotor allows the
separation of soluble (peripheral) from insoluble (integral) protein
fractions. Freshly purified mitoplasts were subjected to proteinase K
treatment. Mitoplasts (1 mg/ml) were incubated with 100 µg/ml
proteinase K in 8.6% sucrose, 50 mM Tris-HCl (pH 7.5) for
30 min at 4 °C. Phenylmethylsulfonyl fluoride was added at the final
concentration of 1 mM to stop the protease activity, and
the mitoplasts were recovered by centrifugation through a 22% sucrose
cushion at 15,000 × g for 15 min.
Import of Radiolabeled Proteins into Isolated
Mitochondria--
Mitochondria were isolated from potato tubers
(Solanum tuberosum, var. Bintje) with a juice extractor as
described (37). Proteins were synthesized from the corresponding
cDNA clones in pBluescript vector by coupled
transcription/translation in the presence of
[35S]methionine according the supplier's instruction
(Promega). Import assays were carried out as described (38).
AtCCME Is a Single-copy Gene in A. thaliana--
The physical and
genetic analysis of a nuclear gene cluster on A. thaliana
chromosome 3 (39, 40), revealed the presence of AtCCME, a
gene encoding a protein showing sequence similarity with the E. coli CcmE (3) and its orthologues for Gram-negative bacteria (4,
41). All cDNAs, isolated from a library prepared from growing cell
suspensions, share the same 5'-end at only 5 nucleotides from the
initiation codon, whereas two different polyadenylated ends were found
at 226 and 268 nucleotides from the stop
codon.2 No transcript could
be detected by Northern experiments, but reverse
transcription-PCR experiments allow detection of AtCCME transcripts in the roots, rosette leaves, stems, stem leaves and flowers of A. thaliana (data not shown). A radiolabeled
probe generated from AtCCME cDNA (ATU72502) was used for
Southern hybridization of A. thaliana total DNA. The probe
hybridized to a single DNA fragment for each of the five restriction
enzymes used. The sizes of the bands correspond to those deduced from
the physical map of the AtCCME genomic locus (Fig.
1, A and B). Therefore, in
A. thaliana, CCME is a single-copy gene located
on chromosome 3 and is expressed at a low level.
AtCCME Is a Mitochondrial Protein and Its Precursor Is Imported
into Mitochondria through an N-terminal Cleavable Targeting
Sequence--
AtCCME cDNA encodes a putative protein of
256 amino acids, which presents an N-terminal extension of about 70-80
amino acids when compared with bacterial proteins (Fig.
2). An extension of a similar size is
also present in the putative protein encoded by a CCME
homologous gene of Oryza sativa (AC025783). In AtCCME, this
extension is enriched in positively charged (Arg) and hydroxylated (Ser) residues and contains few acidic residues. Its first 20 amino
acids could form an amphiphilic AtCCME Is a Peripheral Inner Membrane Protein--
Bacterial CcmE
are mainly hydrophilic proteins except for a short hydrophobic region
at the N-terminal domain, which is predicted to act as a
noncleavable signal sequence and to anchor the protein in the membrane.
Although its amino acid sequence is not conserved, such a hydrophobic
domain is present in AtCCME (Fig. 2), which could, as for its
bacterial counterparts, attach the protein in a membrane. AtCCME
antibody recognized a protein of 27 kDa in the mitochondria
(M) and mitoplasts fraction (MP), whereas no signal could be detected in the outer membrane fraction
(OM), characterized by the presence of porin, a major
protein of the mitochondrial outer membrane (25) and the absence of
cytochrome c1, a subunit of the mitochondrial
inner membrane cytochrome bc1 complex (Fig.
4A). The presence of porin in
the mitoplast fraction indicated that, in the experimental conditions
used, part of the outer membrane remains attached to the inner
membrane, most likely at contact points between the two membranes. The
mitoplasts were broken and further separated into soluble
(S) and membrane (P) protein fractions; AtCCME
was present in the membrane fraction (Fig. 4B). No
contamination between matrix and membrane proteins could be detected,
which was verified using antibodies directed against a matrix protein,
manganese-superoxide dismutase, and antibodies against cytochrome
c1. These results strongly suggest that AtCCME
is located in the mitochondrial inner membrane. To further characterize
the nature of the membrane interaction, extreme pH treatment was used
to extract extrinsic proteins by disruption of electrostatic
interactions (46). After alkali treatment of mitoplast membranes,
AtCCME was found in the soluble fraction (Fig. 4C). AtCCME
protein behaves like NAD9, a protein located in the peripheral arm
(iron-sulfur protein fraction) of the L-shaped complex I (27, 47), and
not like cytochrome c1, a protein with a
C-terminal membrane helix responsible for its intrinsic behavior (Fig.
4C). Our results suggest that AtCCME is attached to the
mitochondrial inner membrane by electrostatic interactions such as
protein-protein interactions rather than direct contact with the lipid
bilayer.
AtCCME Is Oriented toward the Intermembrane Space--
PhoA fusion
analyses of B. japonicum CcmE (BjCcmE) and E. coli CcmE (EcCcmE) proteins have shown that the hydrophilic part of the protein is exposed to the periplasm (18, 19). By analogy, AtCCME
is predicted to be located at the outer face of the inner membrane. To
assess this hypothesis, intact mitoplasts were treated with proteinase
K to strip inner membrane proteins, which are exposed to the
intermembrane space. Mitochondria and mitoplasts incubated in the same
conditions in the absence of protease treatment were used as control
(Fig. 4D). When proteinase K was added, the immunodetection
of AtCCME was lost, whereas it was still observed in untreated
mitoplast. The intactness of the inner membrane after proteinase K
treatment was assayed with NAD9 antibody (27). NAD9 is located in the
iron-sulfur protein fraction of complex I, which is facing the
matrix (47), and therefore protected from proteinase K. The hydrophilic
domain of AtCCME is most likely completely digested in treated
mitoplasts because no signal corresponding to a smaller partially
protected protein could be detected. Therefore in AtCCME, as in its
bacterial counterpart, the main conserved motifs are localized on the
external side of the inner membrane.
Assaying Complementation of a AtCCME Covalently Binds Heme in E. coli--
The cytochrome
c maturation pathway was impaired when trying to complement
a In photosynthetic eukaryotes, two cytochrome c
biogenesis pathways operate in the same cell although in separated
compartments, mitochondria and chloroplasts. In land plants, system I
and system II are proposed to perform cytochromes c
maturation in mitochondria and chloroplast, respectively. Aside from
organelle genes, several nuclear loci are most likely involved in
cytochrome c biogenesis for each system. System I and system
II share some common elements (heme delivery and thioreduction), and
their evolutionary link appears through the conservation of a
tryptophan-rich motif and some histidine residues (3, 22, 48).
Therefore, the subcellular location of nuclear-encoded proteins showing
similarities with bacterial proteins required for cytochromes
c biogenesis must be addressed carefully. AtCCME
is the first ccm orthologue found in a plant nuclear genome.
In A. thaliana this gene is unique. Using different
approaches, in vitro import into isolated mitochondria and
immunodetection into Arabidopsis protein extracts, we have demonstrated clearly that AtCCME is a mitochondrial protein. In addition, in vivo experiments were performed using transient
expression of different AtCCME-GFP fusion proteins in tobacco cells.
All these fusion proteins were detected exclusively in
mitochondria.3 We can exclude
a dual targeting to both organelles, which has been reported for other
proteins such as glutathione reductase (49), ferrochelatase (50), and
aminoacyl-tRNA synthetases (51, 52).
We have shown that AtCCME is associated with the mitochondrial inner
membrane. AtCCME has a typical mitochondrial targeting sequence at its
N terminus, which is able to target a reporter protein to
mitochondria3 and which is cleaved upon in vitro
import. Our attempts to purify the mature protein for N-terminal
sequencing have been unsuccessful, mainly because of its instability
when extracted. The exact N-terminal sequence of the mature protein
remains unknown. Nevertheless, we propose that a domain of
about 16 to 30 residues, conserved in several plant CCME proteins and
rich in charged amino acids, is present at the N-terminal end of the
mature mitochondrial protein, preceding the hydrophobic domain. This
region would constitute a plant-specific motif not found in bacterial
CcmE proteins.
In a number of plants, mitochondrial genes encoding counterparts of
CcmB, CcmC, and CcmF are transcribed and their mRNAs are edited. In
some cases, the putative corresponding proteins could be immunodetected
in the membrane protein fraction of mitochondria (53).4 However, no topology
of any of these proteins has been described, and no functional analysis
of the corresponding mitochondrial genes has been successful up to now.
AtCCME, the product of a nuclear gene, comprises the relevant features
that play a role in mitochondrial cytochrome c
biogenesis and appears a better candidate for functional analysis based
on complementation of E. coli mutant strains.
The complementation of Because of experimental limitations, heme binding to At- CCME in
mitochondria could not be tested with the heme staining methods used
for the overexpressed EcCcmE. The instability of the purified mitochondrial protein and the fact that heme binding, although covalent, is proposed to be transient were the main difficulties encountered in getting direct evidence of heme binding to AtCCME in
plant mitochondria.
The cytochrome c biogenesis pathway, known as system I, is
followed by In eukaryotes, AtCCME is a unique example of a mitochondrial heme
chaperone. In system II, no heme chaperone has yet been described
neither in Chlamydomonas chloroplast nor in Gram-positive bacteria, which are the model organisms for the study of this pathway.
However, it is possible that other types of heme chaperones, perhaps
with a different type of heme linkage, will be discovered in these
systems. In system III, it is also unknown whether an heme chaperone is
needed and, if so, whether this function is held by the cytochrome
c and cytochrome c1 heme lyase
proteins or another unidentified protein.
It is striking that in mitochondria two systems have evolved for
cytochrome c maturation. More detailed knowledge of each of
these systems will help the understanding of why system I has been
retained in the mitochondria of some species and why and from
what source system III has evolved.
- and
-proteobacteria. We report the first
characterization of a nuclear-encoded component, namely AtCCME, the
Arabidopsis thaliana orthologue of CcmE, a periplasmic heme
chaperone in bacteria. AtCCME is targeted to mitochondria, and its
N-terminal signal peptide is cleaved upon import. AtCCME is a
peripheral protein of the mitochondrial inner membrane, and its major
hydrophilic domain is oriented toward the intermembrane space.
Although a AtCCME (Met79-Ser256) is
not fully able to complement an Escherichia coli CcmE
mutant strain for bacterial holocytochrome c
production, it is able to bind heme covalently through a conserved
histidine, a feature previously shown for E. coli CcmE. Our
results suggest that AtCCME is important for cytochrome c
maturation in A. thaliana mitochondria and that its
heme-binding function has been conserved evolutionary between land
plant mitochondria and
-proteobacteria.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminolevulinate, the universal precursor of tetrapyrroles (chlorophyll and heme) is formed in chloroplasts via the C5 (glutamate) pathway (13), whereas in yeast and
mammals, it is synthesized by 5-
-aminolevulinate synthase, a
mitochondrial enzyme (14). The next steps of protoporphyrin IX
synthesis involve trafficking in cytosol, mitochondria, and plastids in
the case of plant cells (15). The trafficking of heme precursors in
eukaryotic cells is poorly understood as is the intracellular transport
of heme in the cytoplasm or to the nucleus where heme is implicated in
transcriptional regulation. In animals and fungi, a short sequence
called the "CPV" motif is conserved in cytochrome heme lyases and
in other heme-binding proteins such as the transcription factor Hap1,
5-
-aminolevulinate synthase or heme oxygenase-2 (16). In cytochrome
c1 heme lyase, this motif has been shown to bind
heme in a reversible manner (17). However, the exact mechanisms of the
transient heme binding to the CPV motif is still unknown.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, the fusion
protein was expressed in E. coli strain BL21 (24) and purified in a denaturing buffer by affinity column chromatography using
Ni-NTA-Sepharose (Novagen). The fusion protein was injected into
rabbits to raise polyclonal antibodies. The sera were purified against
the fusion protein, His6-AtCCME, coupled to CnBr-activated Sepharose according to the supplier (Amersham Pharmacia Biotech).
-tubulin (Amersham Pharmacia Biotech) were used as control.
Antibodies directed against potato porin (25) (provided by H.-P. Braun,
Hannover University, Hannover, Germany), tobacco
manganese-superoxide dismutase (26) (obtained from F. van Breusegem,
Gent, Belgium), wheat subunit 9 of NADH dehydrogenase (27), and yeast
cytochrome c1 (provided by G. Schatz, Basel
University, Basel, Switzerland) were used as control for outer
membrane, matrix, and extrinsic or intrinsic inner membrane protein
fractions, respectively, of the mitochondria. The production of
antibodies against E. coli CcmE and analysis of proteins
expressed in E. coli with alkaline phosphatase-coupled secondary antibodies are described elsewhere (19). The apparent molecular weight of proteins was calculated using middle range molecular weight markers (Bio-Rad) as ladder.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Southern analysis of A. thaliana
genomic DNA. A, A. thaliana genomic
DNA was digested with B, BamHI; Bg,
BglII; E, EcoRI; H,
HindIII; and P, PstI. After Southern
blotting, the membrane was hybridized with an AtCCME
cDNA probe labeled by random priming. The molecular weight ladder
is indicated in kilobase pairs, shown on the left.
B, physical map of the AtCCME locus on A. thaliana chromosome 3 derived from the nucleotide sequence
(GenBankTM accession number AFO49236). The restriction
fragments that hybridize with AtCCME are represented
above the physical map, and their sizes are indicated in
kilobase pairs. Restriction enzymes are the same as used for
panel A.
-helix. These features are
characteristic of the import domain of mitochondrial targeting sequences (42, 43). The localization of AtCCME was checked first by
immunodetection using purified polyclonal antibodies generated against
an overexpressed fusion protein (from Phe111 to
Ser256). The anti-AtCCME antibodies recognized a 27-kDa
protein in mitochondria but not in chloroplast protein fractions
prepared from A. thaliana protoplasts (Fig.
3A). CCME could also be
detected in cauliflower, turnip, rapeseed, and radish mitochondria, but
not in potato, pea, sunflower, wheat, and maize mitochondria,
indicating that anti-AtCCME antibodies are rather specific to
Brassica species (data not shown). To test whether
AtCCME encodes a precursor processed after import, we tried
to import the radiolabeled protein in vitro into purified
mitochondria. A major 32-kDa protein was obtained by in
vitro coupled transcription/translation of AtCCME
cDNA (Fig. 3B). After its incubation with mitochondria,
a signal corresponding to a 27-kDa protein resistant to added
proteinase K appeared. The protection was abolished when mitochondrial
membrane proteins were extracted by Triton X-100 before proteinase K
treatment. AtCCME import was inhibited in the presence of valinomycin,
indicating the requirement of an electrochemical membrane potential,
, to achieve AtCCME translocation (44). A 5-kDa
reduction was observed between the apparent molecular weights of the
precursor and the mature protein. For the mitochondrial processing
peptidase domain, only a weak consensus has been found, mainly a
conserved Arg in position-2 or-3 from the cleavage site. For plant
mitochondrial precursors, two cleavage motifs were proposed (45), of
which one, an "R-2" motif (RX(A/S)(T/S)), is
present at position 47-50 (RLSS, Fig. 2). Although the
actual processing site has not been determined experimentally, the R-2
motif described above is a good candidate. A cleavage at this position
would shorten the precursor protein by 48 amino acids, corresponding to
a 5.4-kDa peptide, which is in agreement with the shift observed in
import assays. The radioactive band of the in
vitro processed protein and the immunodetected A. thaliana endogenous mitochondrial protein migrate at the same
position (Fig. 3C), which strongly suggests that in
vitro processing in potato mitochondria reflects in
vivo maturation of A. thaliana protein.
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Fig. 2.
Amino acid sequence comparison of CCME
proteins. CCME proteins from different organisms are aligned:
AtCCME (39); O. sativa (OsCCME)
(GenBankTM accession number AC025783); -proteobacteria
B. japonicum (BjCcmE) (18); and
-proteobacteria E. coli (EcCcmE)
(GenBankTM accession number U00008). The four protein
sequences were aligned using the GCG PileUp algorithm (57). Residues
identical in more than two sequences are highlighted in
black, and the functionally conserved residues are
highlighted in gray. The conserved motif 1 and
motif 2 regions are indicated. The consensus R-2 plant processing site
in AtCCME is boxed. The amino acids corresponding to the
hydrophobic domain are underlined. The conserved histidine
residue, which binds heme covalently in E. coli, is marked
with an asterisk. The numbering is that of the
AtCCME amino acid sequence.
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Fig. 3.
AtCCME is imported into mitochondria.
A, Western blot analysis of Arabidopsis protein
fractions (30 µg) of protoplast (P), chloroplast
(C), and mitochondria (M), probed with antibodies
directed against AtCCME, NAD9, LSU (large subunit of ribulose
bisphosphate carboxylase), and -tubulin. B, in
vitro translated AtCCME precursor (Pre, p) corresponds
to a 32-kDa band (lane 1). In the presence of mitochondria
(M), a smaller protein corresponding to the mature form
appears at 27 kDa. This mature protein is resistant to proteinase K
(PK) in intact mitochondria but is degraded when
mitochondria membranes are disrupted by Triton X-100 (TX).
The import process is abolished in the presence of valinomycin
(Val). The precursor and the mature polypeptides are
indicated by arrows. Lane 1 contains one-fifth of
the amount of precursor included in each import reaction. 40 µg of
purified potato mitochondria were used for each import assay. Proteins
were separated on a 15% SDS-polyacrylamide gel; proteins labeled with
[35S]Met were detected by autoradiography for 2 weeks at
80 °C. C, in vitro translated AtCCME
precursor (lane 1), import assay of AtCCME in potato
mitochondria (lane 2), and 20 µg of A. thaliana
total mitochondrial proteins (lane 3) were loaded on the
same gel. After Western blotting, the membrane was cut in two pieces:
one was exposed to a film (lanes 1 and 2), and
the second probed with the purified anti-AtCCME antibody (lane
3).
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Fig. 4.
Submitochondrial localization and topology of
AtCCME. A, mitochondria (M), outer membrane
(OM), and mitoplast (MP) protein extracts were
analyzed for AtCCME, porin, and cytochrome c1
(cyt c1). B, mitoplasts were
subjected to freeze/thaw cycles and sonication. Soluble and membrane
proteins were collected after ultracentrifugation. Total mitoplast
(MP), supernatant (S), and pellet (P)
fractions were analyzed with the indicated antibodies. C,
The mitoplast pellet fraction was treated with
Na2CO3, pH 11.5, to extract peripheral proteins
(S), whereas intrinsic membrane proteins remains in a
100,000-g pellet (P). D, mitoplast were treated
with 100 µg/ml proteinase K and analyzed for AtCCME and NAD9. NAD9,
which is exposed to the matrix, is shown as the control for
inner membrane integrity. The Western blots were probed with antibodies
directed against the following mitochondrial proteins: AtCCME, potato
porin, wheat NAD9, yeast cytochrome c1, and
tobacco manganese-superoxide dismutase (Mn-SOD).
ccmE E. coli Strain for
Holocytochrome c Production--
In addition to its location and
topology in mitochondria, the AtCCME sequence similarity with bacterial
proteins suggests that the plant protein could fulfill similar
functions for cytochrome c biogenesis in mitochondria.
Indeed, AtCCME shares 35.5 and 40.6% identical amino acids with
E. coli CcmE and B. japonicum CcmE proteins,
respectively, whereas prokaryotic CcmE proteins share from 26 to 81%
identity. The two major conserved motifs in bacteria, motif 1 and motif
2, are present in AtCCME, although in A. thaliana and in
O. sativum, the insertion of a sequence rich in charged residues increases the distance between them (Fig. 2). In motif 2, the
histidine residue, which was shown to bind heme covalently in E. coli (19), is strictly conserved in all organisms. To test whether
the eukaryotic protein could complement the E. coli
ccmE mutant strain (EC65, Table
I), we constructed the plasmid pAT1,
which expresses a truncated form of AtCCME deleted from its first 78 amino acids. AtCCME (Met79-Ser256) best
corresponds to the EcCcmE size, pI, and hydrophobicity profiles. EC65
strain was transformed with a vector carrying various ccmE
genes together with pRJ3291 (Table I), carrying cycA, the soluble periplasmic cytochrome c550 gene from
B. japonicum used as a reporter for holocytochrome
c maturation (3). The peroxidase activity associated with
covalently bound heme was used to check the presence of holocytochromes
c in periplasmic protein extract (22). The transformants
were grown anaerobically to induce expression of the chromosomal genes
ccmA-ccmH and promote cytochrome c
production. Both CcmE and cytochrome c550 were
expressed from an arabinose-inducible promoter. When EcCcmE was
expressed in EC65 background, three cytochromes c were
detected by heme stain: NrfA and NapB, two endogenous cytochromes
c, and the reporter cytochrome c550
of B. japonicum (Fig. 5,
lane 1). When the complementation was tried with pAT1, no
holocytochrome c could be detected (Fig. 5, lane 2).
Strains and plasmids used in this work
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Fig. 5.
Functional complementation of the
ccmE strain for holocytochrome c
formation. E. coli strains were grown anaerobically in
the presence of nitrite. Periplasmic proteins (100 µg) were separated
by SDS-15% polyacrylamide gel electrophoresis and stained for
covalently bound heme. The EC65
ccmE strain was
cotransformed with pRJ3291, a plasmid expressing B. japonicum-soluble cytochrome c550
(cyt c1) and the following plasmids:
lane 1, pEC412 expressing EcCcmE; lane 2, pAT1
expressing AtCCME(Met79-Ser256); lane
3, pISC-2, the empty vector, as control. NrfA and NapB are two
endogenous cytochromes c of E. coli.
ccmE E. coli strain with AtCCME in anaerobic respiration. We wanted to know at which step heme trafficking was
blocked. For this determination, we tested whether heme transfer to CcmE was possible. Heme stain was used to check the formation of
holo-CCME, i.e. a protein binding heme in a covalent way. We transformed EC06, an E. coli strain deleted in all
ccm genes (3) with pAT1 alone or with pEC101 (Table I). The
assays were performed under aerobic growth conditions in the presence
of the CcmA-D proteins, a condition that is sufficient for heme
incorporation into CcmE. We first checked whether AtCCME was correctly
expressed and inserted in E. coli membranes. After induction
by arabinose, a protein corresponding to the truncated form of AtCCME
was expressed and detected in the membrane fraction (Fig.
6A, lane 1).
Immunodetection using antibodies directed against AtCCME or EcCcmE
suggests that the level of expression of AtCCME is reduced compared
with that of EcCcmE (Fig. 6, A and B). In a
ccm background, AtCCME, as its E. coli
counterpart, did not bind heme (Fig. 6C, lane 1). When the E. coli CcmABCD proteins were expressed with
AtCCME, the mitochondrial protein was able to bind heme in a covalent way (Fig. 6C, lane 2). This shows that the
truncated mitochondrial protein is correctly inserted in the bacterial
membrane and that its conserved heme-binding domain is orientated
toward the periplasm, thus allowing heme attachment. In E. coli, CcmC is the only Ccm protein that is strictly required for
heme transfer and binding to EcCcmE (19). Our results suggest that heme
transfer is possible from E. coli CcmC to A. thaliana CCME. To check whether, in AtCCME, heme is attached to
the conserved histidine of motif 2, we changed the histidine 222 to an
alanine by site-directed mutagenesis. EC06, the
ccm strain, was cotransformed with pAT2 expressing the His222Ala truncated AtCCME and pEC101 expressing
CcmABCD. Although the mutant protein was expressed, no heme-binding
AtCCME could be detected (Fig. 6, A and C, lane
3). A positive control was done with a strain expressing E. coli CcmABCD and CcmE (Fig. 6, B and C, lane
4). Histidine 222 is crucial for heme binding to AtCCME, as it is for the bacterial protein. Together with the complementation assays, these results show that AtCCME is partially able to complement EcCcmE function on the heme trafficking part of cytochrome c
maturation pathway. Indeed, AtCCME is able to bind heme, most likely
through the conserved histidine of motif 2, in a bacterial heterologous background including EcCcmC. Our results suggest that a full
complementation of the E. coli
ccmE strain by AtCCME is
most probably impaired by the absence of heme release to the following
proteins of the pathway (CcmF, -G, or -H) rather than by the low
efficiency with which AtCCME gets heme from CcmC.
View larger version (25K):
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Fig. 6.
Overexpression and heme binding of different
CCME proteins. Membrane proteins were prepared from E. coli cells grown aerobically. The ccm
EC06 strain was transformed with plasmids expressing the following
proteins: lane 1,
AtCCME(Met79-Ser256); lane 2,
E. coli CcmABCD and
AtCCME(Met79-Ser256); lane 3:
EcCcmABCD and AtCCME(Met79-Ser256)
His222Ala mutant protein; lane 4, E. coli
CcmABCD and CcmE. A, Western blot of membrane fractions (50 µg/lane), probed with anti-AtCCME purified serum. B,
Western blot identical to that in panel A, probed
with anti-EcCcmE serum. C, heme stain on membrane proteins
(100 µ g/lane).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ccmE E. coli strains
has been tested at two different steps of cytochrome c
maturation: heme binding to CcmE and heme transfer to apocytochrome
c. AtCCME was detected in E. coli membranes
and could be heme-stained when overexpressed with EcCcmABCD. Because
heme binding occurs only when CCME is translocated to the periplasm
(19), this indicates the correct location of AtCCME in E. coli. Complementation assays of a
ccmE E. coli strain by AtCCME
(Met79-Ser256) were unsuccessful for
holocytochrome c production. The results of the
complementation assays could be explained by the inability of the
AtCCME to release heme to the following bacterial partners of the
maturation pathway, although it is able to catch it from EcCcmC. Within
Ccm proteins, CcmC mitochondrial orthologues are among the closest
relatives of their bacterial counterparts, by their predicted topology
and by the sequence conservation of the Trp-rich motif and the
two flanking essential histidine residues (48, 54, 55). These conserved
motifs were proposed to have a function in heme delivery. They are also
found in the mitochondrial CcmFN orthologue to the
N-terminal part of CcmF. The conserved domains are interspersed by
plant-specific sequences, creating greater divergences between the
plant mitochondrial and their bacterial CcmF counterparts than
for CcmC ones. The potential interactions of AtCCME with EcCcmF could
be less efficient in a heterologous system, explaining the absence of
heme release from CCME. The knowledge of the full set of proteins for
the c-type cytochromes pathway in mitochondria will help in
designing design different combinations of mitochondrial/bacterial
genes for holocytochrome c formation in bacteria.
- and
-proteobacteria and Archae. Genes encoding related proteins were identified by sequence similarities in
mitochondrial genomes of a few protists, one red algae (56),
and land plants. The maximum set of ccm genes (system I)
found in mitochondrial genomes is found in protists like R. americana (9). CcmA, CcmB, CcmC, and CcmF orthologues are encoded
by these mitochondrial genomes, which are among the closest relatives
of the ancestral mitochondrial genome and resemble eubacterial ones. A
ccmH/cycL-like gene (GenBankTM accession number
AC007591) was recently found in A. thaliana (chromosome 1;
28% amino acid sequence identity with the B. japonicum CycL gene product), but the subcellular localization of its
product is unknown. CCME is a new mitochondrial protein proposed to be involved in cytochrome c biogenesis in plant mitochondria.
The existence of such a set of proteins strongly argues in favor of the
conservation of a functional system I in plant mitochondria. In this
paper, for the first time, a function has been attributed to a
mitochondrial Ccm counterpart. We propose that AtCCME is a heme
chaperone of the intermembrane space attached to the inner mitochondrial membrane. Because His222 is essential for
heme binding on AtCCME, we propose that heme is attached by a
single covalent bond to a histidine, as found for E. coli CcmE.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. F. Grellet (Perpignan, France) for the gift of AtCCME cDNA clone. We thank A. Klein for valuable technical assistance and Prof. L. Bonen for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Centre National de la Recherche Scientifique, the Swiss National Foundation for Scientific Research, and the Eidgenössische Technische Hochschule.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.
§ Supported by a grant from the Ministère de l'Education Nationale and by a European Molecular Biology Organization short-term fellowship.
To whom correspondence should be addressed. Tel.:
33-3-88-41- 72-37; Fax: 33-3-88-61-44-42; E-mail:
geraldine.bonnard@ibmp-ulp.u-strasbg.fr.
Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008853200
2 F. Grellet, personal communication.
3 N. Spielewoy, J. M. Grienenberger, and G. Bonnard, unpublished results.
4 G. Bonnard, unpublished results.
![]() |
ABBREVIATIONS |
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The abbreviations used are: PCR, polymerase chain reaction; AtCCME, Arabidopsis thaliana orthologue of CcmE; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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