From the Department of Chemistry and Biochemistry,
UCLA, Los Angeles, California 90095-1569
Received for publication, August 22, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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Three distinct systems (I, II, and III) for
catalysis of heme attachment to c-type apocytochromes are
known. The CcsA and Ccs1 proteins are required in system II for the
assembly of bacterial and plastid cytochromes c. A
tryptophan-rich signature motif (WWD), also occurring in CcmC and CcmF
found in system I, and three histidinyl residues, all strictly
conserved in CcsA suggest a function in heme handling. Topological
analysis of plastid CcsA in bacteria using the PhoA and
LacZ The c-type cytochromes are ubiquitous metalloproteins
which reside on the p-side of energy transducing membrane
systems. They display very little sequence similarity apart from the
occurrence of one to four conserved CXXCH motifs which are
the binding sites for the prosthetic group. The c-type
cytochromes are distinguished from other cytochromes by covalent
attachment of the prosthetic group to the apoprotein via one, or in
most cases, two thioether linkages between the vinyl side chains of
heme and the cysteines from the CXXCH motif. Because all
holocytochromes c acquire their function in a different
compartment from the sites of synthesis of their apoproteins and
cofactor heme, their biogenesis follows a complex pathway with several
steps: (a) transport and processing of the apocytochromes,
(b) transport of heme, (c) transfer of reducing
equivalents across the membrane in order to assure the maintenance of
heme and sulfhydryl groups of the CXXCH motif under a
reduced state and d) stereospecific attachment of the heme group to the
apocytochromes. Remarkably, genetic and biochemical analyses of
c-type cytochromes biogenesis have demonstrated the
existence of three distinct systems for the maturation of these
molecules (for review 1, 2-6).
The first pathway to be defined, now referred to as system III (3), was
discovered originally in fungal mitochondria and is also believed to
operate in vertebrate and invertebrate mitochondria. System III appears
to be the least complex of the three systems as only two factors,
cytochrome c heme lyase and cytochrome
c1 heme lyase were found to be required for the
assembly of the two c-type cytochromes found in the
mitochondrial intermembrane space, cyt1 c and cyt
c1, respectively (7-10). The interaction of
cytochrome c heme lyase with both heme (11), via the
characteristic CPX heme binding motif (12), and apocytochrome (13) led
to the assumption that it functions in the catalysis of thioether bond formation (7, 14-16).
A contrasting situation occurs in system I which operates in
System II was discovered in the green alga Chlamydomonas
reinhardtii in a search for ccs mutants specifically
deficient in membrane-bound cyt f and soluble cyt
c6, the only two c-type cytochromes resident in the plastid (47). All the ccs mutants failed to accumulate the plastid holocytochromes c because of
compromised heme attachment in the lumen (47, 48). Genetic analysis
established that five nuclear loci (CCS1 to 5)
and one plastid locus (ccsA) are required for the biogenesis
of plastid c-type cytochromes (47, 49, 50). At present, only
ccsA (51) and CCS1 (52) have been characterized
at the molecular level and shown to encode previously unknown integral
membrane proteins, for which function can only be inferred based on our
knowledge of the biochemistry required to complete holocytochrome
c maturation. Ccs1 is a pioneer protein lacking any
recognizable motifs which might suggest an obvious biochemical activity
in the pathway (52). CcsA, on the other hand, has a
tryptophan-rich2 "WWD"
motif (WGX In an effort to elucidate the biochemical function of CcsA in the
plastid system, we used the bacterial topological reporters alkaline
phosphatase and Strains and Culture Conditions--
C. reinhardtii
wild type strains CC125, 2137 (Chlamydomonas Genetic Center,
Duke University) and WT59+ (Institut de Biologie
PhysicoChimique, Paris, France) are wild type with respect to
photosynthesis and c-type cytochrome biogenesis and are
accordingly labeled wild type in the figures. The
ccsA-ct59, -B6 (49) and
ccs1-6::ARG7 (59) strains
display a "ccs" phenotype (47, 48) and are labeled accordingly
ccsA and ccs1 in the figures. Strains were grown
at 22-25 °C in TAP medium (60) with or without copper
supplementation under dim light (25 µmol/m2/s) for non
photosynthetic strains or under standard illumination for wild type
strains (125 µmol/m2/s) as described previously (47). The
ccsA site-directed mutants were tested for their ability to
grow photosynthetically on minimum medium under various illuminations
(40-170-500 µmol/m2/s). E. coli strains
XL1-blue and DH5 Construction of ccsA-PhoA and ccsA-LacZ Fusions--
Plasmid
pRGK200 (19) containing the alkaline phosphatase encoding gene
(phoA) was used to generate 9 ccsA-phoA
translational fusions by a PCR-based strategy described previously
(17). Various portions of the ccsA gene were amplified with
Pfu polymerase using the cloned ccsA gene as a
template, F-CcsASac as a forward primer and one of the 9 R-CcsA
oligonucleotides as a reverse primer (see list of primers below).
F-CcsASac was engineered with a SacI site upstream of the
first ATG codon in ccsA ORF (51) and reverse primers were
designed with a SalI site at the desired
ccsA-phoA fusion junction. PCR products thus obtained were
digested with SacI and SalI and cloned into
SacI/SalI digested pRGK200 to yield the
pccsA::phoA series of plasmids. The
pccsA::phoA plasmids express translational fusions of ccsA to phoA with
fusions at positions 14, 65, 138, 168, 200, 247, 288, 321, 352 of CcsA
polypeptide. The ccsA::LacZ
F-CcsASac, 5'-CGCGACTCTATGAATTTTGTTAATTTAG-3';
R-CcsA14, 5'-ACGCGTCGACCGTAAAGAATTTTCAATTT-3';
R-CcsA65, 5'-ACGCGTCGACTTGTTTGCTTGAAACCCGTC-3';
R-CcsA138, 5'-ACGCGTCGACTTGTTTGCTTGAAACCCGTC-3';
R-CcsA168, 5'-ACGCGTCGACTCAACCAATAAAGTAAAACTTG-3';
R-CcsA200, 5'-ACGCGTCGACAATGGTGATGCTTGTTGC-3';
R-CcsA247, 5'-ACGCGTCGACTCATACTTTTGGGTGTACC-3';
R-CcsA288, 5'-ACGCGTCGACCTCCAATATGATCCCCATGC-3';
R-CcsA321, 5'-ACGCGTCGACGGTTTTTCACCTTCCCAACC-3;
R-CcsA352, 5'-ACGCGTCGACTTAAAAAAACCATAACTATG-3'.
Measurement of PhoA and LacZ Activities--
Alkaline
phosphatase assays were performed on whole E. coli cells as
described previously in (63), except that
isopropyl-1-thio- Site-directed Mutagenesis of ccsA--
Site-directed mutants of
CcsA: ATG1 Transformation of the Chloroplast Genome--
C.
reinhardtii vegetative cells grown in TAP liquid culture
(1-2 × 106 cells/ml) were transformed by particle
bombardment (using a home-made particle gun at Institut de Biologie
PhysicoChimique, Paris or a Bio-Rad PDS-1000/He particle delivery
system at UCLA) as described in (51). Particles were coated with DNA as
in (67). We also found in the course of this study that the
transformation of gametes significantly facilitates the recovery of
homoplasmic strains carrying a site-directed mutation in
ccsA (see section below). Gamete cells for transformation
were prepared as follows. Vegetative cells grown in liquid TAP medium
(0.5 × 106 cells/ml) were collected by
centrifugation, washed once with sterile water and resuspended in TAP
without nitrogen source in order to induce gametogenesis (60). Cells
were maintained in the dark with agitation for 16-20 h, then
centrifuged, washed once in water and resuspended in 50 mM
sorbitol (107 cells/ml). 5 × 106 to
107 cells were plated on agar and immediately bombarded.
Bombarded cells were incubated overnight under dim light. For the
complementation experiments, ccsA-B6 or ccsA-ct59
cells were bombarded directly on solid minimal agar. Bombarded cells
were then incubated at a higher light intensity (50 µmol/m2/s). For the generation of SpecR
ccsA site-directed mutants, wild type WT59+or
2137 cells were bombarded directly onto solid fresh TAP medium containing 150 µg/ml spectinomycin. Plates were incubated under dim
light (25 µmol/m2/s) until colonies appeared. The
transformation of gametes was found to be 10-50 times less efficient
with respect to the frequency of SpecR colonies obtained by
transformation of vegetative cells.
Purification of Homoplasmic ccsA Site-directed Mutant
Strains--
Rescued colonies and SpecR transformants were
screened for the presence of the mutant copy of ccsA by
amplification using ccsA specific primers flanking the
mutation of interest. Initial SpecR transformants were
found to be predominantly heteroplasmic when vegetative cells were used
as recipient cells for transformation but homoplasmic for chloroplast
genomes carrying the mutant copy of ccsA for the most part
when gamete cells were transformed instead. Homoplasmy for
ccsA mutation could be fixed by inducing gametogenesis of
heteroplasmic transformants, followed by one round of subcloning to
single colonies on TAP with 700 µg/ml spectinomycin. About 90% of
the purified colonies were found to be homoplasmic and remained stable
through vegetative growth in the absence of spectinomycin in the medium.
Fluorescence Measurements--
The fluorescence imaging system,
Fluorcam 700 MF (Photon Systems Instruments Ltd., Brno, Czech
Republic), was used to record chlorophyll fluorescence induction and
decay kinetics of Chlamydomonas cells in vivo.
Cells were grown on TAP medium under dim light (25 µmol/m2/s) and dark-adapted for at least 5 min before
measurement of the fluorescence emission. Dark-adapted cells were
illuminated for 3 to 5 s under actinic light of 60 µmol/m2/s. Emitted fluorescence was captured by the
camera whose sensitivity and shutter were set up at 80% and 1/500
respectively. The collected data were converted into a graph using
Excel software (Microsoft).
Protein Preparation and Analysis--
For detection of
cytochromes, freeze-thaw fractionation and analysis of
electrophoretically separated supernatant and pellet fractions by
immunodetection or a heme staining procedure have been described
previously (47, 52). Enriched thylakoid membrane fractions were
prepared from sonicated cell lysates according to Howe et
al. (47). The 33-kDa protein previously recognized as CcsA (51)
was later identified as cyt f and all subsequent attempts to
generate specific antisera thus far have failed to detect the
endogenous CcsA protein (B. W. Dreyfuss and S. S. Nakamoto, unpublished). Polyclonal antisera raised against C. reinhardtii cyt c6, cyt f fusion
protein, Trx-Ccs1 fusion protein (59), CF1 and plastocyanin
were used for immunodetection. Bound antibodies were detected by
alkaline phosphatase-conjugated secondary antibodies.
Enriched thylakoid samples were subjected to Blue Native polyacrylamide
gel electrophoresis (68, 69). All steps were carried out at 4 °C.
Immediately after preparation, enriched thylakoid membrane samples
corresponding to 100 µg of Chl were pelleted by microcentrifugation
for 15 min at 4 °C and resuspended in 90 µl of 750 mM
Conservation of CcsA in Plastid and Cyanobacterial Genomes--
An
alignment of 27 CcsA homologs from 22 plastid genomes from plants and
algae and 5 cyanobacterial genomes (see Fig. in supplemental data)
shows that CcsA is a well conserved protein (32-43% identity, 43-54% similarity in pairwise alignment with Chlamydomonas
CcsA) particularly in its C-terminal part. Putative heme-binding
residues in CcsA include the WWD signature motif and three invariant
histidine residues. Histidinyl residues are of particular interest in
the context of heme binding as they are known to be virtually universal proximal and common distal heme ligands in other heme containing proteins such as cytochromes and globins. A key step in understanding the structure and function of CcsA is the determination of the orientation (p-side versus n-side) of
its hydrophobic and hydrophilic domains, in particular the WWD motif
and conserved histidines, with respect to the membrane.
Topological Arrangement of CcsA--
We decided to deduce the
topology of CcsA in a bacterial system using alkaline phosphatase
(phoA) and the
Plasmids expressing CcsA-LacZ Functional Analysis of CcsA in Plastid--
A
preliminary assessment of the effect of site-directed mutations in CcsA
can be obtained rapidly by testing all mutated alleles for their
ability to rescue the photosynthetic deficiency of a ccsA
mutant. Restoration of photosynthesis relies only on the restoration of
holocyt f accumulation. Mutations that have no or only a
marginal effect on function should rescue the photosynthetic deficiency
at frequencies comparable with wild type whereas those that do affect
function should not rescue. In order to obtain a precise and
quantitative evaluation of the function of CcsA, rescued colonies are
then assayed for the unselected phenotype, i.e. the
accumulation of holocyt c6 in copper-deficient
conditions. Mutations that do not rescue are analyzed by replacing,
through homologous recombination in the plastid, the wild type
ccsA gene with the mutated version of ccsA. This
was accomplished either via constructs which have ccsA
physically linked to the aadA gene or by co-transforming the
mutated ccsA together with the SpecR16 S rRNA
gene. Both aadA and SpecR 16 S rRNA genes
confer resistance to spectinomycin.
Determination of the Initiation Codon--
Analysis of the longest
ORF in Chlamydomonas ccsA indicates the presence of two
in-frame ATGs (ATG1 and ATG2), 19 codons apart (51). Because neither ATG is associated with a typical consensus Shine-Dalgarno sequence, it was not clear which codon functions as the
initiation site for the translation of the CcsA polypeptide. To address
this question, both ATG1 and ATG2 codons were
mutated to GAG (aspartic acid) and GCG (alanine) codons and the
resulting sequences tested for their ability to rescue the
photosynthetic deficiency of ccsA-B6 strain. Neither of
these latter codons are known to function as initiation codons in the
plastid context. The ccsA-B6 strain was chosen as a
recipient because it carries a single nucleotide deletion
causing a premature termination of translation at codon 23 of the
ccsA gene (49). We reasoned that the proximity of this
mutation to the introduced mutations at positions 1 and 20 should
preclude a high frequency of recombination events that could result in
reconstruction of a wild type ccsA gene. The frequency of
photosynthetically proficient transformants recovered after
transformation of ccsA-B6 strain is comparable when
transforming with a wild type ccsA gene (116 ± 3 transformants, n = 3 transformations) or with the
ccsA allele carrying the ATG2
We therefore generated SpecR ATG1 Mutagenesis of Candidate Heme-binding Residues--
The tryptophan
residues Trp284, Trp288, and Trp290
lying within the WWD motif of CcsA and the neighboring residues
Trp279, Trp296, and Trp301 (Figs. 1
and 3A) were tested for
function by directed mutagenesis to alanine residues. The
non-photosynthetic strain ct59 carrying a frameshift mutation at
position 269 of the ccsA gene has a low frequency of
reversion to photosynthetic proficiency (<10
The abundance of holoforms of cyt c6 and cyt
f was not affected in homoplasmic W296A and W301A
transformants. However, holocyt f accumulation was reduced
to 50% and holocyt c6 accumulation was reduced
to 20% of the corresponding wild type level in the W290A mutant (Fig.
3B). Although the W290A mutant is able to grow photosynthetically, its ability to assemble c-type
holocytochromes is diminished. We conclude that Trp290 is
required for full function of CcsA whereas Trp296 and
Trp301 are not. Homoplasmic W279A-aadA and
W284A-aadA transformants failed to grow on minimal medium
under all light intensities (40-170-500 µmol/m2/s)
indicating that holocyt f assembly is severely impaired in those mutants. The fluorescence induction and decay kinetics are identical to those of a ccsA mutant and characteristic of
b6f deficient mutants due to
compromised assembly of the b6f
complex in the absence of membrane-anchored holocyt f (data
not shown). Immunoblot and heme stain analysis showed that the W279A
mutation blocks completely the assembly of both holocyts f
and holocyt c6 (Fig.
4). In the W284A-aadA
transformants, a small amount of apocyt f could still be
detected immunochemically (Fig. 4). In the course of our study, we
observed that authentic apocyt f can accumulate to various
levels in strains carrying a ccsA-null allele and also in
other ccs mutants. The amount of apocyt f which
accumulates does not appear to correlate to the molecular lesion but
may be dependent upon genetic background and cell density.
The W288A-aadA mutant was able to grow photosynthetically at
all light intensities, albeit to a lower extent compared with the
W290A-aadA mutant and the wild type-aadA control
strain. (Fig. 5A). Note that
based on the immunoblot and heme stain analysis, it is evident that the
level of holocytochrome c maturation is much less in the
W290A-aadA strains (Fig. 4) compared with the W290A
transformants obtained by rescue of the ccsA-B6 strain (Fig. 3). The genetic background of the two original strains
(ccsA-B6 and WT59+) used to generate the
transformants might account for the difference we observed.
The fact that Trp288 and Trp290 lie close to
one another within the conserved WWD motif and that neither alanine
substitution completely abolished CcsA function led us to assess the
phenotype of the double mutation W288AW290A. The
W288AW290A-aadA strain was unable to sustain photosynthetic
growth under 500 µmol/m2/s illumination although it
displayed some residual growth at 50 and 170 µmol/m2/s
(Fig. 5A and data not shown). The level of photosynthetic
growth on minimal medium of the W288A-aadA and
W288AW290A-aadA mutants correlated with the level of holocyt
f accumulated as shown by immunoblot and heme stain analysis
(Fig. 4). Holocyt c6 conversion seems to be
affected to the same extent as holocyt f by these mutations
(Fig. 4). The W288AW290A cells displayed typical ccsA-null mutant fluorescence transient (Figs. 4, 5B) but the low
residual level of holocyt f (2%) appears to be enough to
sustain photosynthetic growth up to a light intensity of 170 µmol/m2/s.
The aspartate residue Asp291 was also tested for functional
importance by mutating it to glutamate, asparagine or alanine residue. None of the mutations rescued the photosynthetic growth of
ccsA-ct59 arguing for the absolute requirement of the
aspartic residue in the assembly of holocyt f. Indeed, the
D291E-aadA, D291N-aadA and D291A-aadA
strains are similar to a ccsA-null mutant. They were unable
to grow photosynthetically, displayed
b6f mutant fluorescence induction and decay kinetics and did not accumulate holocyt
f (data not shown and Fig. 4). Surprisingly, when analyzed
for the unselected phenotype (i.e. holocyt
c6 assembly), the D291A, D291E, and D291N
mutations still enabled the assembly of appreciable amounts of holocyt
c6 (Fig. 4). It seems that the D291E and D291N mutations have less effect on holocyt c6
maturation than does the D291A substitution.
To assess the importance of the histidinyl residues, the
mutants H212D, H309E, H347E, H347A were constructed. All mutations failed to restore photosynthesis to a ccsA mutant and all
spectinomycin-resistant transformants carrying either mutation were
fully deficient for photosynthesis. As shown by immunoblot and heme
staining analysis, no accumulation of plastid c-type
cytochromes was detectable demonstrating that all three histidinyl
residues are critical for the activity of CcsA (Fig.
6). We conclude that all three conserved
histidine residues as well as the tryptophan-rich region of CcsA are
functionally important for the assembly of plastid c-type
holocytochromes.
A CCS Complex within the Thylakoid Membranes--
Based on the
clustering of ccsA-like and CCS1-like genes in
bacteria, Ccs1 is likely to be a candidate interacting partner of CcsA.
To look at the presence of a CCS complex in the thylakoid membrane, we
chose the technique of BN-PAGE (71, 72). Thylakoid membrane fractions
from the wild type strain, ccs1 and ccsA mutants, and a transformant restored to photosynthetic competence by
complementation with the wild type ccsA gene were analyzed
by this technique. Several discrete complexes including green bands
representing Chl-containing complexes can be observed (Fig.
7). The intensely green band at ~100
kDa was confirmed to be the trimeric form of the major light-harvesting
complex of the photosystem II by subsequent fully denaturing
electrophoresis in the second dimension (data not shown). The dimeric
form of the b6f complex (250 kDa) could be detected by immunoblotting with cyt f specific
antibodies in the wild type and ccsA-complemented
ccsA mutant (data not shown). The migration of both these
complexes is consistent with their known masses and previously observed
migrations of dodecyl maltoside solubilized spinach thylakoid separated
on BN-PAGE (71), indicating proper solubilization and electrophoretic
separation of the Chlamydomonas thylakoids. The presence of
a CCS complex was analyzed by immunoblotting with a Ccs1-specific
antibody. An immunoreactive species was observed in thylakoid membranes
from the wild type strain but was absent in membranes from either the
ccs1 or ccsA strains. This immunoreactive species
had a molecular weight of ~200 kDa, suggesting that Ccs1 occurs in
the thylakoid membrane as a component of a high molecular weight
complex. The observation that Ccs1 is absent in a ccsA mutant strain but present in the same strain complemented with the
ccsA gene suggests that CcsA is required for Ccs1
accumulation and that CcsA is also a component of the 200-kDa
complex.
In an attempt to extend our current knowledge on the mechanisms of
cytochromes c assembly in system II, we explored here the function of plastid CcsA as a prototypical protein. In this paper, we
provide evidence that: i) CcsA is a polytopic protein with the WWD
motif and two conserved histidine residues lying in the lumen and one
histidine residue exposed on the stromal side, (ii) the WWD motif and
histidinyl residues are important for the function of CcsA in
assembling cyt f and cyt c6 in
plastid, (iii) a probable CCS complex containing CcsA and Ccs1 is
present in thylakoid membranes.
The WWD Motif Is Functionally Important--
A recurring theme for
this family of proteins is that the WWD motif acts like a hydrophobic
platform involved in the binding and presentation of heme. This view is
further reinforced by the presence of at least two conserved histidinyl
residues, believed to coordinate the heme and lying in loops which
bracket the WWD motif. Functional studies addressing the question of
the functional importance of the key residues of these WWD proteins in
their topological context largely support their role in a transmembrane heme delivery route. It is noteworthy that all experimentally confirmed
topology studies have placed the WWD motif and two conserved histidines
on the p-side of the membrane where the heme attachment reaction was demonstrated to occur (18, 31, 42). By using the well
established PhoA and LacZ topological markers in bacteria, we were able
to construct a topological model of plastid CcsA. As far as the
histidines and tryptophan signature motif locations are concerned, our
model is in concordance with other candidate topologies such as that
deduced for Mycobacteria leprae CcsA (31). The mycobacterial
model supports the existence of one additional N-terminal transmembrane
domain, compared with its plastid counterpart. This model results in an
extra-membrane loop lying on the n-side of the membrane
which is absent in Chlamydomonas CcsA. However, we believe
that this topological distinction in the mycobacterial CcsA is probably
not functionally meaningful as the region corresponding to the
additional transmembrane segment and loop is not conserved in the
plastid and cyanobacterial homologs. This topological distinction could
reflect the different modes of membrane protein import in bacteria
versus cyanobacteria/plastids and in that view, one possible function for the additional transmembrane segment in mycobacterial CcsA
could be to serve as an uncleaved signal-sequence anchor.
Previous attempts to identify essential residues in polytopic WWD
proteins through site-directed mutagenesis have not led to a clear view
of how important is the tryptophan-rich motif for the assembly of
c-type holocytochromes (31, 42, 44-46). This could be due
to the fact that the effect of the mutations appears different when
examined only at the level of growth (31) or when analyzed for the
accumulation of a subset of endogenous c-type cytochromes
and/or a heterologous cytochrome expressed as reporter of the
maturation process (45, 46). In order to give a consistent view of the
functional importance of the tryptophan rich domain, we have correlated
systematically in our study each site-directed mutation in the
ccsA gene with the level of both holocyts
c6 and f formed in the plastid and
also with its ability to sustain photosynthetic growth. Our
experimental data show that any substitution of the tryptophan residues
results in a similar effect on the formation of both holoforms of cyt
f and cyt c6. Among the six conserved
tryptophan residues we tested (Trp279, Trp284,
Trp288, Trp290, Trp296, and
Trp301), only Trp279 and Trp284 are
strictly essential for c-type cytochrome biogenesis.
Although there is the possibility that tryptophan residues have a
topology-determining role, we favor the idea that they are critical for
CcsA activity and/or structure. Indeed, recent studies have established
that tryptophan residues in membrane proteins are not topological
determinants but could rather fulfill a role as interfacially anchoring
residues when located on the trans-side of the membrane
(73). The finding that neither simple or double alanine substitutions
of the Trp288 and Trp290 residues in the
Trp288-Xaa-Trp290-Asp sequence disrupt
completely the assembly process of both holocyt f and
holocyt c6 substantiates the concept that the
signature motif provides multiple hydrophobic interfaces, possibly for
interaction with heme (3). This is also illustrated by the fact that
only a residue with an aromatic side chain, such as phenylalanine, can
functionally substitute for a tryptophan in a position equivalent to
Trp290 in Pseudomonas fluorescens CcmC whereas
any other substitution at this position results in a complete loss of
c-type cytochrome assembly (42). This is in contrast to
other mutagenesis studies of CcmC and CcmF in E. coli, where
only multiple substitutions of the tryptophan residues within the WWD
motif lead to a complete loss of cytochrome c maturation
(45, 46).
The aspartic residue Asp291 in the signature motif is
absolutely required for the biogenesis of holocyt f as
glutamic, asparagine and alanine substitutions completely abolished its
assembly. Unexpectedly, we found that any of these changes do not
affect to the same extent the formation of holocyt
c6. One likely explanation would be that in
addition to its role as a heme presentation surface, the WWD motif also
provides a site of interaction with apocytochromes as already suggested
by Page et al. (24). At present, the question of why this
particular acidic residue is needed in this position in the tryptophan
motif of CcsA, whereas CcmC function seem to tolerate a glutamic
residue in E. coli and P. fluorescens has no
obvious answer (31, 42).
The Histidine Residues Are Critical for CcsA Function--
That
all histidines residues, when altered, completely disrupt
holocytochromes maturation and that we could not obtain by-pass suppressors (<10
The theme of invariant and essential histidines as heme ligands along
with the idea that tryptophans afford several sites of contact for heme
handling makes in our sense the heme delivery function of CcsA the most
compelling hypothesis. Recently, the implication of the WWD motif and
conserved histidines in heme-binding has been questioned by the
observation that a CcmC mutant with mutated histidines and all
tryptophan and aspartic residues of the motif changed to alanines is
fully deficient for cytochrome c maturation but remains
unaltered in its binding capacity to heme in an hemin-agarose assay
(45). Based on the fact that such a mutant is defective for its
interaction with CcmE, the WWD motif and histidines are believed to be
required for a functional interaction with the heme chaperone CcmE.
However, the closer evolutionary relationship between CcmC and CcsA
(5), suggestive of an equivalent function of these two proteins in
cytochrome maturation, and the absence of a CcmE-like heme chaperone in
system II bacterial genomes makes in our view the role of the WWD motif as a CcmE interaction interface less likely. On the other hand, the
crystal structure of the heme-hemopexin (Hpx) complex revealed that the
heme ligand is bound to two histidines residues in a pocket formed by
aromatic residues including tryptophans (74). This supports the idea
that in the WWD cytochrome c biogenesis proteins, tryptophan
and histidine could also provide a transient binding site for the heme
during the assembly of cytochromes c.
CcsA-Ccs1, A Functional Subcomplex Involved in Heme
Delivery--
How does CcsA operate and function in the pathway?
Evidence for a high molecular weight complex containing Ccs1 within the thylakoid membrane that is no longer detected in the absence of CcsA
suggests that Ccs1 could be a putative partner of CcsA (Fig. 8). An attractive hypothesis is that the
invariant and essential histidines predicted to be exposed to the
stroma in both CcsA, a unique feature of this WWD family
representative, and Ccs1 (59) provide an entry site for heme on the
stromal side. However, our topology and mutagenesis studies do not
elucidate whether these residues are in the membrane or exposed to the
stroma and how heme is captured from the n-side
of the membrane to be delivered to the site of cytochrome c
assembly is not known in any of the three systems (6, 28). Our current
working model postulates that heme is relayed through the histidine
residues from stroma to lumen and is presented on the WWD platform to
the apocytochromes chaperoned by Ccs1 (Fig. 8). Whether CcsA is also
involved in the ligation reaction of heme to the apocytochromes still
remains undetermined. Prevalent models for a heme delivery pathway to apocytochromes in system I favor CcmF as the presumptive heme lyase
versus CcmC which is required earlier in cytochrome
c maturation (28, 46). In Wolinella succinogenes,
an
Other components of the high molecular weight complex detected in the
thylakoid membrane are likely to correspond to the gene products of the
unidentified CCS2 to 4 loci (59). We believe that
this complex acts as a site for c-type cytochromes
maturation and is composed of other factors whose biochemical
activities are required to complete holocytochrome assembly. Such
candidate factors might include components required to maintain
apocytochrome cysteinyl thiols and heme in a reduced state, compatible
with the lyase reaction. Evidence for a membrane-bound thiol-reducing subpathway for apocytochromes c in the "CCS" pathway was
provided recently in bacterial models with the discovery of CcdA/DipZ, a protein first identified in system I and involved in conveying reducing equivalents across the membrane, and CcsX a putative periplasmic thioredoxin (57, 58, 77, 78). The existence of a redox
subpathway in the plastid system is suspected based on the presence of
a CcdA-like gene in the rhodoplast genome of P. purpurea and in the nuclear genome of
both3
Chlamydomonas and Arabidopsis thaliana (6). In
Chlamydomonas, preliminary results showing that some ccs
mutants are restored for photosynthetic growth upon addition of
exogenous reducing thiol compounds in the medium, whereas
ccsA and ccs1 mutants are not, indicate that this
thiol-reducing pathway in cytochrome c biogenesis also
functions in plastid.4
Finally, we established also that the transformation of
Chlamydomonas gametes significantly improves the generation
of homoplasmic chloroplastic transformants. Even though we did not
carry out a comparative study for all the site-directed mutants we
generated, the ease with which homoplasmic transformants are obtained
when gametes are used as recipient cells suggests that this approach could be a useful one. Gametes undergo a reduction of the copy number
of their chloroplast genomes (60) and this probably facilitates the
segregation of the molecules carrying the introduced DNA. Our method
should be preferred over the 5-fluorodesoxyuridine treatment of
vegetative host cells prior to transformation which also reduces the
copy number of the plastid genomes but has a mutagenic effect on the plastome.
reporters placed the WWD motif, the conserved residues His212 and His347 on the lumen side of
the membrane, whereas His309 was assigned a location on the
stromal side. Functional analysis of CcsA through site-directed
mutagenesis enabled the designation of the initiation codon of the
ccsA gene and established the functional importance of the
WWD signature motif and the absolute requirement of all three
histidines for the assembly of plastid c-type cytochromes. In a ccsA mutant, a 200-kDa Ccs1-containing complex is
absent from solubilized thylakoid membranes, suggesting that CcsA
operates together with Ccs1. We propose a model where the WWD motif and histidine residues function in relaying heme from stroma to lumen and
we postulate the existence of a cytochrome c assembly
machinery containing CcsA, Ccs1 and additional components.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
-proteobacteria, Deinococcus, archaea, plant and
protozoal mitochondria. Extensive functional studies in
Rhodobacter capsulatus (17-20), Paracoccus
denitrificans (21-24) and Escherichia coli (25, 26)
microbial models have led to the identification of as many as 12 genes
whose products are recruited to complete holocytochrome c
maturation (for review 2, 27, 28). A detailed view of this pathway has
now emerged on the basis of thorough biochemical investigation of the
different gene products. A complex of multiple membrane and periplasmic
proteins proposed to catalyze heme transport across the membrane (17,
18, 22, 29-32), the transfer of reducing equivalents (20, 23, 33-37),
the reduction and chaperoning of apocytochromes (19, 27, 38-41), the
handling and ligation of heme onto the apocytochromes (26, 28, 42-46)
is now envisioned to function as an apparatus for c-type
cytochrome assembly.
WXWD, where
is an aromatic residue), similar to that
found in the CcmC and CcmF proteins involved in the biogenesis of
c-type cytochromes in system I (17, 25, 28, 31, 53). The
presence of strictly conserved histidinyl residues which are known
ferroheme ligands led to the hypothesis that CcmC, CcmF and CcsA
proteins bind and deliver heme to the apocytochromes (6, 31, 51).
However, despite the conservation of histidinyl and tryptophan
residues, CcsA is not a true homolog of CcmC or CcmF and clearly
defines a separate member of the WWD motif family (5, 31). The
co-occurrence of Ccs1 and CcsA homologues, encoded by genes often
organized in an operon arrangement, in plastid and bacterial genomes
led to the definition of a new pathway for assembling holocytochromes
c (3, 5, 6). In Bacillus subtilis, Synechocystis sp. and Bordetella pertussis,
functional genomics and classical genetics have also implicated the
CcsA and Ccs1 homologues in the assembly of c-type
cytochromes (54-58) but a clear function in the assembly process has
not yet been assigned to either of these two proteins.
-galactosidase to deduce the polytopic arrangement
of Chlamydomonas CcsA and thus to determine the orientation of the WWD motif and conserved histidine residues. The functional significance of these key residues was further demonstrated by site-directed mutagenesis of the ccsA gene in the plastid
context and each mutant was assessed for its ability to assemble the
c-type cytochromes and support photosynthetic growth. We
also provide the first evidence for the existence of a system II
cytochrome assembly machinery by showing that CcsA and Ccs1 are present
in a 200-kDa complex that could be detected within the thylakoid membranes by BN-PAGE. Based on our results, we formulate and discuss the hypothesis that CcsA and Ccs1 constitute a subcomplex functioning in the delivery/ligation of heme to apocytochromes.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
were used as hosts for recombinant DNA techniques
(61). The Lac
Pho
strain CC118 (62) was
used for alkaline phosphatase assays and DH5
for
-galactosidase assays.
fusions
at positions 14, 247, and 321 were generated from the corresponding
pccsA::phoA plasmids by replacing a
2.6-kb SalI-PstI fragment including the entire
phoA gene with a 0.7-kb PCR-amplified lacZ
segment corresponding to the
fragment of
-galactosidase and in
frame with the upstream CcsA reading frame. The lacZ
segment was amplified from plasmid pSKII+
(Stratagene) with M13 reverse primer and a lacZ specific
primer engineered with a PstI site. The PCR product was
digested with SalI and PstI and then cloned into
PstI-SalI digested
pccsA::phoA plasmids.
-D-galactopyranoside was not used to
induce the expression of the fusion proteins.
-galactosidase
activity was measured on SDS-chloroform treated cells essentially as
described in (64). Alkaline phosphatase activity expressed in Miller
units was calculated using the following formula:
(1000·A420 nm)/(time
(min)·A600 nm).
-Galactosidase activity is
expressed in units and calculated by the following formula:
(1000·(A420 nm
(1.75·A550 nm))/(time
(min)·A600 nm).
GAG, ATG2
GCG, H212D, H309E, H347E, H347A, W279A, W284A, W288A, W290A,
W288AW290A, W296A, W301A, D291A, D291E, and D291N were constructed by a
PCR approach or with the Sculptor in vitro mutagenesis kit
(Amersham Biosciences) using as a template plasmid pEBP or M13mp18
vector containing the ccsA gene (51) and the set of primers
listed in Table I. Mutagenic primers were
designed in order to introduce a new restriction site or to alter an
existing one in the sequence of the ccsA gene (see Table I).
All PCR amplified regions of ccsA were sequenced on both
strands. Plasmid pPmP-aadA was obtained by cloning a 5.5-kb
PmeI-PstI chloroplast DNA containing the
ccsA gene into the XbaI (Klenow-filled) and
NsiI-digested pGEM7Z plasmid (Promega). To simplify cloning,
the HindIII site, located at the 3' far end of the
ccsA gene was removed by site-directed mutagenesis. An
EcoRI-SmaI fragment containing the
aadA gene expression cassette from the plasmid pUC-atpX-aad
(65) was cloned into the MluI (Klenow-treated) and
EcoRI digested pPmP vector to yield pPmP-aadA. Mutations H212D, H309E, H347E, H347A, W279A, W284A, W288A, W290A, W288AW290A, D291A, D291E, and D291N in ccsA were transferred
to pPmP-aadA as a 1.9-kb EcoRI-HindIII
or a 1.7-kb EcoRI-PacI fragment. Plasmid
pATG1
GAG was co-transformed with plasmid p228 carrying a mutant allele of the 16 S rRNA gene which confers resistance to
spectinomycin (66).
Primers used for ccsA site-directed mutagenesis
) a
restriction site in the sequence of the ccsA gene
(modification).
-aminocaproic acid, 50 mM BisTris, pH 7.0. The membranes were then solubilized with 10 µl of dodecylmaltoside (1% final) for
5 min, followed by centrifugation at 20,000 g for 30 min to remove
insoluble material. The supernatant was supplemented with 5 µl
Coomassie-blue solution (5% Serva Blue G-250/500 mM
-aminocaproic acid) to produce a detergent/Coomassie ratio of
4:1(g/g) and loaded directly onto the gel. In order to estimate the
size of the CCS complex, high molecular weight markers (Amersham
Biosciences) were prepared in detergent and Coomassie Blue and loaded
onto the gel, along with the membrane samples. Blue Native gels
consisted of a separating gel (gradient of 6-12% acrylamide) and a
stacking gel (4%). Electrophoresis was initiated with cathode buffer
containing 0.02% Coomassie-blue at 100 V for ~16 h (1.5 mm x 14 cm),
after which the buffer was exchanged with cathode buffer lacking
Coomassie-blue and electrophoresis continued for 6 h. Blue-native
gels were electrophoretically transferred to 0.2 µm PVDF membranes
using the cathode buffer as a transfer buffer at 100 V for 90 min at
4 °C. Membranes were immunodecorated with Ccs1 antisera overnight
and bound antibodies detected by alkaline phosphatase-conjugated
antibodies using CDP-Star chemiluminescence reagent (Roche Molecular
Biochemicals). Immunoblots were exposed to Kodak Bio Max film for 10 min prior to development.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide of
-galactosidase (lacZ
) as topological reporters because of their
reliability in many experimental systems. The PhoA fusions were
engineered in frame to all the predicted soluble loops of
the CcsA protein (Fig. 1). A neural
network system was utilized to predict the topological arrangement of
CcsA (70). Regions which are lumenal in the chloroplast correspond to
the p-side of the membrane and are expected to be
periplasmic. Reciprocally, stromal domains are on the n-side
of the membrane and are predicted to be exposed in the cytoplasm.
Because alkaline phosphatase activity requires oxidative-folding of the
protein in the periplasmic space, CcsA-PhoA fusions at periplasmic
positions should exhibit high PhoA activity whereas those which are on
the cytoplasmic side should exhibit lower activity. Conversely,
-galactosidase being active in the cytoplasm, CcsA-LacZ
fusion
constructs should show high activity only if the fusion junction is
cytoplasmic. As shown in Table II, CcsA
fusion products at positions 65, 138, 168, 200, 288 and 352, predicted
to be periplasmic, display high alkaline phosphatase activity. All
activities are about 8-15-fold above the level of vector control
pRGK200 which expresses a cytoplasmically inactive alkaline phosphatase
that is devoid of the signal sequence for translocation in the
periplasm (18). These results confirm the periplasmic localization of
the junctions tested and we can deduce that they have a lumenal
localization in the chloroplast.
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Fig. 1.
Topology of Chlamydomonas
CcsA. The topological arrangement of
Chlamydomonas CcsA was drawn according to PhoA/LacZ fusion
analysis in bacteria. The p-side (positive side) corresponds
to the lumen of the thylakoid and the n-side (negative side)
corresponds to the stroma of the chloroplast. Large rectangles feature
the transmembrane helices of CcsA as predicted from in
silico analysis using the PHDhtm algorithm (70). The
arrows indicate, within the CcsA polypeptide, the positions
of in frame alkaline phosphatase and -galactosidase
peptide
fusions. Fusions at position 14, 65, 138, 168, 200 247, 288, 321 and
352 are indicated on the drawing by arrows. Residues in
gray are strictly conserved in all 22 plastid and 5 cyanobacterial CcsA homologs (see Fig. in Supplemental Data). Candidate
heme binding residues in a square were tested for function by
site-directed mutagenesis of the ccsA gene in
Chlamydomonas.
CcsA-PhoA and -LacZ activities
-galactosidase activities of CcsA fusion
proteins expressed in bacteria were measured as described under
"Experimental Procedures." At least two representatives of each
CcsA fusion that was generated by PCR were tested for activity. The
value is indicated as the mean and S.D. of three independent
measurements for the two representatives of each fusion.
n-Side and p-Side correspond to the negative and
positive sides of the membrane, respectively. ND, not determined.
at positions 14, 247 and 321 show a
significantly higher level of
galactosidase activity compared with
the empty vector control (see Table II). In accordance with these
results, the CcsA-PhoA fusion at position 65, for which we have
assigned a periplasmic location, exhibits a very low
galactosidase activity, about 7-9-fold less than all other CcsA-LacZ
fusions. The
cytoplasmic location of positions 14, 247, and 321 can be confirmed
unambiguously and we are confident in ascribing a stromal location for
these positions in the plastid context. Multiple copies of the FLAG or
His6 epitopes can be introduced via an engineered restriction site between residues 243 and 244 without affecting CcsA in
Chlamydomonas, which is consistent with the extra-membrane stromal
orientation of this region of the polypeptide (data not shown).
Overall, the data establish the polytopic arrangement of plastid CcsA
in the membrane with five transmembrane helices and N and C termini
facing the stroma and lumen, respectively (Fig. 1). Based on this
topology, the WWD motif and the conserved histidinyl residues
His212 and His347 are located on the
lumenal side. The conserved residue His309 appears to be
located on the stromal side, perhaps within a transmembrane segment. We
then addressed the question of the importance of the conserved
residues for Chlamydomonas CcsA function and structure by
site-directed mutagenesis of ccsA.
GCG mutation
(153 ± 23 transformants, n = 3 transformations) whereas no transformants were recovered with the ATG1
GAG ccsA allele (3 independent transformations) or the no
DNA control. Homoplasmic ATG2
GCG transformants were
indistinguishable from wild type transformants in that they displayed
the same fluorescence induction and decay kinetics and accumulated wild
type levels of holoforms of both cyt f and cyt
c6 (data not shown and Fig. 2, A and B). The
ATG2
GCG mutation fully rescues the function of a
ccsA mutant whereas the ATG1
GAG cannot,
suggesting that the first ATG is critical for the function of the CcsA
polypeptide.
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Fig. 2.
Phenotypic analysis of
ATG1 GAG and
ATG2
GCG ccsA mutants.
A, fluorescence kinetics of ATG1
GAG
ccsA mutant. The fluorescence induction and decay kinetics
observed in a dark to light transition for the
ATG1
GAG ccsA mutant is shown in
comparison with those from the wild type (WT) and B6 (ccsA)
strains. Only one representative transformant is presented here. The
continuously rising curve for the ccsA mutant is typical of
a specific block in electron transfer at the level of cytochrome
b6f complex due to its impaired
assembly in the absence of membrane-bound holocytochrome f.
The strains were grown on TAP agar for 3 days before recording the
fluorescence in arbitrary units (AU) over a 3 s
illumination period. B, accumulation of plastid c-type
cytochromes in ATG1
GAG and
ATG2
GCG ccsA mutants. Protein fractions
were prepared from copper-deficient cultures of wild type (2137),
ccsA (B6) and 2 representatives (a and
b) of each ATG1
GAG and
ATG2
GCG ccsA strains generated by
chloroplast transformation. The supernatant fractions were analyzed for
holocyt c6 accumulation (anti-cyt
c6 and heme stain) whereas the pellet fraction
was used to assess holocyt f abundance (anti-cyt
f and heme stain). All the samples were found to be
copper-deficient based on the absence or very low abundance of
immunoreactive holoplastocyanin (data not shown). Hence, cyt
c6 should be expressed in all samples. Samples
corresponding to 5 µg of Chl were separated in SDS-containing
acrylamide (12%) gels or native acrylamide gels (15%) to detect cyt
f and cyt c6 respectively. For an
estimation of the cytochrome abundance, dilutions of the wild type
sample were loaded on the gel. Equal loading of the samples was tested
by Coomassie Blue staining (not shown). Gels were then transferred to
PVDF membranes following electrophoresis before heme staining by
chemiluminescence and immunodecoration with antisera against
Chlamydomonas cyt f and cyt
c6 (79). Heme staining and autoradiographic
exposures were performed simultaneously for all samples.
GAG
mutants under non-selective conditions (i.e.
heterotrophic growth). Fluorescence transients of homoplasmic
SpecR ATG1
GAG transformants appear wild
type, albeit with slightly slower decay kinetics (Fig. 2A)
and the transformants were able to grow photosynthetically under light
intensities from 50 to 170 µmol/m2/s, but very poorly at
500 µmol/m2/s, suggesting that photosynthesis is affected
but not completely compromised (data not shown). Indeed, we showed
that, although significantly reduced, some holocyt f and
c6 accumulation occurs in the plastid of
homoplasmic ATG1
GAG mutants (Fig. 2B).
The reduced accumulation of holoforms of cyt f (<20%) and
cyt c6 (<5%) in these transformants suggests
that some translation of the ccsA gene is rendered possible,
presumably at ATG2 or alternatively at the GAG codon as
there is no other in-frame ATG or non orthodox initiator codon upstream
of ATG1. The first possibility is more likely since the GAG
codon has not been shown previously to function as an initiator codon
in any system investigated so far. Because ATG2 can
function as an initiator when ATG1 is mutated, we infer that the first 20 amino-acids of CcsA are not absolutely essential for
targeting of the polypeptide to the thylakoid membrane, but that the
first ATG serves normally as the preferred initiation codon for the
translation in vivo of the CcsA polypeptide.
9) and
therefore was selected as a recipient strain for complementation experiments. We reasoned that the recovery of photosynthetic
transformants that arise from a recombination event between residues
269 and 279-301 should be a rare event. Thus, all photosynthetic
transformants recovered should harbor the introduced mutation. Plasmids
pW279A, pW284A, pW288A, pW290A and pW296A were introduced into
ccsA-ct59 by chloroplast transformation. Only pW290A, pW296A
and pW301A mutations were able to complement the photosynthetic
deficiency of strain ccsA-ct59 suggesting that
Trp279, Trp284, and Trp288 are
critical for the function of CcsA.
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Fig. 3.
Accumulation of plastid c-type cytochromes in
W290A, W296A, and W301 mutants. Protein fractions were prepared
from copper-deficient cultures of wild type (2137), ccsA
(ct59) strains and 2 representatives (a and b) of
W290A, W296A and W301 transformants generated by biolistic
transformation. The supernatant fractions were analyzed for holocyt
c6 accumulation (anti-cyt
c6 and heme stain) whereas the pellet fraction
was used to assess holocyt f abundance (anti-cyt
f and heme stain). All the samples were found to be
copper-deficient based on the absence or very low abundance of
immunoreactive holoplastocyanin (data not shown). Hence, cyt
c6 should be expressed in all samples. Samples
corresponding to 5 µg Chl were separated in SDS-containing acrylamide
(12%) gels or native acrylamide gels (15%) to detect cyt f
and cyt c6 respectively. Equal loading of the
sample was tested by Coomassie blue staining (not shown). Gels were
then transferred to PVDF membranes following electrophoresis before
heme staining by chemiluminescence and immunodecoration with antisera
against Chlamydomonas cyt f and cyt
c6. The dilution series of wild type for heme
stain and immunodecoration were developed in parallel with the
experimental samples from the mutant strains.
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Fig. 4.
Accumulation of plastid c-type cytochromes in
the tryptophan, aspartic acid, and histidine ccsA mutants. Protein
fractions were prepared from copper-deficient cultures of wild
type-aadA (WT) and 2 representatives (a and
b) of W279A-aadA, W290A-aadA,
W288AW290A-aadA, D291A-aadA,
D291E-aadA and D291N-aadA transformants generated
by chloroplast transformation. The supernatant fractions were analyzed
for holocyt c6 accumulation (anti-cyt
c6 and heme stain) whereas the pellet fraction
was used to assess holocyt f abundance (anti-cyt
f and heme stain) and CF1 of the ATPase (anti
CF1) as a loading control. All the samples were found to be
copper-deficient based on the absence or very low abundance of
immunoreactive holoplastocyanin (data not shown). Hence, cyt
c6 should be expressed in all samples. Samples
corresponding to 15 µg Chl were separated in SDS-containing
acrylamide (12%) gels or native acrylamide gels (15%) to detect cyt
f and cyt c6 respectively. Samples
corresponding to 3 µg Chl were separated in denaturing acrylamide gel
to detect CF1. Following electrophoresis, gels were then
transferred to PVDF membranes before heme staining by chemiluminescence
and immunodecoration with antisera against Chlamydomonas cyt
f, cyt c6 or CF1. For an
estimation of the cytochrome abundance, dilutions of the wild type
sample were loaded on the gel. Heme staining exposure representing
equivalent intensity for wild type controls in all experiments are
shown. The ability and inability of the transformants to grow
photosynthetically is indicated by " " and "X,"
respectively.
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Fig. 5.
Tryptophan 288 and 290 of the WWD
motif are required for photosynthesis. A,
photosynthetic growth of W288A, W290A and W288AW290A mutants. Equal
numbers of cells were grown photosynthetically (Min) under high light
(500 µmol/m2/s) or heterotrophically (Acetate) under dim
light (25 µmol/m2/s) for 10 days at 25 °C.
B, fluorescence induction and decay kinetics of W288A,
W290A and W288AW290A mutants. Fluorescence transients of
W288A-aadA, W290A-aadA,
W288AW290A-aadA, ccsA and wild
type-aadA strains were measured on colonies grown for 4 days
on solid TAP medium using Fluorcam 700 MF from Photon Systems
Instruments. The strains were plated on TAP agar 3 days before
recording the fluorescence in arbitrary units (AU) over a 3 s
illumination period.
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Fig. 6.
Invariant histidine residues are essential
for the assembly of plastid c-type cytochromes.
Protein fractions were prepared from copper-deficient cultures of wild
type-aadA (WT), 2 representatives (a and
b) of H213A-aadA, H309A-aadA,
H347A-aadA, and one representative (a) of
H349E-aadA transformants generated by chloroplast
transformation. The supernatant fractions were analyzed for holocyt
c6 accumulation (anti-cyt
c6 and heme stain) whereas the pellet fraction
was used to assess holocyt f abundance (anti-cyt
f and heme stain) and CF1 of the ATPase
(anti-CF1) as a loading control. All the samples were found
to be copper-deficient based on the absence or very low abundance of
immunoreactive holoplastocyanin (data not shown). Hence, cyt
c6 should be expressed in all samples. Samples
corresponding to 15 µg Chl were separated in SDS-containing
acrylamide (12%) gels or native acrylamide gels (15%) to detect cyt
f and cyt c6 respectively. For an
estimation of the cytochrome abundance, dilutions of the wild type
sample were loaded on the gel. Samples corresponding to 3 µg Chl were
separated in denaturing acrylamide gel to detect CF1.
Following electrophoresis, gels were then transferred to PVDF membranes
before heme staining by chemiluminescence and immunodecoration with
antisera against Chlamydomonas cyt f, cyt
c6 or CF1. Heme staining exposure
representing equivalent intensity for wild type controls in all
experiments are shown.
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Fig. 7.
A "CCS" complex in the thylakoid
membranes. Thylakoid membrane enriched fractions from wild type
CC125 (WT), ccsA-B6 (ccsA),
ccs1-7::ARG7 (ccs1) strains
and a phototrophic transformant generated by complementation of
ccsA-B6 strain with a plasmid carrying the ccsA
gene (pccsA) were solubilized with dodecylmaltoside. Samples
corresponding to 100 µg of Chl were subjected to BN-PAGE (left
side) and transferred to 0.2 µm PVDF membranes before
immunodecoration with an anti-Ccs1p antibody (right side).
The positions of the dimeric form of the cytochrome
b6f complex (cyt
b6f) and the trimeric form of the
light-harvesting complex of photosystem II (LHCII) are indicated by an
arrow on the BN gel. The position of the 200-kDa complex
referred as "CCS" complex and containing Ccs1 is indicated by an
arrow on the anti-Ccs1 immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9, unpublished) underscore their pivotal
role in the assembly process. Strictly conserved histidinyl residues
located on the p-side of the membrane in CcmC and CcmF were
also demonstrated to be critical for the activity of the proteins in
cytochrome c maturation (31, 45, 46) and, similar to our
study, no compensatory mutation of the E. coli CcmC
histidinyl mutants could be isolated (31).
-protebacterium with a possible system II for cytochrome
c maturation, the NrfI protein is proposed to result from a
fusion of a protein predicted to be topologically similar to Ccs1 and a
CcsA-like moiety (75). The fact that NrfI is only dedicated to the
attachment of heme to the unusual CXXCK motif in pentaheme
c-type cytochrome NrfA argues in favor of a heme
handling/ligation activity of CcsA and reinforces the view of a
CcsA-Ccs1 functional subcomplex (76). It remains to be determined if
NrfI is only involved in heme delivery to NrfA or functions also in the
transport of heme from the n-side of the membrane for
ligation to the CXXCK motif.
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Fig. 8.
Model for a c-type cytochromes assembly
machinery in plastid. Our current working model of a
c-type cytochromes assembly apparatus in the thylakoid
membrane includes plastid-encoded CcsA, nucleus-encoded Ccs1 and other
unidentified components (?). Essential conserved histidinyl residues
(H) in CcsA and Ccs1 (see accompanying paper) are positioned in the
lumen and within the membrane close to the stromal side. Heme is
represented as a gray rectangle and we hypothesize it is
handled from the stroma to the lumen side through histidinyl residues
and the WWD motif, for delivery and ligation to reduced apocytochromes
(SH indicate the sulfhydryl groups of the CXXCH motif)
"chaperoned" by Ccs1.
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ACKNOWLEDGEMENTS |
---|
We thank all the present members of the Merchant laboratory for support and intellectual input during the course of this study; J. Leichman for technical assistance; Dr. N.-J. Yu for initiating some aspects of the project; and Dr. J. Moseley, Dr. R. Khanna, and Dr. M. D. Page for critical reading of the manuscript. We particularly thank Dr. M. D. Page for sharing his valuable expertise in the field of cytochrome c biogenesis. P. H. also gratefully acknowledges all the members of the Wollman group (Paris) who introduced him to the experimental system and hosted him during the initial steps of the project.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM48350 (to S. M.) and American Heart Association Post-doctoral Fellowship 0120100Y (to P. H.).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.
The on-line version of this article (available at
http://www.jbc.org) contains a figure.
§ Present address: Biocept, 2151 Las Palmas Dr., Ste. C, Carlsbad, CA 92009.
¶ To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, UCLA, Box 951569, Los Angeles, CA 90095-1569. Tel.: 310-825-8300; Fax: 310-206-1035; E-mail: merchant@chem.ucla.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M208651200
2
The signature motif
"WGXWXWD" will be referred to as WWD motif
throughout the article.
3 GenBankTM accession number AAL84598.
4 M. D. Page and P. Hamel, unpublished material.
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ABBREVIATIONS |
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The abbreviations used are: cyt, cytochrome; BN, blue native; ccs, cytochrome c synthesis; Chl, chlorophyll; ORF, open reading frame; PVDF, polyvinylidene difluoride; SpecR, spectinomycin-resistant; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
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