(Received for publication, July 31, 1995; and in revised form, September 14, 1995)
From the
The biosynthesis of cytochrome f is a multistep process
which requires processing of the precursor protein and covalent
ligation of a c-heme upon membrane insertion of the protein.
The crystal structure of a soluble form of cytochrome f has
revealed that one axial ligand of the c-heme is provided by
the -amino group of Tyr
generated upon cleavage of the
signal sequence from the precursor protein (Martinez S. E., Huang D.,
Szczepaniak A., Cramer W. A., and Smith J. L.(1994) Structure 2, 95-105). We therefore investigated, by site-directed
mutagenesis, the possible interplay between protein processing and heme
attachment to cytochrome f in Chlamydomonas
reinhardtii. These modifications were performed by chloroplast
transformation using a petA gene encoding the full-length
precursor protein and also a truncated version lacking the C-terminal
membrane anchor. We first substituted the two cysteinyl residues
responsible for covalent ligation of the c-heme, by a valine
and a leucine, and showed that heme binding is not a prerequisite for
cytochrome f processing. In another series of experiments, we
replaced the consensus cleavage site for the thylakoid processing
peptidase, AQA, by an LQL sequence. The resulting transformants were
nonphototrophic and displayed delayed processing of the precursor form
of cytochrome f, but nonetheless both the precursor and
processed forms showed heme binding and assembled in cytochrome b
f complexes. Thus, pre-apocytochrome f adopts a suitable conformation for the cysteinyl residues to
be substrates of the heme lyase and pre-holocytochrome f folds
in an assembly-competent conformation. In the last series of
experiments, we compared the rates of synthesis and degradation of the
various forms of cytochrome f in the four types of
transformants under study: (i) the C terminus membrane anchor
apparently down-regulates the rate of synthesis of cytochrome f and (ii) degradation of misfolded forms of cytochrome f occurs by a proteolytic system intimately associated with the
thylakoid membranes.
The presence of quinol-oxidizing proteins is a common feature of
energy transducing membranes. In most cases, these transmembrane
proteins are multisubunit protein complexes with similar
characteristics in bacteria and mitochondria (cytochrome bc complexes) and in chloroplasts (cytochrome b
f complexes). Their activity results
from intraprotein electron transfer reactions between an Fe-S center,
two b-hemes and one c-heme, which couple
translocation of protons across the membranes to oxidation of
lipophilic electron carriers, ubiquinols or plastoquinols, and
reduction of small hydrophilic proteins, a c-type cytochrome
or a copper-containing protein, plastocyanin (Cramer et al.,
1991).
The assembly of such oligomeric proteins is a multistep
process involving the biosynthesis and delivery of several prosthetic
groups to the various subunits whose interactions are required for the
function of the mature protein. One of the major subunits of cytochrome bc (bf ) complexes is a c-type cytochrome which is membrane-bound by a single
hydrophobic
-helix located in the C-terminal domain of the protein
(for reviews, see Gray(1992) and Howe and Merchant(1994)). Most of the
protein, including its N terminus and the heme-binding domain, extends
into an aqueous environment facing the membrane on the side opposite
that where membrane insertion is initiated. The final orientation of
the mature protein is achieved by the cleavage of the N-terminal
extension of the precursor form. Therefore, the cleavable sequence acts
as a signal sequence for the proper localization of the mature
cytochrome.
The chloroplast protein, cytochrome f, is
encoded by the petA gene located on the organelle genome. In Chlamydomonas reinhardtii, the petA gene is located
upstream of the petD gene-encoding subunit IV, another subunit
of the cytochrome bf complex
(Büschlen et al., 1991). Even though the
two can be cotranscribed (Sturm et al., 1994), they are
translated from monocistronic transcripts (Kuras and Wollman, 1994).
The petA message is translated on polysomes which are bound to
the thylakoid membranes in pea chloroplasts (Gray et al.,
1984) as well as in C. reinhardtii. (
)
Conversion
of the petA gene product to a photosynthetic protein fully
competent for electron transport requires four major events: membrane
insertion of the precursor protein, processing of the precursor form,
heme attachment to the polypeptide chain, and assembly in cytochrome bf complexes. The temporal sequence of,
and the possible interplay between, these four events remains largely
unknown. However, several recent studies have provided new insights on
the biogenesis of cytochrome f and enable further dissection
of the biosynthesis pathway.
Membrane translocation of the precursor
protein requires a recognition process of the N terminus presequence
since several mutations in its hydrophobic core were shown to prevent
membrane insertion and maturation of the protein (Smith and Kohorn,
1994). Upon membrane translocation, the transmembrane anchor next to
the C terminus would act as a stop transfer sequence (Willey and Gray,
1988), a proposal substantiated by the fact that truncation to
eliminate this hydrophobic segment results in the complete
translocation of a fully processed and redox-active soluble cytochrome
(Kuras et al., 1995). We observed that the overall rate of
synthesis of cytochrome f is an assembly-controlled process
since the absence of either subunit IV or cytochrome b resulted in decreased rates of synthesis of cytochrome f with no decrease in the lifetime of the holoprotein (Kuras and
Wollman, 1994).
N-terminal processing of cytochrome f and covalent attachment of the c-heme to the conserved protein motif CXX`CH would both occur on the lumen side of the thylakoid membranes (Howe and Merchant, 1994). Therefore, both steps require translocation of part of the polypeptide sequence across the membrane but nevertheless can occur in the absence of translation of the C terminus membrane anchor (Kuras et al., 1995).
The
recent determination of the crystal structure at 2.3-Å resolution
of the soluble domain of cytochrome f from turnip (Martinez et al., 1994) unexpectedly revealed that one axial ligand of
the c-heme is provided by the -amino group of
Tyr
, the amino-terminal residue of the mature protein. This
is at variance with the other cytochromes c whose axial
ligands are usually provided by side chains from methionine residues
within the polypeptide core. It was therefore suggested that heme
attachment to cytochrome f requires, and is subsequent to,
processing of the precursor protein (Cramer et al., 1994; Howe
and Merchant, 1994).
In order to unravel the possible interactions between precursor maturation and heme ligation, we used chloroplast gene replacement in C. reinhardtii to perform site-specific modifications aimed at preventing heme attachment or precytochrome f processing. The biosynthetic consequences of such alterations were further examined on a truncated protein which lacked the membrane anchor in the C-terminal region.
We then used plasmids pAF29L-31L, pAF52L-55V,
pAF29L-31L-283ST, and pF52L-55V-283ST to transform a wild-type (WT)
strain. Transformants were selected on TAP medium for the expression of
the aadA cassette in the presence of 100 µg/ml
spectinomycin. Transformants were then screened for the inactivation of
cytochrome bf complexes based on their
fluorescence induction kinetics after dark adaptation. Transformants
displaying a lack of cytochrome b
f activity were further purified by successive rounds of subcloning
both on TAP-spectinomycin medium under dim light and on minimum medium
(medium without acetate) under high light. When subcloning no longer
generated colonies able to grow on minimal medium, transformants were
considered as homoplasmic for the mutated genome. In parallel, the
transformants were checked for their restriction fragment length
polymorphism based on the presence of the new restriction sites brought
along with the introduced mutations. At least three independent
homoplasmic transformants for each modification were kept on TAP medium
for further biochemical analysis. Their comparison with the wild-type
was performed after having checked that that there were no changes in
the biogenesis of cytochrome b
f subunits
upon insertion of the aadA cassette at the EcoRV site
in the intergenic petA-petD region in an otherwise
wild-type context.
To characterize the multistep process of cytochrome f biosynthesis, we constructed mutant strains of C. reinhardtii bearing specific alterations in the petA gene. Mutations
were introduced as described under ``Materials and Methods''
to alter either heme attachment to the apoprotein (mutant F-52L-55V) or
N-terminal processing of the precursor protein (mutant F-29L-31L). The
same mutations were also introduced into a truncated version of the
polypeptide sequence (mutants F-29L-31L-283ST and F-52L-55V-283ST).
This mutation yields, in an otherwise wild-type context, a cytochrome bf-deficient strain which nevertheless
accumulates a soluble, redox-active, and truncated cytochrome f in the thylakoid lumen (Kuras et al., 1995).
Figure 1:
Analysis of whole cell protein extracts
in heme attachment cytochrome mutants. Cytochrome f (cyt.f) and mitochondrial soluble cytochrome c (h2) viewed after TMBZ staining of a urea-SDS
electrophoresis gel loaded with total protein extracts from the WT (lane 1), F283ST (lane 2), F-52L-55V (lane
3), and F-52L-55V-283ST (lane 4) strains (A).
The same samples were transferred onto nitrocellulose and reacted with
antisera against cytochrome f, the Rieske protein, and subunit
IV (su IV); the former was revealed with iodinated protein A,
whereas the latter were revealed by the ECL method (B).
Immunodetection of cytochrome f, in total protein extracts (tot.) and soluble protein fractions (sol.) from the
WT and F-52L-55V-283ST transformants respectively (C).
Both F-52L-55V and F-52L-55V-283ST transformants
failed to accumulate significant amounts of the other cytochrome bf subunits. This is illustrated on Fig. 1B by the immunoblots reacted with subunit IV and
Rieske protein antisera. These subunits were present in trace amounts,
comparable to that of the F283ST strain, and therefore are indicative
of impaired assembly and/or stabilization of the cytochrome b
f complex subunits (Kuras and Wollman,
1994).
Total protein extracts were further separated into soluble and membrane protein fractions by differential centrifugation. Cytochrome f from the wild-type strain is found exclusively in the membrane protein fraction (Fig. 1C). In spite of its trace amounts, the heme-less cytochrome f in its full-length version could also be detected exclusively in the membrane protein fraction (data not shown). In contrast, the truncated version of the heme-less cytochrome f was present in the soluble fraction (Fig. 1C) as was its heme-containing counterpart (Kuras et al., 1995). The corresponding membrane fractions retained trace amounts of truncated (apo)cytochrome f (experiment not shown), which were probably trapped inside the membrane vesicles created by the French Press treatment as we reported previously (Kuras et al. 1995). Thus, the truncated heme-less cytochrome f behaved as a soluble protein.
We then performed in vivo pulse-chase labeling experiments to determine whether the decreased amounts of heme-less cytochrome f in the mutant strains could be attributed to decreased synthesis or to higher turnover of the apoproteins. Fig. 2shows the electrophoretic region of the autoradiogram where the labeled petA gene product migrates (indicated by arrows on Fig. 2, A, B, and C). During the chase period, the D2 band (see legend of Fig. 2) is often converted in a doublet, whose components D2.1 and D2.2 are produced in variable relative proportions (Delepelaire, 1984). They result from a slow post-translational phosphorylation of polypeptide D2 (Delepelaire and Wollman, 1985). This process, the regulation of which remains unclear, proved more active during the chases shown on Fig. 2C and Fig. 4, A and B, than during the chases shown on Fig. 2, A and B.
Figure 2:
Synthesis and turnover of cytochrome f in the WT (A), F-52L-55V (B), and
F-52L-55V-283ST (C) transformants. Autoradiographs show
cytochrome f forms (arrows) in the 30-48-kDa
region of electrophoretic migration in urea-SDS gels. This region is
bordered on the upper side by the P6 band, which corresponds to a core
subunit of Photosystem II. The lower border of this region displays two
bands: a fuzzy band corresponding to the D1 subunit of the Photosystem
II reaction center, and a sharper band corresponding to the D2 subunit
of the Photosystem II reaction center. The gel was loaded with total
protein extracts from cells labeled with
[C]acetate for 5 min in the presence of
cycloheximide. Relative changes in the amounts of
C-labeled cytochrome f upon chase, as quantified
by phosphorimaging of panels A-C, using the 0 time
points as 100% (D).
Figure 4:
Cytochrome bf complex subunits in cytochrome
processing mutants.
Immunoblots from urea-SDS-polyacrylamide gels loaded with total protein
extracts from the WT (lane 1), F283ST (lane 2),
F-29L-31L (lane 3), and F-29L-31L-283ST (lane 4)
transformants reacted with antisera against cytochrome f (revealed with iodinated protein A), the Rieske protein and
subunit IV (revealed by the ECL method) (A). Same gel system
as for A was loaded with purified membranes and stained with
TMBZ; arrows point to the three heme-binding forms of
cytochrome f in F-29L-31L transformants; h1 and h2 correspond to mitochondrial cytochromes contaminating
thylakoid preparations (Atteia et al., 1992) (B).
Comparison of the pulse time points (0) shows that there
was no decrease in the rate of synthesis of apocytochrome f in
the transformants (compare the bands indicated by arrows in lanes 0, Fig. 2). In contrast, the lifetime of the
newly synthesized forms of cytochrome f in the transformants
was much shorter than in the wild-type. As we previously reported
(Kuras and Wollman, 1994), holocytochrome f remained stable in
the wild-type throughout the chase period (Fig. 2A).
After 15 min of chase, a significant decrease in labeled apocytochrome f was already visible in F-52L-55V transformants (Fig. 2B) but not in F-52L-55V-283ST transformants (Fig. 2C). Quantification of the labeling by
phosphorimaging (Fig. 2D) shows that the apocytochrome f is much more sensitive to proteolytic degradation when bound
to the membrane (approximately t of 30 min) than
when it is released as a soluble protein in the thylakoid lumen
(approximately t
of 120 min).
Figure 3: Synthesis and turnover of cytochrome f in F-29L-31L (A) and F-29L-31L-283ST (B) transformants. Labeling and electrophoresis were as in Fig. 2except that urea was omitted from the gels. Arrows point to the various forms of cytochrome f expressed in the transformants. Quantification, by phosphorimaging, of the changes in relative amounts of the four forms of cytochrome f, f1, f2, f3, and f4, during the chase period are shown in C. The total labeling among the four forms of cytochrome f, taken as 100%, remained constant throughout the chase.
Figure 6: Comparative rates of synthesis of cytochrome f forms. Upon 5 min of pulse-labeling of chloroplast-encoded polypeptides from the WT (lane 1), F-52L-55V (lane 2), F283ST (lane 3), F-52L-55V-283ST (lane 4), F-29L-31L (lane 5), and F-29L-31L-283ST (lane 6) strains. Labeled polypeptides were resolved upon polyacrylamide gel electrophoresis in the presence (A) or absence (B) of urea. Cytochrome f synthesis in each strain was quantified by phosphorimaging, relative to that in the wild-type (C).
The unprocessed form of truncated cytochrome f was heavily labeled during 5-min pulses of F-29L-31L-283ST transformant cells (Fig. 3B, arrow). Most of it turned over within 2 h of chase, with a half-time of about 45 min. We observed upon chase a possible conversion product of lower apparent molecular mass (asterisk, Fig. 3B), the low labeling and fuzzy aspect of which prevented quantification.
We then used
specific antibodies to probe the cytochrome bf complex subunits which were expressed in the two
processing mutants (Fig. 4A). A polyclonal antiserum
raised against cytochrome f detected three bands in the
F-29L-31L transformant (lane 3 on Fig. 4A)
which migrated in this gel system at the same positions as the labeled
bands f1, f3, and f4 from Fig. 3A (experiment not
shown). The three cytochrome f bands in F-29L-31L were also
recognized by an antibody raised against a peptide from the C terminus
of the protein (experiment not shown). Comparison of the
electrophoretic migration positions shows that band f3 has an apparent
molecular mass about 1 kDa higher than that of mature cytochrome f, whereas band f4 migrates slightly ahead of cytochrome f (compare lanes 1 and 3). Interestingly, the
three cytochrome f bands, identified by antibody labeling,
were TMBZ-stainable in purified thylakoid membranes (arrows, lane 3, Fig. 4B). The accumulation of the
Rieske protein and subunit IV were similar in F-29L-31L transformants
to that in the wild-type (compare lanes 1 and 3 in Fig. 4A). A similar observation was made for cytochrome b
upon TMBZ staining of the membrane fraction (Fig. 4B).
Two cytochrome f bands where
immunodetected in F-29L-31L-283ST transformants, the upper one
corresponding to the one observed in 5-min pulse-labeling (lane 4 in Fig. 4A). A further analysis of the total
protein extract from the F-29L-31L-283ST strain showed that the
precursor form was enriched in the membrane fraction whereas the form
of lower molecular weight was released in the soluble protein fraction
(experiment not shown). TMBZ staining of the thylakoid membranes
detected trace amounts of truncated cytochrome f (not visible
on Fig. 4B). The content of other cytochrome bf subunits in the F-29L-31L-283ST
strain (lane 4, Fig. 4A) was decreased to
about the same extent as in the F283ST strain which fails to assemble
the protein complex (lane 2, Fig. 4A).
Cytochrome b
remained below detection upon TMBZ
staining (lane 4, Fig. 4B) a situation similar
to the one we previously reported for F283ST transformants (Kuras et al., 1995).
Since all the major subunits of the
cytochrome bf complex accumulated in the
F-29L-31L mutant, we investigated the state of assembly of the three
heme-stainable forms of cytochrome f. Detergent-solubilized
preparations of thylakoid membranes from the wild-type and F-29L-31L
strains were loaded on a continuous 10-35% sucrose gradient
(Pierre et al., 1995). After centrifugation, the distribution
profiles of cytochrome f, subunit IV, and cytochrome b
were analyzed using specific antibodies.
Cytochrome f, cytochrome b
, and subunit
IV comigrated among the gradient fractions from the wild-type (Fig. 5A) and mutant (Fig. 5B). A
single peak, centered around a sucrose density of 22%, was observed
with the wild-type. It corresponded to the migration of cytochrome b
f dimers which are preserved by this
method of purification (Pierre et al., 1995). The distribution
of cytochrome b
f complex subunits in the
transformant was bimodal, with a major peak similar to that in the
wild-type (22% sucrose) and a minor peak at higher density (28%). The
three forms of cytochrome f (f1, f3, f4) were found in
association with cytochrome b
and subunit IV. The
precursor form f1 is particularly visible in the fractions of higher
sucrose density. We conclude that the three forms of cytochrome f, the precursor form and the two forms of lower molecular
weight, were competent for assembly in cytochrome b
f complexes.
Figure 5:
Distribution of three major cytochrome bf subunits. Cytochrome f,
cytochrome b
, and subunit IV were immunodetected
after 10-35% sucrose gradient centrifugation of
hecameg-solubilized membranes from the wild-type (A) and
F-29L-31L strains (B). Proteins from each gradient fraction
were subjected to urea-SDS gel electrophoresis, transferred on
immunoblots, reacted with specific antisera, and revealed by the ECL
method. *, the fractions of maximal cytochrome b
f content in the two
gradients.
The
interplay between heme attachment and membrane translocation has been
addressed for both the soluble and membrane-bound c-type
cytochromes in the mitochondria. In each case, heme attachment seems to
be required for proper localization of the final protein. It has been
proposed that soluble cytochrome c is trapped irreversibly in
the intermembrane space by covalent ligation of the heme (Dumont et
al., 1991) whereas heme attachment is required for the proper
processing of membrane-bound cytochrome c (Nicholson et al., 1989).
In the case of chloroplast
cytochrome f, a plastome mutant from Oenothera described as defective in heme binding to (pre)apocytochrome f showed a decreased efficiency in processing of the precursor form
(Johnson and Sears, 1990). In contrast, previous studies based either
on nuclear mutants from C. reinhardtii probably defective in c-heme attachment in the chloroplast (Howe and Merchant, 1992)
or upon gabaculin treatment aimed at decreasing heme availability
(Anderson and Gray, 1991; Howe et al., 1995) led to the
conclusion that cytochrome f processing occurs in the absence
of heme binding. The present study, employing a reverse molecular
genetic approach, supports the latter view. We destroyed the heme
binding sites by replacement of the two cysteinyl ligands by a leucine
and a valine. We observed that the altered cytochrome f,
although synthesized at higher rates than in the wild-type (see below
for further discussion), was highly unstable and therefore prevented
significant accumulation of the other subunits of cytochrome bf complexes. However, the mutations
still allowed conversion of pre-apocytochrome f to its
processed form as well as its complete thylakoid translocation as
demonstrated by the release of a soluble protein in a strain lacking
the C-terminal membrane anchor of cytochrome f. Thus, the
biogenesis of membrane-bound cytochrome f resembles that of
soluble cytochrome c
, the other c-type
cytochrome in the chloroplast (Howe and Merchant, 1994); translocation
and processing of precursor forms of the two proteins are independent
of heme binding.
The crystal structure of cytochrome f (Martinez et al., 1994) shows that one axial ligand is
provided by the -amino group of the N terminus released upon
processing of the precursor protein. It was then tempting to suggest
that conversion of pre-apocytochrome f to the mature form
would occur through an orderly process opposite the one advocated for
mitochondrial cytochrome c
(Nicholson et
al., 1989); the protein conformation suitable for heme attachment
would require processing of its precursor form. In contrast to this
view, the delayed processing in F-29L-31L transformants allowed us to
observe heme binding to both the precursor and processed forms of
cytochrome f. Moreover, these three heme-stainable forms of
cytochrome f were detected within assembled cytochrome b
f complexes. Therefore, we conclude
that rapid cytochrome f processing is not required for heme
attachment or for the assembly of the protein into cytochrome b
f complexes. This observation does not
necessarily imply that heme binding occurs prior to protein processing
in the wild-type strain. Delayed kinetics of processing may have
elicited a branched pathway for direct conversion of pre-apo- to
pre-holocytochrome f. It nevertheless implies that, were the
maturation processes to occur in two subsequent steps in the wild-type,
they would be primarily determined by a kinetic competition rather than
by mechanistic constraints. Since the delayed processing in the
F-29L-31L transformants yielded assembled but inactive cytochrome b
f complexes, we presently assume that
an improper liganding environment has prevented the attached heme from
adopting a suitable orientation and that its redox potential may be
modified. Further biophysical studies will be required to elucidate
this point.
If the degradation process was initiated from the stromal face of the thylakoids, we would expect a release of the large amino-terminal domain of the protein in the lumen upon proteolytic attack of the carboxyl terminus anchor. This mechanism would mimic the situation we created by truncating cytochrome f at its carboxyl terminus. Since we observed that the free soluble amino-terminal domain is then rather stable (this work and Kuras et al. 1995), we conclude that the proteolytic system responsible for recognition and degradation of misfolded cytochrome f is intimately associated with the thylakoid membranes and has access to the protein via the lumenal side of the membrane.