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J Biol Chem, Vol. 273, Issue 45, 29315-29320, November 6, 1998
Subunit of Cytochrome
b-559 Is Required for Assembly of the Photosystem Two
Complex in both the Light and the Dark in Chlamydomonas
reinhardtii*
,From Wolfson Laboratories, Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AY, United Kingdom
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ABSTRACT |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The role of cytochrome b-559 in the
photosystem two (PSII) complex has been investigated through the
construction of a psbE null mutant by transformation of the
chloroplast genome of the green alga Chlamydomonas
reinhardtii. No PSII activity could be detected in this mutant
either in oxygen evolution assays or by analysis of variable
chlorophyll fluorescence. Immunoblotting experiments showed that the
absence of PSII activity in the mutant was due to the loss of the PSII
complex in both light-grown and dark-grown cultures. In contrast, the
photosystem one reaction center polypeptide, PsaA, was present at
wild-type levels in the mutant. RNA gel blot assays confirmed that the
transcript levels for the psbA, psbD, and
psbF genes were unaffected by disruption of the
psbE gene, suggesting a post-transcriptional effect on their expression. Pulse-labeling experiments showed that either synthesis of PSII subunits was impaired in the psbE null
mutant or there was extremely rapid degradation of newly synthesized subunits. Interestingly, the PsbE and PsbF subunits accumulated to
wild-type levels in a psbA deletion mutant of C. reinhardtii, FuD7, which fails to synthesize D1 and assemble
PSII. Our results provide evidence for a role for cytochrome
b-559 in the early steps of assembly of the PSII complex,
possibly as a redox-controlled nucleation factor that determines the
level of PSII within the thylakoid membrane.
Photosystem two (PSII)1
is a multisubunit complex within the thylakoid membrane that catalyzes
the light-induced oxidation of water to molecular oxygen as well as the
reduction of plastoquinone to plastoquinol (1). At the heart of PSII is
a the reaction center (RC) complex composed of the polypeptides D1, D2,
PsbI, and a transmembrane b-type cytochrome termed
cytochrome b-559 (2). As yet, the function of this
cytochrome within PSII is unresolved, but it probably plays a role in
protecting the PSII RC from photoinactivation, possibly by acting as a
source of electrons to reduce long-lived chlorophyll cation species
within the PSII RC (3), which unless reduced would oxidize nearby amino
acids within the protein complex (4).
The structure of Cyt b-559 is quite unusual, as it is
thought to be a heterodimer of two subunits arranged in parallel
orientations: the One way to investigate the roles of psbE and psbF
in PSII function is to construct specific null mutants. In the
transformable cyanobacterium Synechocystis sp. PCC 6803, as
with all higher plants, psbE and psbF are
cotranscribed with the downstream genes, psbL and
psbJ (8), which also encode PSII subunits (9, 10). The
effect of mutating either psbE or psbF on PSII
function is therefore complicated by possible polar effects on gene
expression within this operon. This potential problem is highlighted by
the work of Shukla and co-workers (11), which showed that the presence of a stop codon within the coding sequence for PsbE in a spontaneous mutant of Synechocystis 6803 led to loss of the entire
psbEFLJ transcript.
Early studies showed that deletion of the entire psbEFLJ
operon in Synechocystis led to loss of PSII activity (12,
13) and in light-grown cultures to a failure to accumulate D2 and to a
reduction in the levels of D1 and the PSII subunits CP47 and CP43 to
about 50% of WT (11). Whether the loss of PSII activity in this mutant
is due to increased rates of photoinactivation has not been resolved.
Deletion of psbF also leads to loss of PSII activity (14),
suggesting that the In addition to Synechocystis sp. PCC 6803, the green alga
Chlamydomonas reinhardtii is also widely used to study PSII
through genetic means. Because of its eukaryotic nature,
Chlamydomonas is possibly a more appropriate model system
with which to study photosynthesis in higher plants (15). Unlike
cyanobacteria and higher plants, the psbEFJL operon is naturally
disrupted in C. reinhardtii. The psbF gene is
cotranscribed with the downstream psbL gene (16), whereas
psbE is located approximately 7.3 kb away and transcribed in
the opposite direction (16, 17). This gene arrangement should thus
allow the specific disruption of the psbE gene without
affecting transcription of the psbF and psbL genes.
In this paper, we report on the construction and characterization of
the first chloroplast psbE null mutant. Our results
demonstrate the importance of the PsbE protein for assembly of PSII
both in the light and the dark and suggest a model in which cytochrome b-559, besides having a role in electron transfer, also acts
as a nucleation factor in the assembly of
PSII.2
Strains and Growth Conditions--
C. reinhardtii
wild-type strain CC-125 was obtained from the Chlamydomonas
Culture Collection (CGC, Dept. of Botany, Duke University, Durham, NC),
and the chloroplast PSII mutant, FuD7 (19), was provided by Dr. Saul
Purton (University College, London). Cells were grown on plates or in
liquid culture at 20-25 °C under dim light (20-50
µE·m Plasmid Constructions--
Plasmid pF1 is a derivative of
plasmid p78 (which contains the 15.1-kb PstI-4 fragment from
the chloroplast genome of C. reinhardtii cloned into pBR322)
(20) and was constructed by digesting p78 with EcoRI to
remove a 5.8-kb fragment followed by religation. pF1 contains the
psbE gene with upstream and downstream flanking sequences of
approximately 8 and 2 kb, respectively (Fig. 1).
To disrupt the psbE gene, a 1.9-kb spectinomycin-resistance
cassette was isolated from plasmid pUC-atpX-AAD (21) after digestion with EcoRV and SmaI and ligated into the
AlwNI site found within the psbE gene, after the
ends were made blunt with the Klenow fragment of DNA polymerase I, to
yield plasmid pF1aad. Standard methods were used to generate the
recombinant DNA plasmids (22).
Transformation of Chlamydomonas--
Chloroplast transformation
experiments were performed according to Andronis et al. (23)
using a particle gun (Shearline). Transformants were selected on TAP
plates containing 100 µg/ml spectinomycin. Spectinomycin-resistant
colonies were restreaked three to four times on TAP plates containing
spectinomycin before their genotypes were analyzed.
Isolation and Analysis of DNA and RNA--
The isolation of
total Chlamydomonas DNA and DNA gel blot analyses were
performed according to Ref. 23. Total RNA was isolated from 100-ml
cultures of mid-log phase Chlamydomonas cells as described in Ref. 24, in the presence of 100 µM aurintricarboxylic
acid (Sigma). RNA was electrophoresed in a 1.5% agarose gel containing formaldehyde, transferred to nylon membranes (Hybond-N, Amersham International), and hybridized to 32P random-labeled
specific DNA probes using the SSPE/formamide system as described
(22).
To confirm the homoplasmicity of the psbE insertion mutants
3 and 4, a PCR analysis was performed using primers flanking the psbE gene. PCR reactions were performed in a Techne PHC-3
thermocycler following typical protocols (25) in the presence of
Taq DNA polymerase (Promega). The oligonucleotides used as
primers for PCR were SA1 (5'-TTGTTTCAATGGGGCATTAT-3'; located 407 base
pairs upstream the psbE initiation codon), SA5
(5'-ATAGATGGTTTGAAAAGG-3'; located 301 base pairs downstream the
psbE initiation codon), and B (5'-CACTGCCTCTAATAAAGTC-3';
located within the aadA cassette) (Fig. 1). For WT, primers
SA1 and SA5 amplified a 0.75-kb fragment, whereas in strains 3 and 4, a
2.65-kb fragment was generated because of the presence of the 1.9-kb
spectinomycin-resistance cassette (data not shown). No 0.75-kb band was
amplified from strains 3 and 4 consistent with the absence of WT copies
of the psbE gene. Using primers SA1 and B, a 0.5-kb fragment
was amplified from strains 3 and 4 but not from the WT (data not shown).
Measurements of PSII Activities--
Light-induced
O2-evolution activity in whole cells was measured with a
Clark-type electrode. The measurements were performed in TAP medium at
25 °C, under saturating light conditions, in the presence of 1 mM potassium ferricyanide and 1 mM
2,6-dichloro-p-benzoquinone. Fluorescence induction kinetics
of dark adapted cells, on TAP plates, were performed using a
pulse-amplitude modulation fluorometer (PAM 101, Walz) using a white
actinic light of intensity (1000 µE·m Protein Analysis--
Thylakoid membranes were prepared as
described (26) in the presence of a mixture of protease inhibitors (0.1 mM Pefablock (Boehringer Mannheim), 1 mM
phenanthroline, 1 µM pepstatin A, and 10 µM
leupeptin). The isolation was carried out essentially in the dark at
4 °C, and throughout the procedure the chlorophyll concentration was
measured according to the method of Arnon (27). The separation of
protein by SDS-polyacrylamide gel electrophoresis and the
immunodetection of protein was performed using methods described in
Ref. 23. Visualization of bound antibody was performed by using a
secondary antibody conjugated either to alkaline phosphatase (23) or to
horseradish peroxidase, in the latter case followed by detection using
enhanced chemiluminescence (ECL, Amersham).
Antisera specific for the Pulse Labeling of Cells--
Chlamydomonas cells were
labeled with [35S]sulfate essentially as
described.3 Cells were grown
under white light at 20-50 µE·m Construction of a psbE Insertion Mutant--
DNA sequencing was
first performed to identify further genes in the vicinity of
psbE. In agreement with Ref. 32, a putative rps9
gene, encoding the S9 subunit of the plastid ribosome, and the highly
conserved ycf4 gene, which is involved in the assembly of
PSI (32), lie downstream of psbE (Fig.
1). Northern blots indicate, however,
that psbE is not cotranscribed with these genes so that
disruption of psbE would not be expected to affect their expression (16, 32, 33). To generate a psbE null mutant of
C. reinhardtii, a donor plasmid, designated pF1aad, was
first constructed from the parental plasmid, pF1, by insertion of a spectinomycin-resistance cassette into an AlwNI site located
26 codons into the coding region of psbE (Fig. 1). This
construct was introduced into the chloroplast of WT C. reinhardtii using the biolistic technique developed by Boynton and
co-workers (34) (see "Experimental Procedures") and transformants
selected on spectinomycin.
Fig. 2 shows a Southern blot of total DNA
isolated from three spectinomycin-resistant colonies (designated 1, 3, and 4) obtained from the transformation experiment. Two of the
spectinomycin-resistant strains (3 and 4) gave a profile diagnostic for
homologous recombination of the interrupted psbE gene into
the chloroplast genome (Fig. 2A). A psbE-specific
probe hybridized to a 1.9-kb HindIII fragment in the WT but
to 1.4-kb and 0.9-kb HindIII fragments in these mutants. As
expected, these mutants also contained the spectinomycin-resistance cassette (Fig. 2B). Spectinomycin-resistant strain 1 gave a
WT-like profile and did not contain the spectinomycin cassette.
Presumably, spectinomycin resistance arose in this strain through a
spontaneous mutation. The data presented in Fig. 2 also indicate that
mutants 3 and 4 were homoplasmic and did not contain WT copies of the psbE gene. Homoplasmicity of mutants 3 and 4 was also
confirmed by PCR analysis using primers flanking the psbE
gene (see "Experimental Procedures" and data not shown). Strains 3 and 4 therefore lack a functional psbE gene and were
characterized further.
Biochemical Analysis of the psbE-inactivated Mutants--
In
contrast to WT, the psbE insertion mutants 3 and 4 were
unable to grow photoautotrophically on minimal medium either on agar
plates or in liquid (Table I). However,
the psbE null mutants could be propagated in the presence of
acetate either in the light or the dark, although their growth rates in
the light were slightly slower than WT (Table I). Mutant cells grown on
acetate in the light were tested for light-induced oxygen evolution
using a Clark-type electrode. In contrast to WT, no oxygen evolution
was detected in strains 3 and 4 either in the presence of bicarbonate
(whole chain electron transport) or the artificial electron acceptors 2,6-dichlorobenzoquinone and ferricyanide (PSII-mediated electron transport) (Table I).
PSII activity was also investigated by measuring the chlorophyll
fluorescence arising from cells of WT and mutant growing on TAP plates
in the light. The ratio of variable fluorescence to maximum
fluorescence (Fv/Fm) is a measure of the
efficiency of light capture by PSII. The psbE mutants showed
no detectable variable fluorescence, consistent with the absence of
functional PSII RCs, whereas the WT showed a
Fv/Fm ratio of approximately 0.75.
Immunoblotting experiments were performed to assess if the PSII RC
subunits, D1 and D2, accumulated in the thylakoid membrane in the
absence of PsbE when mutant cells were grown in the presence of light.
Fig. 3A shows that both D1 and
D2 could not be detected under these conditions. To exclude the
possibility that in the absence of PsbE, PSII can be assembled but is
more prone to light-induced degradation, cultures grown
heterotrophically in the dark were also examined. Fig. 3B
shows that the levels of D1, D2, CP47, CP43, PsbF, and PsbO (also known
as the 33-kDa extrinsic polypeptide) were drastically reduced in
thylakoid membranes isolated from dark-grown cultures of the
psbE mutants compared with WT. As judged from a dilution
series, CP43 was present at approximately 1-10% of WT levels in
strains 3 and 4 (Fig. 3B), and in overexposed blots D2 was
found to accumulate to 1-5% of WT levels (data not shown). The amount
of D1 protein in the psbE mutants was below the level of
detection, which was estimated to be at 1% of WT levels. As expected
PsbE was absent in the mutants (Fig. 3B). The CP47, PsbO
(33-kDa polypeptide), and PsbF subunits could not be detected in the
mutant thylakoid membranes (Fig. 3B).
Transcript Levels in the psbE Insertion Mutant--
RNA gel blots
were performed to examine whether disruption of the psbE
gene had effects on the accumulation of mRNA from psbF, located 7.3 kb away on the chromosome, or other PSII genes such as
psbA or psbD. As shown in Fig.
4, the levels of the psbA,
psbD, and psbF transcripts were similar in both
psbE mutant 3 and WT when grown in either the light or the
dark in the presence of acetate. As expected, the psbE null
mutant failed to accumulate psbE mRNA (Fig. 4).
Interestingly, in comparison with the psbA and
psbD genes, the level of transcripts arising from the
psbE and psbF genes in the WT appeared to be
greater in the dark than the light.
Pulse Labeling Using
[35S]Sulfate--
Pulse-labeling experiments were
performed to investigate the rate of synthesis of plastid-encoded PSII
subunits in the absence of the PsbE subunit. To allow visualization of
chloroplast-encoded gene products, the pulse experiments were performed
in the presence of cycloheximide, an inhibitor of cytoplasmic protein
synthesis. Immunoblotting was performed to help assign the labeled
bands. Fig. 5 shows the profile obtained
with cells of WT, mutants 3 and 4, and FuD7, which is a psbA
deletion mutant (19). As expected, the WT shows good labeling of the
D1, D2, CP47, and CP43 proteins. For FuD7, no D1 synthesis was
observed, but as previously documented (19, 35), in the absence of D1
synthesis, some incorporation of the D2, CP43, and CP47 subunits into
the thylakoid membrane was detected. Mutants 3 and 4 showed
radiolabeling profiles more similar to FuD7 than WT. Of the major PSII
subunits, D2 and CP43 were the most conspicuously labeled in the
psbE mutants, although the degree of radiolabeling, as in
FuD7, was much less than WT. These results suggested that the rate of
synthesis of PSII subunits is reduced in the psbE null
mutants or that there is rapid degradation of the subunits in the
absence of PsbE. Similar results were also obtained when pulse labeling
of the cells was performed in the dark (data not shown).
Although FuD7 fails to assemble a functional PSII complex and has
depleted levels of D2, CP47, and CP47 compared with the WT (19, 35),
the PsbE and PsbF polypeptides could still be detected at WT levels in
thylakoid membranes using specific antibodies (Fig.
6).
In this paper, we have described the construction of a
psbE insertion mutant of C. reinhardtii. A
particular advantage of using C. reinhardtii over
Synechocystis 6803 to construct such a mutant is that
pleiotropic effects on the transcription of the downstream
psbFLJ genes in Synechocystis 6803 are avoided.
Disruption of the psbE gene in C. reinhardtii
leads to an inability to accumulate subunits of the PSII core complex
within the thylakoid membrane in both dark-grown (Fig. 3B)
as well as light-grown material (Fig. 3A). Loss of PSII is
therefore not due to enhanced photoinactivation of the PSII complex, a
possibility that was not excluded in the analysis of previous
Synechocystis mutants affected in cytochrome b-559 biosynthesis (8, 11, 12).
Northern blots confirm that the psbF, psbD, and
psbA transcript levels are unaffected in the psbE
null mutants so clearly the inability to assemble PSII lies at a
post-transcriptional step. Pulse-labeling experiments revealed that the
synthesis of the major PSII core polypeptides was drastically impaired
in the psbE null mutants (Fig. 5) consistent with either a
block in synthesis or the rapid degradation of newly synthesized
proteins because of a failure to assemble them into a stable core complex.
A number of PSII null mutants have now been constructed in
Chlamydomonas through biolistic transformation. The
psbH (36) and psbK (37) mutants, like the
psbE mutant described here, also fail to accumulate PSII.
However, in pulse-labeling assays, both the psbH and
psbK null mutants show a WT-like profile, indicating that it
is the degradation rather than the synthesis of PSII subunits that is
affected in these mutants. The labeling pattern for the psbE
null mutant on the other hand is very similar to that of FuD7, which
contains a disrupted psbA gene. Our data therefore support a
model in which the A role for cytochrome b-559 in the early steps of assembly
of PSII is also supported by recent in vitro pulse-chase
experiments that have shown that radiolabeled D1 protein can be
assembled into the PSII core complex through the stepwise addition of
subunits (38). Whether D2 or cyt b-559 is the first partner
for D1 following insertion into the thylakoids remains to be
established. However, it is clear that the PsbI subunit found in the
isolated PSII RC (2) is not required for assembly of PSII (39).
Early studies on the psbEFLJ deletion mutant of
Synechocystis indicated that D1 and D2 were absent from the
thylakoid membrane but that CP47 and CP43 accumulated to WT levels
(12). Later work showed that when an additional protease inhibitor,
leupeptin, was used to avoid inadvertant proteolysis of thylakoid
membrane protein and care taken to avoid saturation of the signals in
immunoblotting experiments, the levels of D1, CP43, and CP47 were
approximately 50% of WT, and only D2 failed to accumulate (11). In the
studies reported here, a mixture of protease inhibitors including
leupeptin was used during the isolation of the thylakoid membranes, and in addition whole cell extracts were analyzed (Fig. 5). The absence of
D2 in the Synechocystis psbEFLJ mutant has been interpreted in terms of a specific interaction between D2 and cyt b-559
during assembly of PSII (11). However, as noted in the Introduction, a
psbEFLJ deletion mutant is not an appropriate strain to
study the specific role of PsbE or PsbF in PSII assembly, and so it is
unclear how far the results in the cyanobacterial psbEFLJ
mutant can be compared with the algal psbE null mutant
described here.
It is clear, however, that quite different phenotypes can be displayed
by Synechocystis 6803 and Chlamydomonas for the
same type of mutation. For example, null mutants of the
Chlamydomonas psbH (36) and psbK (37) genes lead
to a destabilization of the PSII core complex, reminiscent of the
phenotype of the psbE null mutants presented here, whereas
Synechocystis 6803 psbH (40) and psbK
(41, 42) null mutants assemble functional PSII and are still capable of
photoautotrophy. The more drastic effect seen in the
Chlamydomonas mutants is consistent with the presence of a
more efficient proteolytic system in the chloroplast for the removal of
mis-assembled PSII complexes.
We have prepared in the course of this work antibodies specific for the
PsbE and PsbF proteins of Chlamydomonas, which has allowed
for the first time unambiguous examination of the expression of these
proteins in other Chlamydomonas PSII mutants. Interestingly, we found that both PsbE and PsbF accumulate to WT levels in FuD7, which
cannot synthesize D1 and lacks detectable levels of D2 and CP47 (19,
35). The cytochrome b-559 apopolypeptides therefore appear
to be stable in the thylakoid membrane of Chlamydomonas in
the absence of many of the PSII subunits but is required for their
accumulation. Cyt b-559 is also present at approximately WT
levels in a psbA null mutant of Synechocystis
6803 (43) and a psbE mutant of the same organism that is
truncated at the C terminus by 31 residues yet contains only a low
level of assembled PSII (44). The accumulation of Cyt b-559
in the absence of D1 and D2 is therefore likely to be a common feature
among oxygenic photosynthetic organisms. Cytochrome b-559
may therefore act as a nucleation factor for assembly of PSII in both
cyanobacteria (44) and chloroplasts (this work). Indeed, studies on the
light-induced development of the chloroplast has indicated that
cytochrome b-559, detected spectrophotometrically,
accumulates in etioplasts in the absence of the majority of the PSII
subunits (45). We suggest that the level of PSII within the thylakoid
membrane may be controlled by the abundance of cyt b-559 and
that its redox state, which is controlled by reactions within the
thylakoid membrane, may be one of the mechanisms by which the assembly
of PSII is regulated. Assembly of PSII may be closely coordinated with
D1 synthesis, which is known to be under redox control (46).
In previous work (47), the stoichiometry of the
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit encoded by psbE and the 
subunit encoded by psbF (5). The haem molecule is likely to
be ligated by the single histidine residues within each subunit (6).
Although an heterodimeric structure for Cyt b-559 is
generally favored, recent work has demonstrated that the genetic fusion
of the N terminus of the subunit to the C terminus of the subunit in Synechocystis 6803 leads to the assembly of a
functional cytochrome b-559 (7). Because the
heterodimer model requires that the N termini of the monotopic and
subunits lie on the same side of the membrane, the fusion of PsbE
and PsbF without loss of PSII activity has been interpreted in terms of
an 2/2 homodimeric structure for Cyt
b-559 with one haem bound to 2 and one to
2 (7). If this interpretation is correct, then Cyt
b-559 would contain 2 haems and 2 copies each of and
.
subunit alone is crucial for assembly of PSII.
However, the stability of the psbEFLJ transcript was not
investigated in the psbF mutant, so it remains possible that
expression of the other genes within the operon had also been affected.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
2 s1) or in the dark in Tris
acetate-phosphate (TAP) or high-salt minimal media (20). The
transformants were maintained on TAP medium containing 100 µg/ml
spectinomycin, 10 µM
3-[3,4-dichlorophenyl]-1,1-dimethyl urea, and 50 µg/ml ampicillin.
2
s1).
and subunits of Cyt b-559
from C. reinhardtii were raised against 17-mer oligopeptides
derived from the deduced N-terminal sequences of PsbE
(AGKPVERPFSDILTSIR) and PsbF (acetyl-TTKKSAEVLVYPIFTVR) (17). Rabbits
were immunized subcutaneously with the oligopeptides in the form of
multiple antigenic peptides (purchased from Alta Bioscience, UK)
following standard methodologies (28). Polyclonal antibodies specific for D1 (N-terminal fragment, spinach, gift of Roberto Barbato (29)), D2
(C-terminal 12 amino acid residues, (30)), CP43 (whole molecule,
C. reinhardtii P6 polypeptide, gift of N.-H. Chua), CP47
(whole molecule, C. reinhardtii P5 polypeptide, gift of
N.-H. Chua), the 33-kDa extrinsic protein (whole molecule, pea (31)),
and PsaA (gift of K. Redding and J.-D. Rochaix) were also used.
2 s1 to
early exponential phase (1-2 × 106 cells/ml,
approximately A750 of 0.2-0.3) in TAP. The
cells were pelleted (10 min, 1500 rpm, Chillspin benchtop centrifuge,
room temperature), washed once in TAP-RS (reduced sulfur) (TAP made with Beijerinck's solution (20) without sulfate but still containing trace elements that contain sulfur), resuspended in the same medium to
107 cells/ml, and incubated overnight under the original
growth conditions. After another wash, the cells were resuspended in
TAP-S (minus sulfur) (TAP containing no trace elements and made with
Beijerinck's solution lacking sulfate) for 2 h, under 1000 µE·m2 s1, and then incubated with
cycloheximide (Sigma) at 10 µg/ml for 10 min. Pulse labeling of cells
was performed with carrier-free Na235SO4 (stock at 0.9 mCi/ml, ICN)
at 0.1 mCi/ml for 5 min. Aliquots of cultures were immediately
centrifuged, and cell pellets were frozen in liquid nitrogen and kept
at 
80 °C. The cells were subsequently washed once with 50 mM MES/NaOH, pH 6.5, containing 5 mM EDTA and a
mixture of protease inhibitors (as described previously) and
solubilized for 1 h in an equal volume of solubilization buffer. Proteins were separated by SDS-polyacrylamide gel electrophoresis containing 6 M urea, transferred onto nitrocellulose
membranes, and exposed to x-ray film.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
Physical map of the flanking regions of the
psbE gene in the chloroplast genome of C. reinhardtii. Arrows above genes indicate
direction of gene transcription. The psbE gene was disrupted
by inserting the aadA cassette at the AlwNI site.
SA1, SA5, and B primers were used for PCR analysis. bp, base
pairs.
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Fig. 2.
DNA gel blot hybridization of the total DNA
from WT and spectinomycin-resistant colonies (1, 3, and 4), following
digestion with HindIII. Controls were ~1 ng each of
pF1, pF1aad, and a 2-kb aadA cassette (A, probed
with a psbE-specific probe; B, probed with an
aadA-specific probe).
Growth properties and oxygen-evolving activities of WT and transformant
(strains 3 and 4) cells
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Fig. 3.
Western blots of thylakoid membrane
polypeptides of WT and psbE null mutants (3 and 4) isolated
from either light-grown (panel A) or dark-grown
(panel B) cultures and immunostained with antibodies
specific for the -Cyt b-559, -Cyt b-559,
D1, D2, 33 kDa (PsbO), CP43 and CP47 subunits of PSII, and the PsaA
subunit of PSI. Serial dilutions of WT proteins are included for
quantification of the blots in panel B.
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Fig. 4.
Northern blots of total RNA isolated from WT
and psbE null mutant 3, grown either in the light or the
dark, probed with different specific probes.
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Fig. 5.
Autoradiogram of total protein from
[35S]sulfate pulse-labeled whole cells of WT,
mutants 3 and 4, and FuD7, following separation by urea
SDS-polyacrylamide gel electrophoresis. Cycloheximide was added to
inhibit cytoplasmic protein synthesis. Proteins assigned to the and
subunits of the ATPase and CP47, CP43, D1, and D2 of PSII are
indicated.
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Fig. 6.
Immunoblots of thylakoid membrane
polypeptides isolated from light-grown cultures of WT, FuD7, and
psbE null mutant 3, immunostained with antibodies specific
for -cyt b-559, -cyt b-559, and D1
polypeptide.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
subunit is important for promoting synthesis of
D1 or for retarding its degradation. As such the subunit appears to
be an important component in the early steps of the assembly of the
PSII complex.
and subunits to
the D1 and D2 subunits within the isolated PSII RC from C. reinhardtii, as determined by 14C labeling, was found
to be approximately 1:1:1:1 with 1 haem present per PSII RC. This
result led to the conclusion that either cyt b-559 was
present as a heterodimer or else there was an equimolar mixture
of PSII RCs containing either 2 or 2
haem-containing complexes (33, 47). Because accumulation of within
the thylakoid membrane is dependent on the presence of (Fig. 3), it
is likely that each PSII RC contains both and . Hence, the
mutagenesis data combined with the isotopic labeling data provide
support for the heterodimer model for cyt b-559. Such
a conclusion is at odds with the recent finding that an active
cytochrome b-559 was synthesized in a strain of
Synechocystis 6803 in which the psbE and
psbF genes were fused (7). One possible explanation is that
the natural His ligand present in the psbF gene product at
codon 23 has been functional replaced by an alternative ligand within
the same subunit. Inspection of the psbF sequence suggests that Met-38 toward the C terminus is a possible candidate. Although replacement of the His ligand by the Met ligand may be expected to
destabilize haem binding, the covalent fusion of the and subunits may enhance its stability.
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FOOTNOTES |
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* This work was supported by The Royal Society, the BBSRC, and the Portuguese Government.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.
Recipient of a JNICT/PRAXIS XXI studentship from Portugal.
§ To whom correspondence should be addressed. Fax: 44-171-594-5267; E-mail: p.nixon{at}ic.ac.uk.
The abbreviations used are: PSII, photosystem two; RC, reaction center; MES, 2-morpholinoethanesulfonic acid; Cyt, cytochrome; WT, wild type; kb, kilobase(s); PCR, polymerase chain reaction.
2 A preliminary report of this work has been presented (18).
3 J.-D. Rochaix, personal communication.
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Abstract
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