From the Department of Biology, University of Turku,
FIN-20520 Turku, Finland and the § Department of
Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm
University, S-10691, Stockholm, Sweden
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Assembly of multi-subunit membrane protein
complexes is poorly understood. In this study, we present direct
evidence that the D1 protein, a multiple membrane spanning protein,
assembles co-translationally into the large membrane-bound complex,
photosystem II. During pulse-chase studies in intact chloroplasts,
incorporation of the D1 protein occurred without transient accumulation
of free labeled protein in the thylakoid membrane, and photosystem II subcomplexes contained nascent D1 intermediates of 17, 22, and 25 kDa.
These N-terminal D1 intermediates could be co-immunoprecipitated with
antiserum directed against the D2 protein, suggesting co-translational assembly of the D1 protein into PS II complexes. Further evidence for a
co-translational assembly of the D1 protein into photosystem II was
obtained by analyzing ribosome nascent chain complexes liberated from
the thylakoid membrane after a short pulse labeling. Radiolabeled D1
intermediates could be immunoprecipitated under nondenaturing
conditions with antisera raised against the D1 and D2 protein as well
as CP47. However, when the ribosome pellets were solubilized with SDS,
the interaction of these intermediates with CP47 was completely lost,
but strong interaction of a 25-kDa D1 intermediate with the D2 protein
still remained. Taken together, our results indicate that during the
repair of photosystem II, the assembly of the newly synthesized D1
protein into photosystem II occurs co-translationally involving direct
interaction of the nascent D1 chains with the D2 protein.
Photosystem II (PS II)1
is a multiprotein membrane complex that catalyzes water oxidation and
reduction of plastoquinone. The reaction center complex of PS II
consists of the D1 and D2 proteins, the The D1 protein has an unusually high turnover rate as compared with
other chloroplast proteins (5, 6). The photodamaged D1 protein in the
PS II centers is replaced constantly with newly synthesized D1 protein
to maintain PS II in a functional state (7).
The D1 protein, encoded by the plastid psbA gene, is
synthesized on membrane-associated ribosomes and inserted into
thylakoid membrane during its synthesis (8, 9). Moreover, ribosomes have been shown to pause at specific sites during translation of
membrane-bound psbA mRNA, and this was hypothesized to
facilitate the binding of cofactors to the D1 protein (10). It has been suggested that the first step of PS II repair is the association of
newly synthesized D1 with the D2 protein, D2-cytochrome
b559 (11), or D2-cytochrome
b559-CP47 (12) in the stroma-exposed thylakoid
membranes. Studies on different PS II gene deletion mutants of
Chlamydomonas reinhardtii have shown that the protein synthesis of D1 and D2 is tightly coupled and that the D2 protein directly or indirectly regulates the synthesis of D1 protein (13). Contrary to the D1 protein, the synthesis and short term accumulation of the D2 protein are relatively independent of light and availability of chlorophyll (14). These results, together with our earlier studies
on D1 synthesis and assembly into PS II (15-17), suggest a crucial
role for the D2 protein in the stabilization of the D1 protein.
In this study, we have addressed the question whether the D1 protein
assembly into PS II is occurring co-translationally. Reports on
co-translational assembly of proteins into multi-protein complexes are
scarce (18, 19). It has been hypothesized, however, that ribosome
pausing, which is known to occur also during D1 translation (10), may
control protein targeting and insertion into the membrane as well as
the concomitant assembly into a protein complex (20). It can be
envisaged that the efficiency and coordination of D1 replacement during
PS II repair would be greatly facilitated by co-translational assembly
of the D1 protein into PS II.
We show that the newly synthesized D1 protein cannot be trapped as a
free protein after termination of translation but is directly
associated with other PS II proteins. Isolation of ribosome nascent
chain complexes further indicated that the D1 nascent chains interact
already during translation elongation with the D2 protein. A scheme
summarizing the assembly pathway of the newly synthesized D1 protein
into PS II during the repair process is presented.
Plant Material and Isolation of Intact Chloroplasts--
Spinach
was grown hydroponically at 23 °C in a light/dark cycle of 10 h/14
h. For all the experiments fully developed leaves were harvested 1 h after the lights were turned on.
Immediately after harvest, spinach leaves were briefly homogenized in
330 mM sorbitol, 5 mM ascorbate, 0.05% bovine
serum albumin, 2 mM EDTA, 1 mM
MgCl2, 50 mM Hepes-KOH, pH 7.6, filtered through Miracloth and centrifuged for 1 min at 1000 × g. The pellets were resuspended in Medium A (330 mM sorbitol, 2 mM dithiothreitol, 50 mM Hepes-KOH, pH 8.0) and loaded on the top of Percoll step gradients (40 and 70% in Medium A) and spun for 5 min at 4500 × g at 4 °C. Chloroplasts in 70% Percoll were diluted with
Medium A, spun for 2 min at 1300 × g, and washed once
with Medium A. The chloroplasts used in this study were more than 90% intact.
In Vitro Translation in Chloroplast--
In vitro
translations in isolated chloroplasts were performed essentially as
described in Ref. 21. After 5 min of preincubation (0.5 µg
chlorophyll/µl) at 23 °C in the light (about 50 µmol of photons
m Sucrose Gradient Fractionation of Thylakoid
Membranes--
Thylakoids (0.5 mg/ml) were solubilized for 5 min on
ice in medium B containing 1% n-dodecyl- Isolation of Ribosome Nascent Chain Complexes--
Chloroplasts
(equivalent to 100 µg of chlorophyll) were pulse labeled with
[35S]methionine for 2.5 min and thereafter solubilized
with 2% polyoxyethylene 10 tridecyl ether in Medium C (50 mM Hepes-KOH, pH 7.5, 5 mM MgOAc, 50 mM KOAc, 250 µg/ml chloramphenicol, 0.5 mg/ml heparin, 2 mM dithiothreitol) for 10 min. Ribosomes were collected by
centrifugation through a 1.0 M sucrose cushion in Medium C
at 270 000 × g for 1 h.
Immunoprecipitation--
Immunoprecipitation of sucrose gradient
fractions was performed by adding the antiserum to each fraction, and
after overnight incubation the IgGs were collected using bovine serum
albumin-saturated protein A-Sepharose. The beads were washed for five
times with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl,
1 mM EDTA, 1% Triton, and the bound antigen was released
in the sample buffer.
For immunoprecipitation of ribosome pellets under denaturing
conditions, the ribosome pellets were solubilized with 1% SDS in 15 mM dithiothreitol, 100 mM Tris-HCl, pH 7.5, diluted with four volumes of 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton, and
immunoprecipitation was performed as above. For co-immunoprecipitation,
the ribosome pellets were solubilized with 1% DM in 50 mM
Tris-HCl, pH 7.5, before addition of antibodies.
Protein Analysis--
Nondenaturing Deriphat-PAGE (5-16%
acrylamide gradient) separation of protein complexes was performed
essentially according to Ref. 22. SDS-PAGE was performed according to
Ref. 23 using 15% acrylamide gels with 6 M urea and
Western blotting by standard techniques using chemiluminescence for
detection. For autoradiography, gels were stained, dried, and exposed
to film (17). Quantification after SDS-PAGE was performed by scanning
and analyzing the autoradiograms with the software program IMAGE
(Imaging Research Inc., AIS 3.0 rev1.4).
Antibodies--
Antisera raised against the N-terminal residues
(58-86) of the D1 protein, against the residues 230-245 of the D2
protein, and against the purified CP47 protein were used in this study.
Insertion of Newly Synthesized D1 Protein into PS II Subcomplexes
in Intact Chloroplasts--
In earlier studies we observed that the
incorporation of the newly synthesized D1 protein into PS II core
complexes proceeded in a stepwise manner (17). Immediately after pulse
labeling (5 min), the newly synthesized D1 protein predominantly
sedimented in the top of the sucrose gradient as unassembled subunits
(16). Therefore, we postulated that the assembly of the D1 protein
occurs mainly in a post-translational pathway in which the full-length D1 first accumulated as an unassembled protein, in parallel to a
possible co-translational pathway in which D1 incorporates directly into PS II reaction centers (16).
In this study, we investigated these two possibilities in more detail.
If co-translational assembly is the dominant mechanism, our earlier
observation of a post-translational assembly route could be due to a
detergent sensitivity of the initial assembly steps. To avoid this, we
reduced the solubilization time to 5 min while still achieving complete
solubilization of the membrane by homogenizing during solubilization.
In addition we further optimized the analytical sucrose gradient
fractionation earlier used to separate the different assembly steps and
PS II subcomplexes (15). The pH of the solubilization and gradient
buffers was lowered to about pH 6.0, and 0.5 M betaine was
included in the gradients to stabilize the water-soluble proteins
associated with PS II (24). The distribution of PS II subcomplexes in
the sucrose gradients of DM-solubilized thylakoid membranes was further
studied by analyzing the sucrose gradient fractions 3-16 (of 20) on
nondenaturing Deriphat gels, followed by Western blotting analysis
(Fig. 1).
From the D1 immunoblot of Deriphat-PAGE (Fig. 1A, top
panel) and previous analysis (25), it is clear that sucrose
gradient fractions 3-6 contained dimeric PS II cores of about 400 kDa. Fractions 7-9 contained monomeric PS II cores. The assignment of PS II
subcomplexes in the sucrose gradient (Fig. 1B) was based on
the immunoblot analysis, molecular mass calibration (data not shown),
and previous studies (25).
Comparisons of anti-D1 and anti-CP43 immunoblots of Deriphat-PAGE also
revealed CP43-less PS II monomers and dimers (Fig. 1A).
However, the presence of free CP43 in sucrose fractions containing PS
II monomer and dimer suggest that CP43-less PS II core complexes were
at least partially due to dissociation of CP43 from PS II complexes
during the run of Deriphat gels.
Analysis of pulse labeled thylakoids (2.5-min pulse and no chase) by a
combination of the optimized sucrose gradient fractionation and
nondenaturing Deriphat-PAGE (Fig. 2)
revealed a rapid assembly of nearly all newly synthesized proteins into
PS II subcomplexes. Radiolabeled proteins appeared predominantly in PS
II reaction center complexes (fractions 10 and 11) and in PS II
monomers with and without CP43 protein (fractions 7-9) (Fig.
2A). Importantly, only traces of unassembled radiolabeled
proteins were detected (fractions 12-16).
SDS-PAGE analysis of the same sucrose gradient fractions (Fig.
2B) showed that the precursor and mature D1 proteins ((p)D1) were the major radiolabeled proteins. After this short pulse, about
95% of all radiolabeled (p)D1 proteins were found to be incorporated
into PS II reaction centers and monomers. Thus, with our optimized
procedures only minor amounts of free newly synthesized (p)D1 proteins
could be detected in fractions 12-15 (Fig. 2), in contrast to our
earlier observations of a significant transient population of
unassembled D1 protein (16). Therefore, it is likely that D1 assembly
into PS II occurs in a co-translational manner via detergent-sensitive
assembly stages.
Despite our improved protocols, stable assembly of D2 and CP47 still
did not occur in our studies (Fig. 2B) and is therefore likely to depend on newly synthesized nuclear-encoded factors or
biosynthesis of cofactors. Without exception, however, some of the
newly synthesized CP43 protein was always found assembled into PS II
core monomers (Fig. 2B). It is likely that after replacement of the D1 protein, a newly synthesized CP43 or the one released during
earlier stages of PS II repair cycle (25, 26) can reassociate with PS II.
Direct Evidence for D1 Nascent Chains in PS II
Subcomplexes--
Because nearly all newly synthesized D1 protein was
immediately (after a 2.5-min pulse) found associated with the PS II
reaction center and monomeric complexes, we addressed the question of
whether the nascent D1 protein interacts with other PS II polypeptides already during translation elongation. As a first approach, we searched
for D1 protein synthesis intermediates in PS II monomers and reaction centers.
To allow the accumulation of more radioactivity into the D1 protein and
its translation intermediates, a 5-min chase was applied after a
2.5-min pulse in intact chloroplasts. The autoradiogram of the sucrose
gradient fractions from such thylakoids (Fig.
3A) revealed radiolabeled
polypeptides of about 25 kDa concentrated in PS II monomers and
reaction centers (fractions 8-11). The labeled polypeptides of 17 and
22 kDa were mostly concentrated in fractions 12-15 and were thus
mainly present as unassembled polypeptides.
Direct immunoprecipitation of sucrose gradient fractions with D1
antiserum produced, in addition to the mature D1 protein, the 25-kDa
and the 17-kDa labeled polypeptides in PS II monomers and PS II
reaction centers (fraction 8-11) (Fig. 3B). However, the
17- and 25-kDa polypeptides as well as the full-length D1 protein in
fractions 9-11, but not in fractions 12-14, were also efficiently
co-immunoprecipitated with the D2 antibody under the nondenaturing
conditions of sucrose gradients (Fig. 3B). Full-length labeled D2 protein could be immunoprecipitated from sucrose fractions 12-14 with the D2 antibody but not with D1 antibody, indicating that
indeed newly synthesized D2 was not assembled with D1.
To determine that the 25- and 17-kDa labeled polypeptides were indeed
D1 intermediates, the PS II reaction center and monomer complexes were
cut out from Deriphat-PAGE, subsequently separated by SDS-PAGE, and
subjected to Western blotting analysis. Both the 25- and 17-kDa
polypeptides were immunodetected with the N-terminal D1 antibody (Fig.
3C), indicating that they must represent the N-terminal
fragments of the D1 protein. No cross-reaction of the 25- and 17-kDa
polypeptides with the D2 antiserum was detected (data not shown).
Analysis of Membrane-bound Ribosome Nascent Chain
Complexes--
In a second approach, we searched for further direct
evidence of co-translational interaction of D1 nascent chains with
other PS II core proteins. To this end, ribosome nascent chain
complexes were isolated, and immunoprecipitation was carried out with
D1-, D2-, and CP47-specific antibodies.
Fig. 4A shows the
autoradiogram of the immunoprecipitated products from ribosome nascent
chain pellets solubilized with SDS. Different intermediates of the D1
protein (15-30 kDa) could be immunoprecipitated with the N-terminal D1
antibody. Immunoprecipitation with the D2 antibody did not precipitate
the 17-kDa intermediate efficiently but to our surprise precipitated
the 25-kDa polypeptide. Western blotting of this 25-kDa polypeptide
indicated that indeed the 25-kDa polypeptide is a D1 protein
intermediate (Fig. 4A). Furthermore, these labeled
polypeptides first precipitated with the D2 antibody were efficiently
reimmunoprecipitated with the N-terminal D1 antibody (Fig.
4A). It should be noted that the D2 antibody shows no
cross-reactivity with the D1 protein or vice versa.
Interestingly, the 17-kDa (as well as the 25-kDa) labeled D1 nascent
chain could be efficiently co-immunoprecipitated with the D2 antibody
from the ribosome pellets solubilized with nonionic detergent DM (Fig.
4B). Likewise, different intermediates of the D1 protein
could also be co-immunoprecipitated with the CP47 antibody (Fig.
4B). However, as shown in Fig. 4A, incubation of
ribosome nascent chain complexes with CP47 antibody under denaturing
solubilization conditions did not result in the precipitation of any
labeled polypeptides.
In this study we have addressed the initial assembly steps of the
D1 protein into the PS II complex. The high natural turnover of the D1
protein and the experimental possibility to preferentially label the D1
protein during in organello translations provides a unique
model system to address the relation between translation, insertion,
and assembly.
Because the elongation of the D1 protein in isolated chloroplasts is
completed within 10 min (15), we applied only short (2.5 min) pulse
labeling to trap the newly synthesized D1 protein immediately after
termination of translation. Despite the short pulse, most of the
labeled thylakoid proteins, which almost exclusively consisted of the
precursor and mature forms of the D1 protein, had already been
incorporated into PS II reaction center and PS II core complexes. Not
even a transient pool of free D1 protein could be trapped in the
thylakoid membrane. This strongly suggests that the elongation and
assembly processes of the D1 protein are tightly coupled.
Labeled D1 synthesis intermediates were frequently found in gradient
fractionated DM-solubilized thylakoids. The 17- and 22-kDa intermediates migrated mostly on the top of the gradient as free polypeptides. The main D1 intermediate of 25 kDa, however, was strongly
associated with PS II subcomplexes, suggesting that the D1 protein
already during translation elongation interacts with other PS II core
components (Fig. 3). Moreover, this interaction is likely to be tight
because it was not disrupted during the sucrose gradient centrifugation.
More direct evidence for co-translational assembly of the D1 protein
into PS II was obtained by studying the interaction of D1 nascent
chains with other PS II core proteins in the ribosome nascent chain
complexes. Immunoprecipitation with the N-terminal D1 antibody revealed
D1 intermediates of 15-30 kDa (Fig. 4). The labeled D1 intermediates
in ribosome pellets (and PS II core complexes) could be efficiently
chased into the mature form,2
and they could also be released from ribosomes by
puromycin,3 indicating that
they do represent D1 nascent chains, not the degradation products of
D1. The D1 intermediates corresponded to the ribosome pausing sites in
membrane-bound psbA mRNA that have been mapped through
toe print analysis (10). The 17-kDa D1 synthesis intermediate is likely
to originate from ribosome pausing occurring when two transmembrane
domains (TMs) have been inserted into the membrane, with the third TM
having just emerged from the ribosome tunnel/groove. The 22-kDa D1
intermediate was, as opposed to other studies (10), only faintly
labeled in our experiments. The other major D1 synthesis intermediate
of 25 kDa most likely results from ribosome pausing after four TMs have been translated and inserted into the membrane.
Co-immunoprecipitation of D1 nascent chains from ribosome pellets with
the D2 antiserum strongly supports the idea of co-translational interaction of the D1 nascent chains with the D2 protein (Fig. 4).
After the D1 nascent chain has started insertion into the thylakoid
membrane during its early synthesis, interaction with the D2 protein
becomes obvious after two TMs have been inserted into the membrane
(17-kDa pausing intermediate) (Fig. 5).
This interaction, however, is not very tight and can easily be
disrupted with SDS. With further elongation of the D1 nascent chain to
25 kDa the interaction with the D2 protein becomes more tight and can
no longer be disrupted by SDS. Incomplete unfolding by SDS is typical
for most hydrophobic membrane proteins (27), and hydrophobic
interactions between TMs have been shown to persist even under
denaturing conditions (SDS) (28). A similar tight protein-protein
interaction, possibly due to the hydrophobicity of both interacting
proteins, has also been observed in other co-immunoprecipitation
studies under denaturing conditions (29). The co-immunoprecipitation of
the 25-kDa D1 nascent chain with the D2 antibody was not due to any
unspecific aggregation of membrane proteins or unspecific
immunoprecipitation, because no D1 intermediates could be precipitated
with D2 preserum and CP47 antiserum under similar denaturating
conditions. Besides, no cross-reaction of the D2 antibody used in our
experiments with the D1 protein has ever been observed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
subunits of
cytochrome b559, and the psbI and
psbW gene products (1). The PS II reaction center binds all
the components necessary for the primary charge separation (2). Oxygen-evolving PS II complexes additionally contain the intrinsic chlorophyll a binding proteins (CP43 and CP47), the oxygen-evolving complex, and several small proteins of unknown function (3). Recent
structural studies indicate that the PS II core complexes can exist as
a dimer (4).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1), carrier-free
35S-labeled methionine was added in a final concentration
of 0.5 µCi/µl, and chloroplasts were pulse labeled for 2.5 min,
followed by an additional 5-min chase in the presence of 10 mM unlabeled methionine where indicated. The translation
was stopped by adding a 10-fold volume of ice-cold lysis buffer (7 mM MgOAc, 118 mM KOAc, 46 mM
Hepes-KOH, pH 7.6). Subsequently the thylakoids were washed twice in 5 mM MgCl2, 10 mM NaCl, 25 mM Mes-NaOH, pH 6.0 (Medium B). A mixture of protease
inhibitors (antipain (2 µg/ml), leupeptin (2 µg/ml),
phenylmethylsulfonyl fluoride (100 µg/ml)) was added to all solutions.
-D-maltoside
(DM). Homogeneous solubilization was ensured and accelerated by using a
Potter homogenizer. Subsequently the suspension was loaded on a linear
sucrose gradient of 11 ml (5-35% sucrose in 5 mM
MgCl2, 10 mM NaCl, 0.5 M betaine, 0.03% DM, and 25 mM Mes-NaOH, pH 5.7) made with Gradient
MasterTM (model 106, Biocomp Instruments, Inc., New
Brunswick, Canada) and spun for 26 h at 180 000 × g at 4 °C. After centrifugation, the sucrose gradients
were divided into 20 fractions of equal volume using a Piston Gradient
Fractionator (model 151, Biocomp Instruments, Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Distribution of PS II complexes in
sucrose gradients. A, immunoblot analysis of sucrose
gradient fractions after Deriphat-PAGE. The thylakoid membranes were
solubilized in 1% DM at 0.5 mg chlorophyll/ml for 5 min on ice and
loaded onto 5-35% sucrose gradients. Sucrose gradients were
centrifuged for 26 h at 180,000 × g and divided
into 20 fractions of equal volume. Fractions 3-16 (of 20) were run on
Deriphat-PAGE and immunodetected with D1 (upper panel) and
CP43 antisera (lower panel). B, assignment of PS
II subcomplexes in the sucrose gradient. The assignment was based on
the immunoblot analysis (A), molecular mass calibration
(data not shown), and previous studies (15). RC, reaction center.
View larger version (48K):
[in a new window]
Fig. 2.
Incorporation of
[35S]methionine into thylakoid protein complexes. A
short 2.5-min pulse was carried out in intact chloroplasts. After
translation, chloroplasts were lysed, and the thylakoid membranes were
washed, solubilized in 1% DM for 5 min on ice, and centrifuged on a
sucrose gradient. After fractionation, the thylakoid protein complexes
in fractions 3-16 (of 20) were separated by Deriphat-PAGE
(A) and SDS-PAGE (B) and visualized by
autoradiography. Quantification of the radioactivity in the D1 protein
is indicated at the bottom of the autoradiogram. RC,
reaction center.
View larger version (49K):
[in a new window]
Fig. 3.
Direct evidence for D1 intermediates in PS II
subcomplexes. Translation in intact chloroplasts was carried out
for 2.5 min followed by a 5-min chase with cold methionine. After
translation, chloroplasts were lysed, solubilized, and spun on a
sucrose gradient. A, autoradiogram of the newly synthesized
thylakoid membrane proteins after sucrose gradient fractionation.
Fractions 7-16 were separated by SDS-PAGE. B, autoradiogram
of sucrose gradient fractions immunoprecipitated with the D1 or D2
antibody. Sucrose gradient fractions 7-14 were immunoprecipitated with
the antisera raised against the N-terminal residues (58-86) of the D1
protein and the residues 230-245 of the D2 protein. The precipitated
products were separated by SDS-PAGE. C, D1 immunoblot of PS
II subcomplexes. Sucrose gradient fractions 8-11 were concentrated and
run in Deriphat-PAGE. The PS II monomer and reaction center complexes
on Deriphat-PAGE were cut, rerun in SDS-PAGE, and immunodetected with
the antiserum raised against the N-terminal residues (58-86) of the D1
protein.
View larger version (49K):
[in a new window]
Fig. 4.
Interaction of D1 intermediates with other PS
II core proteins in ribosome pellets. After a 2.5-min pulse in
intact chloroplasts, the thylakoids were isolated and solubilized in
2% polyoxyethylene 10 tridecyl ether for 10 min on ice.
Thylakoid-bound polysomes were then isolated by centrifugation through
a 1.0 M sucrose cushion. A, autoradiogram of
immunoprecipitated products from SDS-solubilized ribosome pellets. The
ribosome nascent chain complexes were solubilized by SDS and
immunoprecipitated with the antisera against the D1, the D2, or the
CP47 protein and with the respective presera (panel on the
left). The identity of the labeled polypeptides precipitated
with the D2 antiserum was verified by Western blotting with the
N-terminal D1 antiserum and with the D2 antiserum (panel on
the right). Also, the products precipitated by the D2
antiserum were released in the sample buffer, diluted, and
reprecipitated with the antiserum raised against the N-terminal
residues of the D1 protein (panel in the middle).
B, autoradiogram of immunoprecipitated products from
DM-solubilized ribosome pellets. The ribosome nascent chain complexes
were solubilized in DM, and immunoprecipitation was performed as
described for panel A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (29K):
[in a new window]
Fig. 5.
Model for the proposed steps in
co-translational assembly of the D1 protein into PS II.
I, insertion of the elongating D1 protein into the thylakoid
membrane. The D1 protein is inserted into the thylakoid membrane by a
yet unknown mechanism. II, the D1 nascent chain of 17 kDa
has started interacting with other PS II core proteins (D2 or D2-CP47).
At this stage, the interaction between D1 nascent chain and the D2
protein can be easily disrupted. III, tight interaction of
D1 nascent chain of 25 kDa with the D2 protein. The interaction between
D1 nascent chain and the D2 protein at this stage cannot be disrupted
under denaturing conditions (SDS). Based on immunoprecipitation
experiments, CP47 is located adjacent to the D2 protein. IV
and V, reassociation of CP43 into PS II monomers. After
termination of D1 translation, a newly synthesized CP43, or the one
earlier released, reassociates with PS II. Reassociation may accelerate
the C-terminal processing (arrow) of the D1 protein or vice
versa.
Although the nascent D1 chains seems to interact co-translationally with the D2 protein, it is not known whether a translocon channel (i.e. SecY) is involved during D1 synthesis and membrane insertion. Studies on translocation of membrane proteins have indicated that the folded TMs can laterally exit the translocon channel and enter the lipid environment either before or after termination of translation (30-32). A possible lateral exit of TMs during synthesis might be important in repair of photodamaged PS II centers when only the D1 protein has to be replaced by de novo synthesis.
Structural studies have revealed close interaction between TM D and E of the D1 and D2 proteins, which may account for the tight association of the 25-kDa D1 nascent chain with the D2 protein. Moreover, most of the ligands for pigments and cofactors reside in the amino acid residues in the D and E helices of the D1 and D2 proteins (see Ref. 33). Therefore, the role of ribosome pausing is probably not only limited to D1 protein folding and assembly during the synthesis but may also ensure the co-translational ligation of cofactors to PS II under repair as suggested by Kim et al. (10).
Co-immunoprecipitation of D1 nascent chains under nondenaturing conditions occurred not only with the D2 antibody but also with CP47 antiserum, suggesting that CP47 may also interact with the synthesizing D1 protein during the repair process or, more likely, that D1 can assemble into a complex consisting of both the D2 protein and CP47. Structural studies of PS II core locate CP43 and CP47 on opposite sides of the D1/D2 reaction center (4, 34). Positioning of these two proteins adjacent to the D1 or the D2 protein is, however, not clear from the structural data (35). Our present results on co-immunoprecipitation of D1 nascent chains with the D2 and the CP47 antisera lend strong support for a close association of D2 and CP47. CP43, on the other hand, is easily released from PS II during the repair process (25, 26) to make the replacement of the damaged D1 protein possible.
Taken together, our results indicate that during the repair of PS II,
the assembly of the newly synthesized D1 protein into PS II occurs
co-translationally and the nascent D1 chains start interacting with the
D2 protein already during translation elongation. At this stage, CP47
may also be associated with the D2 protein, whereas CP43 reassembles
with the PS II core complex only after termination of D1 translation.
![]() |
ACKNOWLEDGEMENTS |
---|
The antibodies against the D1 protein and CP47 were kindly provided by Drs. J. E. Boynton and R. Barbato, respectively.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Academy of Finland, Nordiskt Kontaktorgan för Jordbruksforskning, and the Carl Trygger Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biology, University of Turku, Lab Plant Physiology, Biocity A, 6th Floor, FIN-20520 Turku, Finland. Tel.: 358-2-3338079; Fax: 358-2-3338075; E-mail: evaaro{at}utu.fi.
2 L. Zhang, V. Paakkarinen, K. J. van Wijk, and E.-M. Aro, manuscript in preparation.
3 L. Zhang, V. Paakkarinen, K. J. van Wijk, and E.-M. Aro, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PS II, photosystem
II;
DM, n-dodecyl -D-maltoside;
PAGE, polyacrylamide gel electrophoresis, TM, transmembrane domain;
Mes, 4-morpholineethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|