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J. Biol. Chem., Vol. 276, Issue 41, 37809-37814, October 12, 2001
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From the Department of Biology, University of Turku,
FIN-20014 Turku, Finland
Received for publication, June 15, 2001, and in revised form, July 24, 2001
We have used the photosystem II reaction
center D1 protein as a model to study the mechanisms of targeting and
insertion of chloroplast-encoded thylakoid membrane proteins. The
unusually high turnover rate and distinct pausing intermediates during
translation make the D1 protein biogenesis particularly suitable for
these purposes. Here we show that cpSecY, a chloroplast homologue of bacterial essential translocon component SecY, interacts tightly with
thylakoid membrane-bound ribosomes, suggesting its involvement in
protein translocation and insertion. Co-immunoprecipitation and
cross-linking experiments indicated that cpSecY resides in the vicinity
of D1 elongation intermediates and provided evidence for a transient
interaction of cpSecY with D1 elongation intermediates during the
biogenesis of D1. After termination of translation, such interactions
no longer existed. Our results indicate that, in addition to a well
characterized role of cpSecY in posttranslational translocation of
nuclear-encoded proteins, it seems to be also involved in
cotranslational membrane protein translocation and insertion in chloroplasts.
The thylakoid membrane consists of four major protein complexes
that function in electron transfer to produce NADPH and ATP. Approximately 50% of the thylakoid membrane proteins are encoded by
the chloroplast genome, and the other half by the nuclear genome. The
biogenesis of chloroplast thylakoid membranes, therefore, requires
coordinated synthesis and assembly of both the nuclear-encoded and
chloroplast-encoded proteins. Nuclear-encoded thylakoid proteins, synthesized in the cytoplasm, translocate into chloroplast stroma across the general import pathway at the outer and inner envelope membranes (1, 2). Posttranslational insertion or translocation of the
imported proteins across the thylakoid membrane has been studied
intensively and shown to follow at least three distinct routes (2-4).
On the other hand, the mechanisms of targeting and insertion of
chloroplast-encoded proteins into the thylakoid membrane are largely
unknown despite their central role in all thylakoid protein complexes.
Since chloroplasts have evolved from free-living prokaryotic
cyanobacteria, it is likely that the protein transport pathways of
chloroplast-encoded proteins show similarities to those in bacteria.
The essential core of the bacterial translocase is composed of SecYEG
together with SecA (5-9). SecYEG forms a tetrameric transmembrane
channel through which the protein is exported (8, 9), and SecA is a
preprotein-stimulated ATPase (10). Biochemical studies have revealed
that a reversible membrane insertion and deinsertion of SecA at the
SecYEG complex, coupled with SecA ATP binding and hydrolysis, pushes
the translocating preprotein through the membrane channel (11).
Polytopic bacterial membrane proteins were, however, recently reported
not to require SecA for targeting, but are instead dependent on a
homologue of eukaryotic signal recognition particle
(SRP)1 for integration into
the inner membrane (12, 13). Although the SRP-dependent
insertion into the membrane and the SecA-mediated translocation of
secretory proteins across the inner membrane remain mechanistically
distinct, they seem to converge at the SecY translocase, yet engaging
different domains of SecY and different components for translocation
(13). A homologue of both the bacterial SecY and of the eukaryotic
endoplasmic reticulum Sec61p translocon protein has been identified in
chloroplasts (cpSecY) and localized to the thylakoid membrane (14).
cpSecY has been shown to be required for translocation of
nuclear-encoded Sec pathway substrates across the thylakoid membrane;
antibodies raised against cpSecY blocked the Sec-dependent
translocation, but not the delta pH-dependent or
SRP-dependent protein translocation (15, 16). Moreover, the
cpSecY null mutant of maize has been shown to exhibit a severely reduced thylakoid membrane network (17), suggesting a pivotal role for
cpSecY in thylakoid biogenesis.
By analogy with the bacterial export system, cpSecY is an interesting
candidate for a translocon component mediating the translocation and
insertion of chloroplast-encoded proteins into the thylakoid membrane.
So far, no such function has been established for cpSecY, nor has any
functional interaction of cpSecY with the 70 S ribosomes/nascent polypeptides been demonstrated. To elucidate the possible involvement of cpSecY in targeting and insertion of chloroplast-encoded proteins into the thylakoid membrane, we have used the biogenesis of the D1
reaction center protein of photosystem II (PS II) as a model system.
The D1 protein undergoes light-dependent damage and
degradation and is thus the most abundantly synthesized protein in
mature chloroplasts. In order to maintain PS II function, the D1
protein is constantly turning over and assembled into PS II even under normal growth conditions (18). The D1 protein has five transmembrane domains with its N terminus in the stroma and the lumenal loops are
composed of approximately 50 and 30 amino acid residues, respectively. The D1 protein, encoded by the plastid psbA gene, is
synthesized on thylakoid membrane-bound ribosomes and cotranslationally
inserted into the thylakoid membrane. Moreover, ribosomes have been
shown to pause at specific sites during translation of membrane-bound psbA mRNA (19, 20). The observation of an interaction of
such D1 elongation intermediates with the D2 protein, another PS II reaction center protein (20), points to a cotranslational assembly of
the newly synthesized D1 protein with other PS II proteins, thus
ensuring fast and efficient restoration of PS II function during the
repair process. The molecular mechanisms of the insertion of the D1
protein, and the other chloroplast-encoded proteins, into the thylakoid
membrane, however, have remained unknown. Here we demonstrate the
involvement of cpSecY in the insertion of the D1 protein into the
thylakoid membrane and indicate a functional interaction between cpSecY
and the chloroplast ribosomes.
Plant Materials--
Spinach (Spinacea oleracea) was
grown hydroponically at 23 °C with a 10-h photoperiod at a photon
flux density of 300 µmol·m Isolation of Intact Chloroplasts and in Vitro
Translation--
Intact chloroplasts were isolated in a Percoll
gradient, and translation in vitro in intact chloroplasts
was performed essentially according to Ref. 21, with modifications as
described in Refs. 20 and 22. After a 5-min preincubation (0.5 mg of
chlorophyll·ml Isolation of Ribosome-Nascent Chain Complexes (RNCs)--
Intact
spinach chloroplasts were lysed and thylakoids solubilized with 1%
(w/v) dodecyl- Release of cpSecY from Ribosomes--
RNCs were further
solubilized in 1% DM together with different concentration of KOAc
(0.05, 0.5, and 1.0 M KOAc) in the presence or absence of 1 mM puromycin. After incubation first on ice for 10 min and
then at 30 °C for 20 min, the samples were loaded onto a 300-µl
sucrose cushion in the solubilization buffer and centrifuged for 1 h at 270,000 × g at 20 °C.
Subfractionation of the Thylakoid Membrane--
Subfractionation
of the thylakoid membrane was performed principally as described in
Ref. 23.
Blue Native (BN)-PAGE, SDS-PAGE, and Immunoblot Analysis--
BN
gel (5-12%) of RNCs solubilized in 1% DM was carried out as
described in Ref. 24. After electrophoresis, the lane of separated
protein complexes was excised from the gel and immersed in the sample
buffer containing 5% mercaptoethanol for 30 min at room temperature
before the second dimension SDS-PAGE (25), using 15% acrylamide gels
with 6 M urea. Western blot analysis was performed by
standard techniques using chemiluminescence for detection. Extraction
of proteins from specific bands in the BN gel was performed by
incubating the band of interest overnight with 0.1% SDS, 10 mM DTT, and a mixture of protease inhibitors.
Cross-linking--
After 2.5-min pulse labeling in intact
chloroplasts, RNCs were isolated, suspended in 250 mM
sucrose, 50 mM Hepes-KOH, pH 7.4, 5 mM MgOAc,
50 mM KOAc, and centrifuged for 2 min at 5000 × g to remove any aggregated material. Cross-linking reactions were then performed at 25 °C for 30 min by adding the
hetero-bifunctional cleavable cross-linker
N-succinimidyl-3-[2-pyridyldithio]propionate (SPDP)
(sulfhydryl/amine), to a final concentration of 1 mM. The samples were solubilized with SDS before being subjected to immunoprecipitation.
Immunoprecipitation--
For denaturing immunoprecipitation, the
samples were solubilized in 1% SDS, 15 mM DTT, 50 mM Tris-HCl, pH 7.5, and a mixture of protease inhibitors,
and were subsequently diluted with four volumes of 50 mM
Tris-HCl, pH 7.5, containing 150 mM NaCl, 1% Triton X-100,
and 1 mM EDTA. For native immunoprecipitation, the samples
were solubilized in 1% DM, 150 mM NaCl, 50 mM
Tris-HCl, pH 7.5, and a mixture of protease inhibitors. After
solubilization, an antibody cross-linked to Protein A-Sepharose CL-4B
beads was added, and the samples were incubated at room temperature for 2 h. The Sepharose beads were then washed four times with 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, once with 50 mM
Tris-HCl, pH 7.5, and subsequently the bound antigen was released in
the sample buffer. For autoradiography, gels were stained, dried, and
exposed to film. Antibodies raised against the N-terminal residues
(58-86) of the D1 protein (Research Genetics), and against the 18 C-terminal residues of spinach cpSecY (Research Genetics) were used for
immunoprecipitations in this study.
Presence of D1 Elongation Intermediates in
RNCs--
Characteristic of D1 translation elongation is the pausing
of the thylakoid membrane-bound ribosomes at specific sites (19, 20).
To follow D1 translation elongation and concomitant translocation and
insertion into the thylakoid membrane, we isolated RNCs and quantitatively immunoprecipitated the D1 nascent chains with an excess
of D1 antibody. As presented in Fig.
1A, after 2.5 min of pulse
labeling in intact chloroplasts, the D1 intermediates of 17, 22, and 25 kDa were detected in isolated RNCs. By the end of the 5-min chase, D1
elongation was nearly completed and most of the radioactivity had
disappeared from D1 elongation intermediates (particularly from that of
17 kDa) in RNCs (Fig. 1A). At this point the label had been
chased into the full-length D1 protein, which had released from
ribosomes and was detected in the thylakoid membrane (Fig.
1B). This indicates that the labeled and immunoprecipitated D1 intermediates are indeed linked to biosynthetic ribosomes, and thus
represent true translation intermediates. Considering the
cotranslational insertion of the D1 protein into the thylakoid membrane, these intermediates can also be regarded as translocation intermediates. The high natural turnover of the D1 protein together with distinct pausing intermediates during in organello
translation makes the D1 protein biogenesis a convenient model system
to examine specific protein-protein interactions while being translated
on thylakoid membrane-bound ribosomes, translocated across the
membrane, and integrated into the lipid bilayer.
Association of cpSecY with Chloroplast Ribosomes--
Antibody
raised against the 18 C-terminal amino acids of spinach cpSecY (26)
recognized one protein of ~35 kDa in the thylakoid membrane (Fig.
2A, lane
T). Fractionation of thylakoid membrane revealed the
location of cpSecY almost exclusively in the stromal membranes, with
only traces in grana margins, and with an almost complete absence from
the appressed granal membranes (Fig. 2A). This is consistent
with the observations that stromal membranes are the sites of protein
translocation/integration (27-29). Western blot analysis further
demonstrated that a portion of cpSecY (~10%) in mature leaves is
constantly recovered from the thylakoid membrane-bound RNCs (Fig.
2A, lane P). As the synthesis of
chloroplast-encoded thylakoid membrane proteins takes place on
thylakoid membrane-bound ribosomes, it was intriguing to test whether
such co-location means an involvement of cpSecY in chloroplast-encoded
protein biogenesis.
We next tested the interactions between ribosomes and cpSecY to reveal
whether the ribosome binding of cpSecY is occurring solely via its
interaction with nascent polypeptides, or whether there is a distinct
interaction between cpSecY and the ribosomes. As a control, we tested
that nascent D1 polypeptides, associated with ribosomes in chloroplast
homologous translation system (30), were completely released by
puromycin (data not shown). Addition of puromycin to RNCs, however, did
not release cpSecY (Fig. 2B), indicating that the release of
nascent polypeptides from ribosomes is not sufficient for detachment of
cpSecY from the ribosomes. Incubation of RNCs with high salt, either
alone or together with puromycin, induced almost complete release of
cpSecY from ribosomes; this treatment is known to induce a dissociation
of ribosomes (31), and this was a prerequisite for a release of cpSecY.
A similar tight interaction with ribosomes is known for the main translocation component Sec61p in the endoplasmic reticulum (32).
Interaction of cpSecY with D1 Intermediates--
To elucidate the
possible interaction of cpSecY with D1 elongation intermediates,
isolated RNCs were first purified in BN gel, allowing a release of
contaminating protein complexes. After the first-dimensional separation
in the presence of Coomassie G-250 dye, the separated protein complexes
were further subjected to denaturing SDS-PAGE and silver staining (Fig.
3A). The proteins of various
thylakoid membrane complexes were identified from the similar gels by
Western blot analysis with distinct antibodies (Fig. 3B).
From the immunoblots, the positions of monomeric and dimeric cytochrome
b6/f complexes in the BN gel,
migrating at approximately 120 and 240 kDa, respectively, could be
deduced (Fig. 3, A and B). CP43-less and intact
PS II cores at approximately 250 kDa were also present, as indicated by
immunoblots with anti-D1 (Fig. 3, A and B) and
anti-CP43 (data not shown). Contaminating thylakoid protein complexes
were efficiently released from RNCs by BN gel separation, as indicated
by immunoblots with anti-D1 and anti-cytochrome f.
Most importantly, anti-cpSecY recognized two distinct complexes in the
BN gel (Fig. 3, A and B): one migrating at
approximately 100 kDa and the major one at approximately 900 kDa, both
clearly resolved in the separating gel. The major cpSecY complex in
RNCs was distinct from other thylakoid protein complexes, and
co-migrated in the BN gel with ribosomes, as evidenced by
immunodetection with anti-ribosome L21 (data not shown).
The major protein complex containing most of cpSecY together with
ribosomes in the BN gel was extracted from the gel and subjected to
immunoprecipitation with anti-D1. The D1 intermediates of different lengths were efficiently precipitated from this cpSecY-ribosome complex
(Fig. 3C, indicated by filled stars).
This result clearly demonstrates that both the D1 nascent polypeptides
and the cpSecY complex are tightly associated with ribosomes.
To gain further insight into the interaction of D1 with cpSecY, the
DM-solubilized thylakoid membrane was sedimented through a sucrose
cushion and then immunoprecipitation was performed separately from the
supernatant sucrose cushion and from the RNC pellets under denaturing
conditions with anti-D1. As shown in Fig.
4A (lane
2), a large quantity of the full-length D1 could be
immunoprecipitated from the supernatant sucrose cushion (Fig.
4A, lane 1) with anti-D1 but no D1
intermediates were precipitated. D1 elongation intermediates, on the
other hand, were present in RNC pellets (Fig. 4B,
lane 1). To test the interaction and possible
functional role of cpSecY during D1 protein biogenesis, the supernatant
sucrose cushion and RNC pellets were first subjected to
immunoprecipitation under non-denaturing conditions with anti-cpSecY
(co-precipitation), followed by precipitation under denaturing
conditions with anti-D1 (Fig. 4, A (lane
3) and B (lane 2)) and
anti-cpSecY (Fig. 4, A (lane 4) and
B (lane 3)). The labeled full-length
D1 protein was not co-precipitated with anti-cpSecY (Fig.
4A, lane 3), ruling out the
interaction of full-length D1 with cpSecY. As presented in Fig.
4B (lane 2), the labeled D1
intermediates in RNCs could be co-precipitated with the cpSecY
antibody. This result indicates that cpSecY was in the vicinity of D1
intermediates during the elongation process.
Further evidence for the interaction between D1 elongation
intermediates and cpSecY was obtained by subjecting the RNCs to a
cleavable cross-linker (SPDP), followed by solubilization with SDS and
immunoprecipitation with anti-cpSecY. Precipitated cross-linked products were then cleaved with a reducing agent (DTT) and subjected to
second immunoprecipitation under denaturing conditions (Fig. 5). D1 elongation intermediates of 17, 22, and 25 kDa were precipitated with anti-D1, but not with anti-cpSecY
(Fig. 5, lanes 1 and 2). Indeed, the
D1 elongation intermediates can be cross-linked to cpSecY. Incapability
of anti-cpSecY to immunoprecipitate labeled D1 intermediates after
solubilization of RNCs with SDS indicates the specificity of the
cross-linking and immunoprecipitation procedures (Figs. 4 (A
(lane 4) and B (lane
3)) and 5 (lane 3)).
The mechanisms of targeting and cotranslational insertion of
chloroplast-encoded proteins into the thylakoid membrane are poorly
understood, largely due to the lack of suitable systems to dissect the
different subprocesses involved. Recent studies on D1 protein
biogenesis have, however, advanced our understanding of the mechanisms
concerning the translation, insertion and assembly processes (20, 22).
In this study we therefore used D1 protein biogenesis as a model to
address the mechanism of cotranslational insertion of
chloroplast-encoded proteins into the thylakoid membrane. The
observation of functional interaction of cpSecY with chloroplast 70 S
ribosomes first suggested an involvement of cpSecY in the process (Fig.
2). More direct evidence for a transient interaction of cpSecY with D1
translocation intermediates was obtained by immunoprecipitation and
cross-linking experiments with isolated RNCs (Figs. 3-5). After
termination of translation, such interactions no more existed (Fig. 4),
pointing to a specific involvement of cpSecY in cotranslational
insertion of chloroplast-encoded D1 protein into the thylakoid membrane.
Two populations of cpSecY complexes are apparently present in thylakoid
membranes (Fig. 3). One population of approximately 100 kDa detected by
the BN gel was consistent with the observation by gel filtration
analyses (16). This cpSecY complex is likely to be involved in the
posttranslational translocation of nuclear-encoded Sec-dependent substrates across the thylakoid membrane (15, 16). A novel cpSecY population was found to be associated with ribosomes (Fig. 3). Importantly, this portion of cpSecY was interacting with D1 elongation intermediates and most probably forms the
translocation channel where the chloroplast-encoded proteins integrate
into the membrane.
It was recently shown that cpSRP54, a chloroplast homologue of
bacterial SRP, interacts and can be efficiently cross-linked to the D1
nascent chains (33). Thus, in addition to the role in posttranslational
targeting (34, 35), cpSRP54 probably also functions in cotranslational
targeting of the chloroplast-encoded D1 protein into the cpSecY
translocon in the thylakoid membrane. It is reasonable to assume that
cpSRP54 starts to interact with the nascent D1 chain when it emerges
from the ribosome tunnel and targets the nascent chain to the cpSecY
translocon channel. Ribosomes and the cpSecY translocase form a large
complex of approximately 900 kDa, which clearly penetrates to the BN
separation gel (Fig. 3). It is conceivable that cpSecY exists in two
different complexes, one functioning in posttranslational
SecA-dependent translocation (15, 16) and the other
interacting with ribosomes in the cotranslational translocation and
insertion process. This further suggests that the
Sec-dependent posttranslational translocation and
SRP-dependent cotranslational protein insertion into the
thylakoid membrane might converge at the cpSecY translocase. Similar
convergence has already been demonstrated in yeast where Sec61p, a
homologue to SecY, is a component of two different translocons, one of
which is involved in posttranslational protein translocation, the other in cotranslational translocation (36).
It is known that the translocation of nuclear-encoded proteins into or
across the thylakoid membrane takes place via a number of different
pathways (2-4). Do the chloroplast-encoded proteins also use several
translocation pathways, or is it more likely that they share only one
common translocation apparatus? There is not much experimental evidence
to answer this question. However, mutations in the cytochrome
f signal sequence that prevented the translocation of this
chloroplast-encoded protein also interfered with the translocation of
D1 (37). These results point to a possibility of D1 and cytochrome
f sharing at least one common component for translocation.
This component should be found either in the targeting or in the
translocation machinery. Targeting of cytochrome f, however,
seems to use the SecA-dependent mechanism as deduced from
chloroplast SecA deletion mutant analyses (38) and insertion
experiments through biochemical reconstitutions (39, 40). Accordingly,
it is likely to be the chloroplast cpSecY that is common for the two
targeting systems, the chloroplast-encoded SRP-dependent D1
insertion and SecA-mediated cytochrome f insertion. From an
evolutionary point of view, it is interesting to note that in
Escherichia coli the SRP-dependent
insertion and the SecA-mediated translocation have been shown to
converge at the SecY translocase (12, 13).
A distinct portion of cpSecY is tightly associated with membrane-bound
ribosomes even after solubilization of the thylakoid membrane (Fig. 3).
This interaction is not occurring primarily via the nascent chain, as
evidenced by the stability of the ribosome-cpSecY interaction even in
the presence of puromycin (Fig. 2B). Strong interaction of
cpSecY with chloroplast ribosomes resembles that observed in
E. coli inner membrane and in eukaryotic
endoplasmic reticulum membranes, and thus suggests that the
cotranslational translocation mechanism is highly conserved (6, 7, 32). The nature of the tight interaction between cpSecY and ribosomes is as
yet not clear. Recently it was shown in E. coli
that the 28/23 rRNA of the large ribosome subunits participates in the early stage of ribosome-SecY interaction (41).
Earlier studies on D1 protein biogenesis have revealed that the
translation intermediate of 25 kDa is interacting strongly with D2, the
other reaction center protein of PS II, whereas only loose interaction
with D2 was observed for the 17-kDa D1 nascent chain (20). It is
therefore conceivable that, after targeting and initial interaction
with cpSecY in the thylakoid membrane, the transmembrane domains of the
nascent D1 chains exit laterally from the translocon and start
interacting with other PS II core proteins. The cpSecY translocon thus
seems to play an active role in directing the D1 nascent chains to
their site of function in PSII. This seems not to be unique for the D1
protein and the assembly of the multiprotein PS II complex in
chloroplasts. Indeed similar observations have been made on bacterial
membrane protein biogenesis and also on eukaryotic endoplasmic
reticulum membrane, suggesting a lateral release of transmembrane
domains from the translocon before termination of translation (42, 43).
Structural and functional flexibility of the translocon is probably
required for efficient protein folding and assembly during the
biogenesis of multi-protein complexes, such as PS II.
We are grateful to Drs. K. Cline and H. Mori
for their stimulating discussions and for critical reading of the
manuscript. Anti-cytochrome f and anti-ribosome L21 were
generous gifts from Drs. F.-A. Wollman and A. R. Subramanian,
respectively. The other antibodies were kindly provided by Dr. R. Barbato.
*
This work was supported by grants from the Academy of
Finland, INCO-Copernicus (ERB IC 15-CT98-0126), and Nordisk
Kontaktorgan för Jordbruksforskning.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.
Published, JBC Papers in Press, July 25, 2001, DOI 10.1074/jbc.M105522200
The abbreviations used are:
SRP, signal
recognition particle;
BN, blue native;
DM, dodecyl-
A SecY Homologue Is Involved in Chloroplast-encoded D1
Protein Biogenesis*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2·s1. For
all the experiments, fully developed leaves were harvested 1 h
after the lights were turned on.
1) at 23 °C in the light (50 µmol·m2·s1), carrier-free
35S-labeled methionine was added in a final concentration
of 0.5 µCi·µl1. After pulse labeling, which
occasionally was followed by an additional chase in the presence of 10 mM unlabeled methionine, translation was stopped by adding
a 10-fold volume of ice-cold lysis buffer consisting of 50 mM Hepes-KOH, pH 8.0, 5 mM MgOAc, 50 mM KOAc, 250 µg·µl1 chloramphenicol,
0.5 mg·ml1 heparin, 2 mM DTT, and a mixture
of protease inhibitors (100 µg·ml1 Pefabloc, 2 µg·ml1 antipain, and 2 µg·ml1 leupeptin).
-D-maltoside (DM) in the lysis buffer at
0.5 mg of chloroplasts·ml1 for 5 min on ice, and RNCs
were collected by centrifugation through a 1.0 M sucrose
cushion in lysis buffer at 270,000 × g for 1 h (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
D1 protein translation elongation in intact
chloroplasts. After 2.5-min pulse labeling followed by 5-min chase
in the presence of 10 mM unlabeled methionine, the
thylakoid membrane and the thylakoid membrane-bound RNCs were isolated.
The thylakoid membrane-bound RNCs were solubilized with SDS, and D1
elongation intermediates were immunoprecipitated with an excess of
N-terminal D1 antibody. The precipitated products from RNCs
(A) and the labeled thylakoid membrane proteins
(B) were separated by SDS-PAGE and visualized by
autoradiography. The D1 elongation intermediates of 17, 22, and 25 kDa
are indicated as well as the mature (D1) and precursor
(pD1) forms of D1.
View larger version (26K):
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Fig. 2.
Interaction of cpSecY with ribosomes.
A, intact spinach thylakoid membrane (T), stroma
(S), margin (M), and grana (G)
membrane fractions (equivalent to 1 µg of chlorophyll) as well as
RNCs (P) isolated from thylakoid membranes (equivalent to 50 µg of chlorophyll) were separated by SDS-PAGE, and immunodetected
with the antibody raised against the 18 C-terminal amino acid residues
of spinach cpSecY. B, release of cpSecY from ribosomes. RNCs
were suspended in different KOAc conditions in the presence and absence
of puromycin and centrifuged through a sucrose cushion. The pellet
(Pe) and supernatant (Su) fractions were analyzed
by immunoblotting with anti-cpSecY.
View larger version (41K):
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Fig. 3.
Analysis of RNC preparation by a BN gel.
A, two-dimensional resolution of protein complexes in RNC
preparation. RNCs were solubilized with 1% DM and separated by a BN
gel on a acrylamide gradient of 5-12%. The designations on the top of
the BN separation gel indicate the molecular masses of standards
(kDa). A lane of a BN gel was run in a second dimension on a
15% SDS-urea-PAGE and silver-stained. Designations for the identities
of protein complexes, as resolved by the first dimension BN gel, are
given at the bottom of the silver-stained gel. Broken
lines indicate the protein complexes, which can be detected
by immunblotting against specific proteins (see B), but
which are invisible in the silver-stained gel. B,
representative immunoblots from a second dimensional SDS-PAGE of RNCs.
Horizontal strips of immunoblots with anti-cytochrome f,
anti-D1, and anti-cpSecY are shown. C, association of cpSecY
and nascent D1 chains with ribosomes. After a short pulse-labeling of
2.5 min in intact chloroplasts, RNCs were isolated and purified in a BN
gel. The proteins, extracted from the high molecular mass complex
containing cpSecY and ribosomes, were subjected to immunoprecipitation
with anti-D1. Filled stars indicate D1 nascent
chains of 14, 17, 22, and 25 kDa. Molecular mass markers (kDa) are
indicated to the right of the autoradiogram.
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Fig. 4.
Interaction of D1 nascent chains with
cpSecY. After 10-min pulse labeling in intact chloroplasts, the
thylakoid membrane-bound RNCs were isolated through a 1.0 M
sucrose cushion. After centrifugation, the supernatant sucrose cushion
(A) and the RNC pellets (B) were collected
separately, solubilized with SDS, and immunoprecipitated under
denaturing conditions with anti-D1. The precipitated products
(A (lane 2) and B
(lane 1)) and also the proteins from the sucrose
cushion before immunoprecipitation (A, lane
1) were subjected to SDS-PAGE and visualized by
autoradiography. Lanes 3 and 4 in
panel A and lanes 2 and
3 in panel B represent the
precipitated products after two-step immunoprecipitation. The sucrose
cushion (A, lanes 3 and 4)
and the DM-solubilized RNC pellets (B, lanes
2 and 3) were first immunoprecipitated with
anti-cpSecY and then the precipitated products were solubilized with
SDS and subjected to second immunoprecipitation with anti-D1
(A (lane 3) and B
(lane 2)) and anti-cpSecY (A
(lane 4) and B (lane
3)), respectively. Molecular mass markers (st) in
kDa are indicated to the left of the autoradiogram
(A). The elongation intermediates of 17, 22, and 25 kDa as
well as the mature (D1) and the precursor (pD1)
forms of D1 are indicated to the left of the autoradiogram
(B).
View larger version (65K):
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Fig. 5.
Cross-linking of D1 nascent chains with
cpSecY. After a 2.5-min pulse-labeling in intact chloroplasts,
RNCs were isolated and a cleavable hetero-bifunctional cross-linker
(SPDP) was added. After 30 min of incubation at 25 °C, the samples
were denatured with SDS and immunoprecipitated with anti-cpSecY. The
precipitated products were cleaved with 15 mM DTT,
solubilized with SDS, and subjected to further immunoprecipitation with
anti-D1 (lane 1) and anti-cpSecY (lane
2). Lane 3 shows the
immunoprecipitation of SDS-solubilized RNCs with anti-cpSecY without
cross-linking. The precipitated products were separated by SDS-PAGE and
subjected to autoradiography. The D1 intermediates of 17, 22, and 25 kDa are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Biology,
Laboratory of Plant Physiology, University of Turku, Biocity A, 6th
Fl., FIN-20520 Turku, Finland. Tel.: 358-2-3338074; Fax: 358-2-3338075;E-mail: evaaro@utu.fi.
ABBREVIATIONS
-D-maltoside;
PS, photosystem;
RNC, ribosome-nascent chain complex;
SPDP, N-succinimidyl-3-[2-pyridyldithio]propionate;
PAGE, polyacrylamide gel electrophoresis;
DTT, dithiothreitol.
REFERENCES
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DISCUSSION
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