From the Department of Plant Sciences, Weizmann
Institute of Science and the
Department of Agricultural
Botany, The Hebrew University of Jerusalem, P. O. Box 12, Rehovot 76100, Israel
Received for publication, July 6, 2000, and in revised form, November 13, 2000
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ABSTRACT |
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Translation of psbA mRNA in
Chlamydomonas reinhardtii chloroplasts is regulated by a
redox signal(s). RB60 is a member of a protein complex that binds with
high affinity to the 5'-untranslated region of psbA
mRNA. RB60 has been suggested to act as a redox-sensor subunit of
the protein complex regulating translation of chloroplast psbA mRNA. Surprisingly, cloning of RB60 identified
high homology to the endoplasmic reticulum-localized protein disulfide
isomerase, including an endoplasmic reticulum-retention signal at its
carboxyl terminus. Here we show, by in vitro import
studies, that the recombinant RB60 is imported into isolated
chloroplasts of C. reinhardtii and pea in a transit
peptide-dependent manner. Subfractionation of C. reinhardtii chloroplasts revealed that the native RB60 is partitioned between the stroma and the thylakoids. The nature of
association of native RB60, and imported recombinant RB60, with
thylakoids is similar and suggests that RB60 is tightly bound to
thylakoids. The targeting characteristics of RB60 and the potential implications of the association of RB60 with thylakoids are discussed.
The chloroplast contains a small circular genome that encodes
about 5-10% of the chloroplast proteins (1). The rest are encoded by
the cell nucleus. This two-compartment gene organization dictates a
close coordination between nuclear and organellar gene expression
(2-4). Chloroplast mRNAs accumulate in fully developed chloroplasts to relatively high levels in both light- and dark-grown plants and algae. Translation of these mRNAs occurs at a much higher rate during the light-growth phase, thus identifying translation as a key regulatory point (reviewed in Refs. 2 and 4-6). The molecular
basis of light-regulated translation in the chloroplast has been shown
to be dependent on the function of a growing list of nucleus-encoded
proteins (7-14). These factors are thought to mediate translational
regulation by interacting with the 5'-untranslated region
(5'-UTR)1 of chloroplast
mRNAs (11, 12, 15-17). Nucleus-encoded chloroplast proteins are
typically directed to the chloroplast by a transit peptide located at
the amino terminus of the protein (18).
A set of mRNA-binding proteins that bind to the chloroplastic
psbA mRNA 5'-UTR with high affinity and specificity has
been identified and purified from Chlamydomonas reinhardtii
cells (19). psbA mRNA 5'-UTR-binding proteins are
composed of four major proteins, RB38, RB47, RB55, and RB60. These form
a complex (psbA 5'-PC) that appears to bind the mRNA via
the RB47 protein. The level of binding of psbA 5'-PC to the
mRNA parallels the level of psbA mRNA translation
and its association with polyribosomes in light- and dark-grown
wild-type C. reinhardtii (19). Moreover, several nuclear
mutants have been isolated in which the loss of RB47 is accompanied by
the absence of D1 synthesis due to a block in the association of
psbA mRNA with polyribosomes (10, 20). This suggests
that light regulates polyribosome association and translation of
psbA mRNA by modulating the binding of psbA
5'-PC to the 5'-UTR. Cloning of RB47 revealed its high homology with
poly(A)-binding proteins (10). Characterization of the intrachloroplast
localization of RB47 showed that it is associated with thylakoids in
the C. reinhardtii chloroplast (21).
RB60 has been implicated as a regulatory subunit of psbA
5'-PC which is subject to light control via phosphorylation and redox signals in the chloroplast (22, 23). Cloning of RB60 identified high
homology to protein disulfide isomerase (PDI) (24). PDI-like proteins
typically catalyze the formation, reduction, and isomerization of
disulfide bonds during protein folding in the endoplasmic reticulum (ER). However, in addition to their enzymatic role, PDI-like proteins have also been found to be indispensable subunits in protein complexes such as prolyl hydroxylase and triacylglycerol transfer protein (25).
Furthermore, PDI-like proteins have recently been implicated in the
regulation of E2A transcription factor (26) and the shedding of
L-selectin (27). PDIs are most abundant in the lumen of the ER. They
are directed to the ER by a signal peptide at the amino terminus and
then are retained there by virtue of a second signal, -(K/H)DEL, at the
carboxyl terminus (25, 28). The open reading frame of the recombinant
RB60 (rRB60) contains an amino-terminal extension, the targeting
identity of which has yet to be determined. Notably, despite the
implicated function of rRB60 in the chloroplast, its open reading frame
contains a carboxyl-terminal signal for ER retention (24).
Therefore, we first set out to determine whether the cloned rRB60 gene
product is targeted to chloroplasts, and thereafter to study the
subchloroplast localization of the native RB60. We show that rRB60 is
imported into isolated C. reinhardtii and pea chloroplasts
in a transit peptide-dependent fashion. Subfractionation of
chloroplasts showed that whereas a portion of the native RB60 is
present in the stroma, it is also found tightly bound to thylakoids. Moreover, following uptake by chloroplasts, the recombinant rRB60 associates with thylakoids in a manner similar to the native RB60. The
association of RB60 to thylakoids was resistant to EDTA and RNase
treatments, indicating that it is probably not mediated by binding to
polysome-associated psbA mRNA.
Preparation of Intact Chloroplasts--
C. reinhardtii
cw15 cells were grown in Tris/acetate/phosphate medium (29), under
a 12-h light/12-h dark period at 25 °C, to a density of ~1 × 107 cells/ml. Intact chloroplasts were collected from the
45/70% interface of discontinuous Percoll gradient according to a
protocol based on Goldschmidt-Clermont et al. (30) and
Belknap (31).
Pea seedlings (Pisum sativum var. Alaska) were grown under
standard conditions (32). Intact chloroplasts were isolated on Percoll
gradients as described (32, 33).
Chlorophyll concentration was determined spectrophotometrically
according to method of Arnon (34).
In Vitro Import Assays--
In vitro protein
synthesis reactions were performed with a T3
TNT-coupled reticulocyte lysate system according to manufacturer's instructions (Promega) using 2 µg of DNA from RB60,
In vitro import assays into intact chloroplasts were
performed as described previously (32, 35). The import assay was conducted for 30 min at 25 °C in the light in the presence of 10 mM ATP, unless otherwise indicated. Competition import
assays were performed in the presence of nonlabeled pea OEE1 protein, which was expressed and purified as described by Betts et
al. (36). Following import, chloroplasts were pelleted by
centrifugation and then resuspended in HS buffer (50 mM
Hepes-KOH, pH 8, 0.33 M sorbitol). Each import reaction was
divided into 3 aliquots treated with or without thermolysin (0.3 mg/ml)
or with thermolysin (0.3 mg/ml) containing 1% Triton X-100 for 45 min
at 4 °C. Intact chloroplasts were then re-isolated by centrifugation
through a 40% Percoll cushion, washed with HS buffer containing 5 mM EDTA, and resuspended in HS buffer.
Localization of Imported rRB60 and Native RB60--
To localize
RB60, we used isolated chloroplasts (10 µg of chlorophyll) or
isolated chloroplasts containing imported radioactive rRB60 that were
re-isolated using a 40% Percoll cushion and washed with HS buffer
containing 5 mM EDTA. Stromal and thylakoid fractions were
obtained by freezing and thawing, followed by a 1-min centrifugation at
15,000 × g at 4 °C (37). The resulting supernatant
contained the stroma proteins, and the pellet contained the thylakoid
fraction. The pellet was washed with 10 mM Hepes-KOH, pH
7.6, and centrifuged as above. Thylakoids were resuspended in 50 µl
of wash solution (HS with or without 1 M NaCl or 1 M NaCl, 0.05% Triton X-100, or 0.3 mg/ml thermolysin, or
0.1 M sodium carbonate pH 11, or 10 mM EDTA, or
10 mM DTT) and incubated on ice for 30 min. Membranes were
then centrifuged as above for 5 min. The supernatant was saved, and
thylakoids were washed with HS buffer, centrifuged, and resuspended in
50 µl of HS.
Protein Electrophoresis and Immunoblotting--
For localization
and import assays, ~2 µg chlorophyll was incubated in SDS sample
buffer (3% SDS, 2.25% rRB60 Is Imported into C. reinhardtii Chloroplasts--
The
targeting of proteins synthesized by cytoplasmic ribosomes to the
chloroplast is typically determined by an amino-terminal transit
peptide. The amino-terminal sequence of RB60, preceding the conserved
sequence of PDIs, is quite different from other PDIs in both length and
composition (Fig. 1) and contains a
putative cleavage sequence in position 26-28 (39). To test whether the amino-terminal sequence of rRB60 could direct import into chloroplasts, radiolabeled rRB60 was synthesized in vitro and incubated
with C. reinhardtii chloroplasts isolated by Percoll step
gradient. Since in contrast to import by the ER, protein uptake by
chloroplasts occurs post-translationally, we added the radiolabeled
rRB60 only after termination of in vitro translation.
Following incubation, protein that had not entered the chloroplast was
degraded by treatment with the protease thermolysin. Intact
chloroplasts were then repurified on a Percoll cushion and lysed with
denaturing buffer, and the protein extracts were fractionated by
SDS-gel electrophoresis. As seen in Fig.
2, rRB60 was imported into the
chloroplasts and was protected from protease treatment (Fig. 2,
lane 3), similar to a control import reaction containing
in vitro synthesized chloroplast LHCII protein (Fig. 2,
lane 11). Disruption of chloroplast membranes by treatment
with nonionic detergent resulted in the degradation of both imported
rRB60 (Fig. 2, lane 4) and LHCII proteins (Fig. 2,
lane 12). These results verified the effectiveness of the
protease treatment and that the radiolabeled protein was indeed
protected from degradation by being taken up into chloroplasts.
To assay whether the import into chloroplasts was determined by its
amino-terminal sequence, a leaderless version of rRB60 (lacking the
first 28 amino acids, The Targeting of rRB60 Is Conserved in Chloroplasts of Higher
Plants--
Next, we assayed whether rRB60 would also be taken up by
the well established system of highly purified pea chloroplasts. We
chose pea polyphenol oxidase (Ps-PPO) as a control for the import
activity of the isolated pea chloroplasts and C. reinhardtii small subunit of ribulose-1,5-bisphosphate carboxylase (Cr-SSU) as a
control for import of a heterologous protein. All proteins were
incubated with chloroplasts post-in vitro translation. As seen in the autoradiogram in Fig.
3A, both pea PPO and C. reinhardtii SSU were imported into the chloroplasts and were
protected from protease treatment (Fig. 3A, lanes 3 and
7). Disruption of chloroplast membranes by treating with
nonionic detergent resulted in degradation of the imported Ps-PPO and
Cr-SSU proteins (Fig. 3A, lanes 4 and 8). These
results demonstrated the intactness and capacity of the isolated pea
chloroplasts to import C. reinhardtii chloroplast-targeted proteins in vitro. In a manner comparable to Cr-SSU, rRB60
of C. reinhardtii was imported into the isolated pea
chloroplasts, as reflected by its protection from protease degradation
in the presence of intact chloroplast membranes (Fig. 3B,
compare lanes 3 and 4). As with rRB60 imported
into C. reinhardtii chloroplasts (Fig. 2), the mobility of
rRB60 imported into pea chloroplasts corresponded to that of its
precursor (Fig. 3B, lanes 1 and 3). The import of
rRB60 into pea chloroplasts was dependent upon its amino terminus, as
removal of the rRB60 leader abolished import (Fig. 3B, lanes
6 and 7).
To reaffirm the import of rRB60 into chloroplasts, we tested whether
its import exhibits additional characteristics typical of chloroplast
import. The findings that the uptake of rRB60 was enhanced in the
presence of ATP (Fig. 3C) and was competed by the pea
chloroplast protein OEE1 (Fig. 3D) corroborate this. To rule
out the possibility of contaminating ER in the import reactions, we
assayed the purity of the isolated chloroplasts. Fig. 3E
shows that antibodies raised against the tobacco ER protein BiP (41) reacted against pea BiP in total protein extract of leaves but not of
isolated chloroplasts. Similar results were obtained for C. reinhardtii chloroplasts using antibodies raised against the yeast
BiP (Fig. 3E). This indicates that the isolated chloroplasts used were devoid of ER and that rRB60 was taken up by the chloroplasts. The import of rRB60 into both C. reinhardtii and pea
chloroplasts suggests that it is directed to chloroplasts in
vivo and that this capacity is conserved in higher plants.
RB60 Is Partitioned between Stroma and Thylakoids--
Import to
C. reinhardtii and pea chloroplasts, in a transit
peptide-dependent manner, indicated that the
nucleus-encoded RB60 contains a chloroplast targeting signal. To
identify the subchloroplast localization of native RB60, we lysed
isolated C. reinhardtii chloroplasts, and we separated the
thylakoid fraction from the supernatant containing the stroma proteins
by centrifugation. The purity of the stromal fraction was determined by
the absence of thylakoid-associated OEE2 and CF1 proteins, and the
purity of the thylakoid fraction was verified by the lack of stromal ClpC protein (Fig. 4A). RB60
was present in both the stroma and thylakoid fractions of C. reinhardtii chloroplasts (Fig. 4A, lanes 2 and
3). The proportion of RB60 in these fractions fluctuated in
several replications of this assay (data not shown). However, we
routinely observed about 50% of the pool of C. reinhardtii chloroplast RB60 to be associated with thylakoids.
To check whether following import rRB60 is also directed toward the
thylakoid membranes, we assayed the association of imported rRB60 with
thylakoids. Both C. reinhardtii and pea chloroplasts containing imported radiolabeled rRB60 were disrupted and fractionated as in Fig. 4A. Most of the imported rRB60 was found
associated with thylakoids of C. reinhardtii chloroplasts,
whereas the proportion of soluble rRB60 was higher in pea chloroplasts
(Fig. 4B). Together, these results suggest that at least
some of the pool of chloroplast RB60 is associated with thylakoids.
The Nature of the Association of RB60 with Thylakoids--
Next,
we studied the nature of the association between native RB60 and
C. reinhardtii thylakoids by washing purified thylakoids with high salt (1 M NaCl) with or without 0.05% Triton
X-100, or with alkali buffer (0.1 M
Na2CO3, pH 11), or by treating with the
protease thermolysin (0.1 mg/ml) (Fig.
5). Thylakoid membranes were then
isolated by centrifugation, and the resistance of thylakoid-associated RB60 to each of these treatments was determined by immunoblot assay
comparing the amounts of thylakoid-associated and released soluble
RB60. The nature of the association of RB60 with thylakoids was
compared with that of the peripheral thylakoid membrane proteins CF1
and OEE2. Similar to CF1 and OEE2, most of the RB60 remained bound to
the thylakoids after a wash with 1 M NaCl or 0.1 M Na2CO3, pH 11 (Fig. 5,
lanes 2, 3, 6, and 7), suggesting that
RB60 is tightly bound to the thylakoids. The inclusion of 0.05% Triton X-100 in the salt wash completely removed RB60 from the thylakoids, whereas it had only a mild impact on CF1 and OEE2 (Fig. 5, lanes 4 and 5). These results suggest that RB60 is a
peripheral protein and that its association with thylakoids may be
mediated, at least in part, by hydrophobic interactions. The protease
treatment removed RB60 exclusively from the thylakoids leaving the CF1
and OEE2 proteins intact (Fig. 5, lanes 8 and 9),
suggesting that RB60 is located on the stromal face of the
thylakoids.
Furthermore, we tested whether imported rRB60 displays the same type of
thylakoid association as the native RB60. Following import of
radiolabeled rRB60 to C. reinhardtii chloroplasts, thylakoid membranes were challenged with the same treatments as in Fig. 5.
Similar to the native RB60 (Fig. 5), most of the imported
thylakoid-associated rRB60 was resistant to high salt and alkali washes
(Fig. 6, lanes 2, 3, 6, and 7) and sensitive to a wash
with 1 M NaCl including 0.05% Triton X-100 (Fig. 6,
lanes 4 and 5) and to treatment with thermolysin
(Fig. 6, lanes 8 and 9). The same type of
association with thylakoids was observed with rRB60 imported into pea
chloroplasts (Fig. 6, lanes 1-9). Together, these data
further corroborate the authenticity of rRB60 and suggest that at least
a portion of chloroplast RB60 is tightly bound to the stromal face of
the thylakoids.
RB60 has been shown to contain reactive thiols (23). This raises the
possibility that the thylakoid association of RB60 is mediated by
formation of a mixed disulfide bond with a thylakoid protein. In this
case, the association of RB60 with thylakoids would be expected to be
sensitive to reduction. To test this, we washed isolated thylakoids
with buffer containing 10 mM dithiothreitol (DTT) (Fig.
7, lanes 2 and 3).
The resistance of thylakoid-associated RB60 to reduction by DTT did not
corroborate the assumption. RB60 was initially isolated and
characterized as a component of a protein complex showing high affinity
to the 5'-UTR of psbA mRNA (19). Because translation of
psbA mRNA is by thylakoid-bound polyribosomes (42, 43),
RB60 may associate with thylakoids by binding to the 5'-UTR of
polysomes-associated psbA mRNA. The resistance of the
RB60-thylakoid association to a wash with 10 mM EDTA (which dissociates ribosomes) (Fig. 7, lanes 4 and 5)
and to treatment with RNase A (data not shown) indicates otherwise.
Therefore, it is conceivable that RB60 associates with thylakoids
either directly or as part of a protein complex.
RB60 was first isolated as a component of a protein complex
(psbA 5'-PC) which assembles with high affinity on the
5'-UTR of C. reinhardtii chloroplast psbA
mRNA (19). Consistent with the predicted location of RB60, antisera
raised against RB60 cross-reacted with a single protein in C. reinhardtii chloroplasts (23). However, the cloning of rRB60
depicted a PDI-like protein containing a putative amino-terminal leader
sequence, whose targeting information was unknown, and an ER-retention
signal, -KDEL, at the carboxyl-end of the protein (24). This prompted
us to investigate the authenticity of the recombinant rRB60 by testing
whether its amino terminus can direct import by chloroplasts. We showed
that rRB60 is imported into both C. reinhardtii and pea
chloroplasts in a transit peptide-dependent process (Figs.
2 and 3), indicating that RB60 is directed by its amino-terminal
sequence to chloroplasts in vivo. The import of rRB60
displayed several characteristics typical of chloroplast import, such
as post-translational import, ATP-dependent import, and
sensitivity to a competing import of a nonlabeled chloroplast protein
(Fig. 3 and Fig. 4, B and C). The authenticity of
rRB60 was further substantiated by the similar intrachloroplast
localization of the native RB60 and the imported rRB60 (Fig. 4). The
reason for the presence of a -KDEL signal in the carboxyl terminus of rRB60 is yet unknown. It may be cryptic and may represent a potential signature of the evolution of RB60 from an ancestral PDI gene. Alternatively, RB60 may have dual functions as follows: one in the
chloroplast and a second in the ER. Such polytopic proteins, present in
both the mitochondrion and an additional cell compartment, have been
described (44). Polytopic targeting of mitochondrial proteins has been
suggested to arise either from export mechanisms from the mitochondria
(44) or, alternatively, via dual targeting by a unique amino terminus
signal peptide (45). We are currently studying whether RB60 is also an
ER protein.
In the chloroplast, RB60 is partitioned between the stroma and the
thylakoids (Figs. 4-6). A second component of psbA 5'-PC, the RB47 protein, has also been shown to be tightly bound to thylakoids (21). The finding of RB60 and RB47 as membrane-associated proteins suggests a regulatory membrane-associated step in the expression of
psbA mRNA. Translation of chloroplast mRNAs encoding
integral thylakoid proteins is via membrane-bound polyribosomes (42, 43). Therefore, it is possible that RB60 attaches to thylakoids by
binding to the 5'-UTR of polysome-associated psbA mRNA.
However, the association of RB60 with thylakoids is resistant to washes containing EDTA and treatment with RNase A, suggesting that the binding
is not mediated by membrane-bound ribosomes. This, however, does not
rule out the possibility that some amount of RB60 was associated with
psbA mRNA and was washed away from the thylakoids in our
Mg2+-free washes.
An alternative explanation for the membrane association of
5'-UTR-interacting proteins has been invoked by genetic studies showing
many parallels in translational regulation in C. reinhardtii chloroplast and Saccharomyces cerevisiae mitochondrion (2, 3, 46). In the latter, the cox3 mRNA translation
activator proteins, PET54, PET122, PET494, form a complex that has the
capacity for three-way interaction with the 5'-UTR, the small ribosomal subunit, and the inner mitochondrial membrane (47). This three-way association suggests that the yeast translational activators tether the
mitochondrion mRNA to the inner membrane (46). Similar function has
been proposed for the proteins binding the 5'-UTR of chloroplast mRNAs, encoding thylakoid membrane proteins (2, 3). The detection
of tight binding of RB60 (Figs. 4-6) and RB47 (21) to thylakoids
corroborates this model. Furthermore, the characterization of RB60 as a
peripheral protein localized on the stromal face of the thylakoids
(Fig. 5) is consistent with this proposed function of 5'-UTR-binding
proteins in the chloroplast. However, thylakoid association is not
common to all translational regulators: CRP1, a regulator of
petA and petD mRNAs in maize, was found to be
part of a soluble high molecular weight complex and not to be
associated with chloroplast membranes (14).
The finding of specific oxidizing activity of RB60 (23) suggests an
additional reason for the RB60-thylakoid association. Recently, a novel
yeast protein, ERO1, whose function is to oxidize PDI, has been
identified and shown to be membrane-localized (48, 49). Likewise, in
Escherichia coli, the source of oxidizing equivalents
necessary for protein disulfide catalysis has been shown to be
membranal electron transport (50, 51). By analogy with these studies,
one may hypothesize that the photosynthetic electron transport of the
thylakoid is the source of oxidizing equivalents for RB60. If so, this
might necessitate the close association of RB60 with thylakoids.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
28RB60, LHCII, or SSU, or pea PPO cDNA containing plasmid constructs.
Translation products were fractionated by SDS-PAGE, and protein
incorporation of [35S]methionine was determined by
trichloroacetic acid precipitation.
-mercaptoethanol, 6.7% glycerol, 0.133 M Tris-HCl, pH 6.8) and then fractionated by SDS-PAGE on
12% (w/v) polyacrylamide gels (38). Proteins were electroblotted onto
nitrocellulose membranes (Schleicher & Schuell). Imported radiolabeled
proteins were detected by autoradiography. Immunoblots were performed
according to Trebitsh et al. (23). Membranes were incubated
with specific antibodies (as detailed in the figure legends) and
visualized by the enhanced chemiluminescence technique (SuperSignal, Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Multiple sequence alignment of the amino
terminus of C. reinhardtii RB60 and related protein
disulfide isomerases from mammals, plants, and yeast. Multiple
alignment of the polypeptides was generated using ClustalW. Homologous
amino acids are shaded. The putative cleavage site for RB60
(marked by an arrow) was identified using the computer
program of Nielsen et al. (39).
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Fig. 2.
rRB60 is imported into isolated C. reinhardtii chloroplasts. Import into C. reinhardtii chloroplasts was performed with in vitro
synthesized, 35S-labeled RB60 (lanes 1-4),
28RB60 (a deletion spanning amino acids 1-28) (lanes
5-8), and LHCII (lanes 9-12) recombinant proteins.
2% of the input translation products of RB60 (lane 1),
28LRB60 (lane 5), and LHCII (lane 9) is
presented. Import into chloroplasts (lanes 2, 6, and
10) was protected from thermolysin (0.3 mg/ml) degradation
(Therm, lanes 3, 7, and 11). Treatment
with 1% Triton X-100 (Triton, lanes 4, 8, and
12) ensured that the thermolysin-treated proteins were
indeed taken up by chloroplasts. Proteins were fractionated by SDS-PAGE
and electroblotted onto nitrocellulose membrane. Radiolabeled proteins
were detected by autoradiography. Molecular masses of reference
proteins (kDa) are shown on the left.
28RB60) was prepared and subjected to
chloroplast import assays. The leaderless rRB60 was not imported by
isolated chloroplasts (Fig. 2, lane 7), corroborating the
proposed function of the amino-terminal sequence of RB60 as a
chloroplast transit peptide required for import into chloroplasts. The
mobility of the chloroplast-imported rRB60 was similar to that of its
in vitro translated precursor (Fig. 2, lanes 1 and 3), and it corresponded to the mobility of the native
RB60 in immunoblot assays (data not shown). In contrast, the mobility
of the
28RB60 was slightly higher (Fig. 2, lane 5),
suggesting that the transit peptide may not be cleaved after import as
shown for the thylakoid-associated kinase, TAK1 (40).
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Fig. 3.
rRB60 is imported into isolated pea
chloroplasts. A, autoradiogram showing import of control
proteins into pea chloroplasts. Ps-PPO denotes pea
polyphenol oxidase; Cr-SSU denotes small subunit of Rubisco
of C. reinhardtii. B, autoradiogram showing
import of C. reinhardtii rRB60 recombinant proteins into pea
chloroplasts. 28RB60 is a deletion of amino
acids 1-28 of RB60. In vitro synthesis of
35S-labeled recombinant proteins, import into pea
chloroplasts, and detection of the imported proteins were performed as
described in Fig. 2. C, autoradiogram of
ATP-dependent import of recombinant RB60 into pea
chloroplasts. Thermolysin-protected import into pea chloroplasts was
performed as in Fig. 2, lane 3, except that import was
performed in the absence (lane 1) or in the presence
(lane 2) of 10 mM Mg-ATP. D,
autoradiogram showing competition by import of nonradiolabeled pea OEE1
protein. Thermolysin-protected import into chloroplasts was performed
as in Fig. 2, lane 3. Import reactions were performed in the
presence of increased amounts (nanomoles) of the precursor pea OEE1
protein, as indicated above each lane. E,
immunoblot analysis of BiP, an ER protein. Total proteins extract of
tobacco leaves (Tobacco, lane 1), pea leaves
(Pea Total, lane 2), and isolated pea
chloroplasts (Pea Chlps, lane 3) or cells of
C. reinhardtii (Cr Total, lane 4) and
isolated C. reinhardtii chloroplasts (Cr Chlps,
lane 5) were fractionated by SDS-PAGE, electroblotted onto
nitrocellulose membrane, and decorated with antibodies specific to
tobacco BiP (41) and pea OEE1 (52) (lanes 1-3) and
antibodies specific to yeast BiP and C. reinhardtii OEE2
(lanes 4 and 5).
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Fig. 4.
Subchloroplast localization of C. reinhardtii RB60. A, immunoblot analysis of
native RB60. Percoll-purified C. reinhardtii or pea
chloroplasts (Chlps, lanes 1 and 4)
were lysed by freezing and thawing. Stromal (St, lanes
2 and 5) and thylakoid fractions were separated by
centrifugation. Thylakoid membranes were washed with 10 mM
Hepes-KOH, pH 7.6 (T, lanes 3 and 6).
Immunoblot analysis of C. reinhardtii (C.r.
panel) and pea (Pea panel) chloroplast proteins was
performed using antibodies directed against C. reinhardtii
RB60 (23), OEE2 and CF1 proteins, and against pea OEE1 (52) and ClpC
protein (53), and spinach CF1 (54). B, autoradiogram of
recombinant RB60 imported into C. reinhardtii (C.r.
panel) or pea (Pea panel) chloroplasts.
Thermolysin-protected import into chloroplasts (Chlps,
lanes 1 and 4) was performed as in Fig. 2,
lane 3. Stromal (St, lanes 2 and
5) and thylakoid fractions (T, lanes 3 and 6) were obtained as in A. Imported
radiolabeled proteins were visualized as in Fig. 2.
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Fig. 5.
Association of native RB60 with C. reinhardtii thylakoid membranes. Thylakoid membranes
(obtained as in Fig. 4A) were washed with buffers containing
1 M NaCl, 1 M NaCl, and 0.05% Triton X-100 or
0.1 M Na2CO3, pH 11, or subjected
to a protease treatment (thermolysin (Therm), 0.1 mg/ml) and
each separated into membrane-associated (P) and soluble
(S) fractions. Immunoblot analysis of each fraction was
performed with antibodies against C. reinhardtii RB60, OEE2,
and CF1.
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Fig. 6.
Association of imported rRB60 with C. reinhardtii and pea thylakoid membranes.
Thermolysin-protected import into C. reinhardtii (C.r
panel) and pea (Pea panel) chloroplasts was performed
as in Fig. 2, lane 3. Following import, thylakoid membranes
were isolated and treated as in Fig. 4 producing membrane-associated
(P) and soluble (S) fractions. Radiolabeled
proteins were detected by autoradiography.
View larger version (83K):
[in a new window]
Fig. 7.
Thylakoid association of native RB60 is not
mediated by ribosome or mixed disulfide interactions. Thylakoid
membranes (T, lane 1) and washes (lanes
2-5) were performed as in Fig. 4 except that wash solutions
included 10 mM DTT (lanes 2 and 3) or
10 mM EDTA (EDTA, lanes 4 and
5) producing membrane-associated (P) and soluble
(S) fractions. Immunoblot analysis of each fraction was
performed with antibodies against C. reinhardtii RB60, OEE2,
and CF1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank E. Harel for help with the protein chloroplast import assays and for providing us with the polyphenol oxidase construct; S. Mayfield for the C.r. OEE2 and CF1 antisera; Z. Gromet-Elhanan for the spinach CF1 antisera; A. Vitale for the tobacco BiP antisera; and G. Galili for critical reading of this manuscript. We thank T. Danon for assistance with antiserum production.
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FOOTNOTES |
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* This work was supported in part by grants from the Israel Science Foundation and the Minerva 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.
§ Recipient of a Feinberg postdoctoral fellowship.
¶ Recipient of a Feinberg Graduate School fellowship.
** Holds The Judith and Martin Freedman Career Developmental Chair. To whom correspondence should be addressed. Tel.: 972-8-934-2382; Fax: 972-8-934-4181; E-mail: avihai.danon@weizmann.ac.il.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M005950200
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ABBREVIATIONS |
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The abbreviations used are: UTR, untranslated region; ER, endoplasmic reticulum; PDI, protein disulfide isomerase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; PPO, polyphenol oxidase; SSU, small subunit of ribulose-1,5-bisphosphate carboxylase; PC, protein complex.
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REFERENCES |
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