From the Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Germany
Received for publication, July 19, 2000, and in revised form, November 28, 2000
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ABSTRACT |
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The light-harvesting proteins in plastids of
different lineages including algae and land plants represent a
superfamily of chlorophyll-binding proteins that seem to be
phylogenetically related, although some of the light-harvesting complex
(LHC) proteins bind different carotenoids. LHCs can be divided into
chlorophyll a/b-binding proteins found in green algae, euglenoids, and
higher plants and into chlorophyll a/c-binding proteins of various
algal taxa. LHC proteins from diatoms are named fucoxanthin-chlorophyll a/c-binding proteins (FCP). In contrast to chlorophyll a/b-binding proteins, there is no information so far about the way FCPs integrate into thylakoid membranes. The diatom FCP preproteins have a bipartite presequence that is necessary to enable transport into the four membrane-bound diatom plastids, but similar to chlorophyll a/b-binding proteins there is apparently no presequence present for targeting to
the thylakoid membrane. By establishing an in vitro import assay for diatom thylakoids, we demonstrated that thylakoid integration of diatom FCP depends on the presence of stromal factors and GTP. This
indicates that a pathway involving signal recognition particles (SRP)
is involved in membrane integration just as shown for LHCs in higher
plants. We also demonstrate integration of diatom FCP into thylakoids
of higher plants and vice versa SRP-dependent targeting of
LHCs from pea and Arabidopsis into diatom thylakoids. The
similar SRP-dependent modes of thylakoid integration of
land plant LHCs and FCPs support recent analyses indicating a common origin of chlorophyll a/b- and a/c-binding proteins.
Photosynthetic organisms possess protein complexes that harvest
light energy and transfer it to the reaction centers. The light-harvesting complexes
(LHC)1 from eukaryotic algae
and land plants probably evolved from a common ancestor that is not
present in modern cyanobacteria (1, 2). They form a superfamily of
thylakoid membrane-intrinsic chlorophyll-carotenoid proteins. The LHCs
include the chlorophyll a/b-binding proteins from green algae and land
plants as well as the chlorophyll a/c-binding proteins from
chromophytic algae and dinoflagellates that may bind different
carotenoids like fucoxanthin (FCPs in diatoms, phaeophytes, and others)
and peridinin (intrinsic PCPs, many dinoflagellates) (2). Red algae
only have chlorophyll a and use phycobilisomes as the major photosystem
II antenna. The chlorophyll a-binding complexes isolated from the
thylakoids of these organisms are also related to the LHCs of other
eukaryotes (3, 4). To avoid confusion we will use the term "LHC
protein" for the light harvesting complexes in general, and "CAB
protein" will refer to the chlorophyll a/b-binding proteins of green
algae and higher plants.
The major PSII-associated chlorophyll a/b-binding proteins (LHCII) from
higher plants have three CAB proteins are inserted into the thylakoid membrane in a reaction
requiring GTP and stromal factors (10). The integration process of the
LHCs from land plants into thylakoid membranes involves binding to a
chloroplast signal recognition particle (SRP), which consists of at
least two proteins named cpSRP54 and cpSRP43, and to a further protein,
FtsY (11-13). cpSRP54 and cpSRP43 are related to homologous proteins
that are major factors of the cotranslational protein import pathway
into the endoplasmic reticulum of eukaryotes (14). Overexpression and
integration experiments in Escherichia coli show that
integration does not depend on binding of chlorophylls and carotenoids
to the LHC proteins (15).
Diatoms are representatives of the heterokont algae, which are assumed
to have evolved by secondary endocytobiosis. This means that a
eukaryotic host cell took up a photoautotrophic eukaryotic cell,
probably an ancestor of modern red algae, followed by transformation of
the endosymbiont into a plastid (16-18). This process resulted in
plastids having four bounding membranes. It has been demonstrated that
protein import into these so-called complex plastids depends on a
bipartite presequence and occurs in at least two steps (19).
Although diatoms and other heterokont algae represent an essential part
of aquatic ecosystems, contributing significantly to the total oxygen
and biomass production, little is known about the photosystems of these
organisms. Recent genetic analyses of Fcp genes show
that similar to genes encoding CAB proteins Fcp genes belong
to a multigene family. Several different Fcp genes have
already been found in diatoms, and it is likely that there are even
more present (20, 21).
So far there are no reports about functional aspects of FCPs with
respect to integration into thylakoid membranes and cooperation with
the diatom photosystems. Therefore, the characterization of integration
characteristics of FCP proteins might reveal the history and the
relationship of FCPs with other carotenoid-binding light-harvesting
proteins. Here we present the first data on thylakoid membrane
integration of a member of the FCP family using a respective protein
from the diatom Odontella sinensis. We have established an
in vitro thylakoid import assay from purified diatom
plastids enabling the study of FCP integration. Our results suggest
that FCPs are integrated mainly by an SRP-dependent
integration mechanism as found for LHCs. Complementary integration
experiments were performed using LHC and FCP proteins as well as
thylakoid membranes from land plant and diatom plastids.
Constructs Made from Fcp--
The Fcp gene was
derived from a cDNA library of the diatom O. sinensis
inserted as EcoRI and XhoI fragments into the
vector
Deletions of coding regions from Fcp were made by polymerase
chain reaction using Pfu polymerase (Stratagene) according
to the manufacturer's instructions. All modifications were verified by
double-strand sequencing using the T7 sequencing kit (Amersham Pharmacia Biotech). The deletion of the signal sequence resulting in
the intermediate FCP form, iFCP (FCP Preparation and Subfractionation of Pea Chloroplasts and Import
Experiments--
Chloroplasts from pea were isolated (19) from 8- to
12-day-old seedlings. Posttranslational import reactions, washing, and subfractionation of the plastids were performed as described. For
protease treatment thermolysin (Fluka) was added to a final concentration of 0.2 mg/ml together with 2 mM
CaCl2. Thylakoids were isolated according to Ref. 24.
Preparation of Chloroplasts and Thylakoids from Diatoms--
The
diatoms O. sinensis and Coscinodiscus granii were
cultured as described (25). Plastids were isolated by gentle breakage of the cells in a Yeda press and subsequent separation of broken and
intact plastids by centrifugation on a 40% Percoll cushion. Chlorophyll concentrations were determined as described (25). Isolated
plastids were routinely checked for integrity by measuring oxygen
evolution under illumination (25).
For thylakoid preparations the plastids were collected by a short
centrifugation step (3,000 × g, 1 min) and incubated
subsequently for 2 min in HM medium (50 mM HEPES, pH 8, 5 mM MgCl2) to break the plastids osmotically (at
a concentration of 1 mg of chlorophyll/ml). Just before recovering the
thylakoids by centrifugation at 15,000 × g for 1 min,
1 volume of H2SM buffer (50 mM HEPES, pH 8, 680 mM sorbitol, 5 mM MgCl2) was added.
The resulting supernatant (stromal fraction) was kept on ice. The
pelleted thylakoids were washed two or three times in HSM buffer (50 mM HEPES, pH 8, 340 mM sorbitol, 5 mM MgCl2) to remove remaining stromal
contaminations (15,000 × g, 1 min) and were thoroughly
resuspended in the stroma fraction or in HSM buffer at a concentration
of 1 mg of chlorophyll/ml.
EDTA-washed thylakoids were obtained by osmotic breakage of the
plastids with HME buffer (50 mM HEPES, pH 8, 5 mM MgCl2, EDTA 10 mM) and washing
of the thylakoids with HSME (HSM plus 10 mM EDTA).
Protease-treated thylakoids were obtained by incubating osmotically
broken thylakoids in HSMC buffer (50 mM HEPES, pH 8, 340 mM sorbitol, 5 mM MgCl2, 2 mM CaCl2) including the protease thermolysin at
a concentration of 0.2 mg/ml for 15 or 30 min. The protease was
inactivated by adding 2.5 mM EGTA and was removed by
washing in a larger volume of HSME2 (HSME including 2.5 mM EGTA).
In Vitro Translation, Import Reactions, and Protease
Treatment--
Genes encoding LHC and FCP proteins were transcribed
and translated in a coupled transcription/translation system (TNT
System, Promega) as described in the manufacturer's introductions
using [35S]methionine (Amersham Pharmacia Biotech).
The conditions for the thylakoid integration experiments with pea or
diatom thylakoids were similar except for the different osmotic
conditions. For import experiments 45 µl of thylakoids (0.5-1 mg of
chlorophyll/ml) and 10 µl of translation reaction were used. To
distinguish between Sec- or SRP-dependent integration, thylakoids were resuspended in the stromal fraction. Inhibitors for
different import pathways were added as described. ATP and GTP were
supplied at concentrations of 8 mM. The integration
reactions were started by adding the translated protein and incubating
the assays at 25 (pea thylakoids) or 16-18 °C (diatom thylakoids) for 25-30 min. Some samples were illuminated (150 µmol of
photons/m2 s). The reactions were stopped by placing the
samples on ice.
All samples were divided into three parts. The first part was used
directly for electrophoretic analysis and was taken as control (100%).
The second and third parts were centrifuged, and the thylakoids were
resuspended in HSM or HSMC, respectively, depending on optional further
protease treatment. The conditions for protease treatment were
identical as for the preparation of protease-treated thylakoids. All
nonintegrated proteins were digested by thermolysin during an
incubation on ice for 30 min. After deactivation of the protease with
2.5 mM EGTA, the thylakoids were collected and immediately
prepared for the electrophoresis.
The third part of the import assay was washed in different solutions to
verify the integration of LHC/FCP proteins. According to Breyton
et al. (26), the thylakoids were incubated with five volumes
of 2 M KSCN, 2 M NaCl, 6.8 M urea,
or 85 mM Na2CO3/DTT, respectively,
for 10 min at room temperature, were vortexed, and were subjected to
two freeze/thaw cycles including additional vortexing. The washed
thylakoids were recovered by a 10-min centrifugation (22,000 × g). To test the stability of FCP integration, this washing procedure was done three times, whereas in standard experiments all
samples were washed once with 2 M KSCN. Afterwards, the
thylakoids were washed in 100 µl of HSM to remove the potassium ions
before preparing the samples for electrophoresis.
To check the membrane specificity of FCP integration, 3 µl of
translation mixture and 10 µl HSM were added to 4 µl of microsomes. After incubation at 18 °C for 30 min, the samples were washed with
100 µl of 2 M KSCN as described above. Before recovering the membranes the samples were diluted to 3.5 ml with washing buffer.
The microsomes were washed by centrifugation at 115,000 × g for 45 min.
Electrophoresis, Fluorography, and Quantification--
Samples
were denatured by adding sample buffer and incubation for 3 min at
90 °C and analyzed on 15% SDS-PAGE (27). For visualization of the
radioactively labeled proteins, the gels were fixed in 30% ethanol,
10% acetic acid and soaked in Amplify (Amersham Pharmacia Biotech)
before drying. Fluorographic signals were detected utilizing Kodak
X-Omat x-ray film (Eastman Kodak Co.). Quantifications were performed
on a PhosphorImager (fluoro-imager BAS-1800, Fuji). For calculation of
integration efficiency, the protein bands in the control sample were
set to 100%.
Construction of FCP Precursor Proteins--
Different constructs
were made from the diatom FCP precursor protein by deleting regions of
the Fcp gene coding for different parts of the presequence
(Fig. 1). iFCP was constructed by
deleting the signal peptide domain necessary for cotranslational
transport across the chloroplast ER (the outermost of the four diatom
plastid-bounding membranes) and therefore represents the
putative intermediate preprotein possessing a transit peptide domain
only. The position of the new start codon was chosen on the basis of
prediction of possible signal peptidase cleavage sites according to the
" Isolation of Functionally Intact Diatom Thylakoids--
To analyze
the integration mode of FCPs into the thylakoid membranes from diatoms,
we established an in vitro import assay for diatom
thylakoids. Functionally and morphologically intact plastids were
isolated from the marine centric diatoms O. sinensis and
C. granii and purified by centrifugation through a Percoll cushion as described (25), followed by osmotic rupture of the plastids.
The integrity of the resulting thylakoids was analyzed by measuring
electron transport capabilities (the isolated thylakoids were routinely
checked and typically yielded ~80 µmol of O2·mg chlorophyll Analysis of Membrane Integration of FCP Proteins--
LHC proteins
do not get proteolytically processed during or after membrane
integration; therefore, integration cannot be visualized by a shift in
molecular mass. Structural comparisons between LHCs and FCPs indicate
that this might be similar in FCPs as there is obviously no cleavable
presequence present for thylakoid targeting (21). In this study we used
varying stringent washing procedures of the membranes, and we compared
the results with protease protection assays. These procedures were
established to distinguish between loosely membrane-bound protein and
membrane-integrated protein. To exclude the possibility that parts of
the presequence needed for plastid import might be involved in the
integration reaction, we analyzed integration of mFCP as well as of
iFCP. Both precursor proteins show similar integration characteristics.
According to Breyton et al. (26) we checked different
procedures for removing peripheral proteins from thylakoid membranes at
conditions where no FCP integration should occur (no addition of
stromal extract and GTP). Fig.
2A demonstrates that urea and KSCN have the best potential to elute loosely bound FCP protein from
diatom thylakoid membranes after incubation for 10 min followed by
harsh vortexing and freeze/thaw cycles. After addition of radiolabeled FCP protein to the thylakoids and subsequent incubation, they were
treated as indicated. The amount of residual protein was determined by
separating thylakoid membrane proteins by SDS-PAGE and subsequent
quantitative analysis of the residual radioactivity of the FCP band
using a PhosphorImager. Thylakoids that were washed with buffer only
served as control (100%). First experiments were performed by
incubation of thylakoids and radiolabeled FCP protein without addition
of further substrates, allowing only minor integration of FCP.
Subsequent washing of the membranes with 6.8 M urea, 2 M KSCN, and 2 M NaCl, respectively, resulted in
a residual amount of radiolabeled FCP protein of 7-18% (mFCP) of
thylakoid-associated protein (Fig. 2A). In contrast washing
with 85 mM Na2CO3/DTT was not
suitable to extract loosely bound proteins (77% residual protein). To
improve the KSCN washing procedure, we tested the integration stability
of mFCP by additional washing steps including vortexing and freeze/thaw
cycles (Fig. 2B). First washing of the membranes with 2 M KSCN resulted in a remaining radioactivity of 27%,
whereas during the following washing steps only a marginal further
extraction of the membrane protein occurred. Repeated washing steps
with urea, however, resulted in a nearly complete removal of FCP
probably due to membrane disintegration by the harsh conditions. None
of the washing procedures resulted in a clearly defined amount of residual membrane-bound protein under varying conditions. Based on our
results repeated washing steps with 2 M KSCN were the best choice to remove most of unspecifically bound proteins and to allow
reproducible results for analyzing the effectiveness of FCP integration
in the following experiments.
In addition to the washing steps the incorporation of the FCPs into
thylakoid membranes was monitored by utilizing the protease thermolysin. It has been demonstrated that some of the CAB proteins from higher plants are protected against the proteases trypsin and
thermolysin when integrated into the thylakoid membrane (24). In our
experiments a limited incubation of thylakoids following FCP
integration assays (0.2 mg/ml thermolysin, 2 mM
CaCl2) on ice for 20 min was sufficient to degrade
peripheral and free FCP, while leaving integrated protein intact (data
not shown). Therefore protease protection assays were used to verify
the results obtained by washing with KSCN.
Integration of FCP into Diatom Thylakoids--
Factors needed for
FCP integration into diatom thylakoids were analyzed by incubation of
mFCP and iFCP, respectively, from O. sinensis with diatom
thylakoids under a variety of different experimental conditions. Both
proteins indicated identical integration features as shown for mFCP
(Fig. 3). Comparable results were also obtained with thylakoid preparations from both diatoms O. sinensis and C. granii. By using the washing procedure
with 2 M KSCN as described above, we found a low amount of
membrane-integrated FCPs when only thylakoids and radioactively labeled
FCP proteins were incubated. A clear enhancement of FCP integration was
obtained by addition of stromal extracts and GTP (8 mM)
during incubation, after washing, or protease treatment. In this case
an average of 2.5-3 times higher amounts of FCP remained in the
thylakoid membranes. Variations of the concentration of GTP during
incubation show a clear GTP dependence of FCP integration (Fig.
4). A GTP concentration of about 12 mM turned out to be sufficient for maximum FCP integration.
Thylakoid washing and protease treatment resulted in comparable results
(Fig. 3).
Different integration conditions were checked to exclude the
possibility that other known protein integration pathways might also be
involved in FCP integration. The Sec system is involved in the import
reaction of the lumenal OEE33 protein (31) and depends on the presence
of stromal factors and of ATP. Addition of stromal extract and/or ATP
to the integration assay did not result in an obvious enhancement of
FCP integration above the level obtained without additions, which would
be expected if the Sec system would also be involved (Fig. 3). To make
sure that the low level of FCP integration obtained without any
additions was not due to the presence of ATP or stromal factors carried over with the thylakoid preparation, we added an inhibitor of the Sec
pathway, sodium azide (5 mM), as well as the
ATP-hydrolyzing enzyme apyrase. In both cases integration was
comparable to experiments without any additions. In land plants lumenal
proteins like OEE23 and OEE16 are transported into the thylakoids by a
pH gradient (31). Apparently there is no additional
The integration of the FCPs is not disturbed by the presence of the
N-terminal presequence which is necessary for targeting of pre-FCP into
the plastids in vivo. The full-length FCP precursor integrates into the thylakoid membrane as well as the intermediate iFCP
or the mature form mFCP. This is consistent with investigations of the
higher plant LHCs (32).
Spontaneous Integration of FCP?--
After repeated washing steps
or protease treatment as described above, we always found a certain
amount of FCP to be associated with the thylakoid membrane even in the
absence of stromal factors or GTP. This might be due to factors carried
over from the in vitro translation system (rabbit
reticulocyte lysate) or insufficient removal of unspecifically bound
proteins. However, a stronger dilution of the added radiolabeled FCP
protein in the integration assay still resulted in membrane-associated
FCP protein. Most other translocation pathways known from higher plant
thylakoids have been ruled out by our experiments; however, it is
possible that other so far unknown transporters might assist FCP
integration. Therefore, we treated diatom thylakoids with thermolysin
(0.1 mg/ml) prior to addition of FCP protein to degrade any protein translocators that might be actively involved in protein insertion. Additionally, we tried to extract electrostatically bound proteins from
the thylakoids by pretreatment with 5 mM EDTA. In both
cases the same amount of associated FCP protein was detected as in
control assays (data not shown). This might suggest that a part of the FCP proteins might be able to integrate spontaneously as reported for
the CF0II subunit of chloroplast ATPase from higher plants or ELIP proteins (24, 33). To verify this we analyzed FCP integration
into a completely different type of membrane, pancreatic ER microsomes.
We incubated radioactively labeled iFCP protein with microsomal
vesicles for 30 min. After washing the membranes repeatedly with 2 M KSCN including freeze/thaw cycles, we still found 13% of
the radioactivity after recovering the membrane pellet (not shown).
Integration of FCP Protein into Higher Plant Thylakoid
Membranes--
To compare CAB and FCP integration in the same
experimental system, we incubated LHC proteins from pea and from
Arabidopsis and FCP from Odontella individually
with thylakoids isolated from pea plastids. As shown in Fig.
5, the integration characteristics of LHC
from pea into diatom thylakoid membranes under various conditions are
similar to the respective experiments with FCP integration (Fig. 3).
Only the addition of GTP and stromal proteins resulted in a high rate
of FCP integration. Similar results were obtained for LHC from
Arabidopsis. In reciprocal experiments we could show that
integration of diatom FCP into thylakoid membranes from pea also
depends on GTP and stromal factors (Fig.
6).
LHC proteins in all organisms analyzed so far have to cross at
least two membranes before being inserted into the thylakoid membrane.
In higher plants and in green and red algae, two plastid envelope
membranes have to be traversed, whereas in heterokont algae FCPs have
to be transported across four membranes to enter the stromal
compartment. In both cases N-terminal presequences are utilized for
correct targeting. Presequences of FCPs and other nucleus encoded
plastid preproteins from diatoms have a bipartite structure. The
individual properties of these two domains have been demonstrated
in vitro (19) and in
vivo.2 In plastids of
higher plants several pathways have been described for insertion of
proteins into the thylakoid membrane or targeting to the thylakoid
lumen (for review see Ref. 31). Lumenal proteins like plastocyanin or
the subunit of the oxygen evolving system OEE33 were found to be
transported by a system homologous to the bacterial Sec system. In
contrast to the Sec pathway, a To analyze the conditions for integration of FCP from a diatom in a
homologous system, we have established a procedure to isolate
functional thylakoids from isolated diatom plastids. In contrast to
thylakoids of higher plant plastids in diatoms, no grana structures are
found except lamellae consisting of triple thylakoid stacks. The main
problem obtaining functionally intact thylakoids from marine diatoms,
indicated by the capability to build up stable proton gradients, was to
find osmotic conditions to break up the plastids but to avoid osmotic
rupture of the thylakoids. Previous thylakoid preparations from diatoms
resulted in thylakoid membranes that showed electron transport
activities, but no stable proton gradients have been demonstrated yet
(36). Isolated plastids from marine diatoms need approximately twice
the sorbitol concentration as used for chloroplasts from higher plants
to avoid osmotic rupture and to keep plastidic functional integrity,
which has been demonstrated by light-dependent oxygen
evolution (25). Therefore, for thylakoid preparations we limited the
osmotic rupture of the plastids in a low osmotic buffer to a very short
time before increasing the osmolarity again to stabilize the
thylakoids. Another advantage of the preparation of thylakoids from
purified intact plastids is the possibility to obtain pure stromal
extracts for supplementation during integration assays.
It turned out to be difficult to determine the exact amount of FCP
integrated into the thylakoid membrane. Most washing procedures were
strong enough to remove unspecifically bound FCP protein, but the
procedures turned out to be very harsh resulting in a removal of most
of the protein when employed repeatedly. However, using one single
washing step with KSCN and standardization of the procedure resulted in
reproducible results that were supported by the protease protection
assays allowing the analysis of effectors on protein insertion. Our
integration experiments revealed a very similar membrane integration
behavior for FCPs and CAB proteins. Integration of FCP was depending on
stromal factors and on the presence of GTP. Variations in the GTP
concentration clearly show a dependence on GTP and resulted in a
saturation of FCP integration at concentrations of 10-12
mM GTP. This result is in agreement with integration
experiments of CAB proteins into land plant thylakoids (10). A slight
effect of ATP on insertion was observed repeatedly in the protease assays.
Despite harsh washing procedures and protease treatment after
incubation, we always found a certain amount of FCP protein cosedimenting together with the thylakoid membranes even in the absence
of GTP and stromal proteins. The same result was obtained even after
pretreatment of the thylakoids with protease to destroy proteins
possibly being involved in protein translocation. Even in heterologous
membranes like ER vesicles from canine pancreas, a certain amount of
FCP was recovered after incubation and subsequent washing/centrifugation steps. The membrane washing procedures with
chaotropic salts we applied have been demonstrated to be effective in
removing loosely bound proteins (26). It remains unclear whether they
were effective enough to remove all of the unspecifically bound FCPs in
our experiments. Protease treatments of the thylakoids after the
integration reaction indicate that in fact a certain amount of FCPs
might be protease-protected. On the other hand CAB integration into
diatom thylakoids and vice versa also resulted in a certain amount of
residual FCP protein in the absence of stimulating factors. This could
mean that so far we are not able to distinguish between spontaneous FCP
integration and unspecifically bound FCP protein. A spontaneous
integration of FCPs would be in contrast to CAB proteins, which clearly
need GTP and SRP proteins. LHC proteins were suggested to have evolved from duplication and fusion of monospanning high light-inducible proteins, an ELIP-related protein (37). The result that ELIPs can
integrate spontaneously into the thylakoid membrane as well as assisted
by the SRP complex (24) indicates that these proteins might be able to
follow parallel insertion pathways. If this should turn out to be true
for FCPs, both pathways might have existed for insertion of the
ancestor of present day LHC proteins. Perhaps such spontaneous
integration capabilities were lost during the evolution of CAB
proteins. Analysis of the integration mode of CABs from more
"primitive" green algae might give further insight into this question.
Phylogenetic analyses have shown that FCP proteins are more closely
related to LHC proteins from red algae and to PCP proteins from
dinoflagellates than to CAB proteins (1), which reflects the putative
phylogenetic origin of heterokont plastids from red algal ancestors
(16). Separation of red and green algae (the latter leading to the land
plants) from a common ancestor is supposed to have occurred early in
the evolution of autotrophic eukaryotes. We demonstrated that the
diatom FCP can integrate into pea thylakoids as well as pea LHC into
diatom thylakoids. This is very interesting because it shows that both
protein types use the same apparently very conserved import machinery.
This cross-functionality in such divergent organisms as diatoms and
land plants indicates that the SRP-dependent integration of
light-harvesting proteins may have been developed very early in the
evolution of plastids and has essentially remained unchanged. This is
especially surprising as cyanobacteria, which are thought to be related
to the ancestors of plastids, apparently do not have light-harvesting
proteins, but phycobilisomes instead. Although it is still possible
that the cyanobacterial ancestor of plastids differed from modern
cyanobacteria, this indicates that the light-harvesting proteins as
well as the needed thylakoid-targeting system might have evolved
shortly after primary endocytobiosis. The genetic history of the
transfer of Fcp genes during secondary endocytobiosis from
the endosymbiont genome to the nucleus of the host cell is also
unclear. As FCPs seem to be generally nucleus-encoded, it is likely
that they might have been transferred to the nucleus after primary
endocytobioses and therefore had to be transferred in a second step
after secondary endocytobiosis, this time from the nucleus of the
endosymbiont to the nucleus of the second host. An intron within the
Fcp gene of the related brown algae Laminaria
(38) between signal and transit peptide domain of the FCP precursor and
phylogenetic analyses of light-harvesting proteins from cryptomonads
and chloroarachniophytes in fact suggest a two-step gene transfer
(39).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical, membrane-spanning regions as
demonstrated by crystal structure analysis by Kühlbrandt and
co-workers (5). The first and the third helices are held together by
ion pairs to ensure a compact complex to bring the carotenoids and the
chlorophylls in close contact. Some CABs form trimeric homocomplexes
(6). All genes encoding LHCs that are known so far reside in the
nucleus. In plants, an N-terminal targeting signal ("transit
peptide") allows the transport of the preproteins into the plastid
stroma (7) and is exchangeable with other stroma-targeting sequences
from plants (8). Targeting to the thylakoid membrane is accomplished by
the mature protein itself. The third transmembrane domain is probably
involved in targeting of CAB proteins to the thylakoids, whereas all
three helices are needed for correct membrane insertion (9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII (22). For a more effective expression the
Fcp gene was cloned downstream of the SP6 promoter of the
pSP73 vector (Promega) using the BamHI and KpnI
sites. The Lhc gene (psAB80) from pea in pSP64 was kindly
provided by Prof. Kenneth Cline (University of Florida) (23). The
Lhca1 gene (Lhca1 is the gene encoding chlorophyll a/b-binding protein of LHCI, type 1) from Arabidopsis thaliana in pGEM4 was a gift from Dr. Bernhard Grimm (IPK
Gatersleben, Germany).
1-15 F16M), was achieved by
amplifying the Fcp gene including the vector and the
Fcp gene but excluding the coding region for the signal
sequence (primers 5'GCATCGATCATGGCCCCGGCTCAGTCC3' and
5'CATGATCGATGCAGAGCCGGCGAGGAG3'). For
intramolecular ligation of the polymerase chain reaction product a
ClaI restriction site was introduced by the primers
(underlined). For constructing the mature form of the FCP, mFCP
(FCP
1-29), the cloned Fcp gene was cut with the
restriction enzymes StuI and SmaI. After
intramolecular ligation, the remaining gene was transferred into
BamHI and KpnI sites of SP72 (Promega). All
modified Fcp constructs were transcribed with SP6 RNA
polymerase. Within the primer for the intermediate Fcp form
(iFCP) a new start codon was included (bold letters). For expression of
the mFCP translation product, the original ATG codon at position
31 was utilized.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3,
1" rule of von Heijne (28) indicating a cleavage site at
Ala15 and Phe16. mFCP represents the
mature form as found within the plastids (20).
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Fig. 1.
N-terminal presequence domains of FCP
precursors and derived constructs used in this study showing predicted
signal peptides and transit peptides as indicated. For
construction of iFCP the signal peptide domain and for mFCP the
complete presequence have been removed and substituted by new
methionine residues to enable the respective new starts of translation.
Dots indicate that only a partial sequence of the mature
proteins is shown.
1·min
1
in the presence of methyl viologen and ADP/Pi) and the
stability of the light-induced proton gradient using the fluorophore
9-aminoacridine (29). The
pH-dependent fluorescence
quench was measured as described (30). The thylakoid membranes were
able to build up a stable proton gradient after illumination. This
proton gradient was sensitive to low amounts of the uncoupler nigericin
(data not shown). One important factor for the ability of the
thylakoids to build up a stable pH gradient are the conditions used for
osmotic breakage of the plastids. We found that osmotic rupture of the plastids is possible with low osmotic buffer for 1 min, but
subsequently the thylakoids need to be replaced in a buffer containing
at least 300 mM sorbitol to avoid swelling and increase of
proton permeability.
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[in a new window]
Fig. 2.
Analysis of residual FCP protein within
thylakoid membranes from the diatom C. granii after
different washing procedures. A, radiolabeled mFCP
protein was incubated with isolated thylakoids from
Coscinodiscus and stromal extracts without other additions
as described under "Experimental Procedures." After incubation the
thylakoids were washed with 85 mM
Na2CO3/DTT (1), 2 M KSCN
(2), 2 M NaCl (3), and 8.5 M urea (4) after vortexing and freeze/thaw
cycles. The residual amount of radioactively labeled protein attached
to the membranes was determined by separating thylakoid membranes by
SDS-PAGE and measuring the amount of radioactivity of the obtained
protein bands (shown on top of the figure) using a
PhosphorImager. Values show percentage of radioactivity compared with
the residual activity of the control reaction without washing steps.
B, analysis of residual amounts of radioactively labeled FCP
proteins within the thylakoids after repeated washing steps with 2 M KSCN and freeze-thaw cycles (1-3 times).
View larger version (45K):
[in a new window]
Fig. 3.
Analysis of integration of radioactively
labeled FCP protein into thylakoid membranes from the diatom O. sinensis under various conditions. Radiolabeled mFCP
protein was incubated with isolated thylakoids from
Odontella as described under "Experimental Procedures."
After incubation the thylakoids were either washed with 2 M
KSCN (Wash) as described or treated with the protease
thermolysin (0.2 mg/ml, 30 min on ice) (Protease) before
separation by SDS-gel electrophoresis. The amount of residual
radioactively labeled proteins within the bands (shown in
window on top) was analyzed by a PhosphorImager.
Values show percentage of radioactivity compared with the residual
activity of the control reaction without washing steps
(Total). The following reagents were present during
incubation: lane 1, stromal extract; lane 2, stromal extract and GTP (8 mM); lane 3, stromal
extract and apyrase (0.25 units); lane 4, stromal extract
and ATP (8 mM); lane 5, no additions; lane
6, nigericin (4 µM). In lane 7 the
thylakoids were pretreated with EDTA (5 mM).
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Fig. 4.
GTP dependence of FCP integration.
Integration of mFCP from Odontella into thylakoids from
Coscinodiscus was measured by incubating thylakoids with
radiolabeled FCP protein, stromal extract, and GTP concentrations as
indicated. Thylakoids were subsequently washed with 2 M
KSCN and separated on a 15% SDS gel. The amount of radioactively
labeled proteins within the bands was analyzed by a PhosphorImager.
Values show percentage of radioactivity compared with the residual
activity of the control reaction without washing steps. Standard
deviations are shown.
pH-depending FCP
integration because performing FCP integration in the light or in the
dark as well as the addition of nigericin, which abolishes
transmembrane proton gradients, did not show obvious effects.
View larger version (39K):
[in a new window]
Fig. 5.
Analysis of integration of radioactively
labeled LHC protein from pea into thylakoid membranes from the diatom
O. sinensis under various conditions.
Radiolabeled LHC proteins from pea were incubated with isolated
thylakoids from Coscinodiscus as described under
"Experimental Procedures." After incubation the thylakoids were
washed with 2 M KSCN as described before separation by
SDS-gel electrophoresis. The amount of radioactively labeled proteins
within the bands (shown in window on top) was
analyzed by a PhosphorImager. Values show percentage of radioactivity
compared with the residual activity of the control reaction without
washing steps. The following reagents were present during incubation:
lane 1, stromal extract; lane 2, stromal extract
and GTP (8 mM); lane 3, stromal extract and ATP
(8 mM); lane 4, stromal extract and ATP (8 mM) and sodium azide (5 mM); lane 5, stromal extract and apyrase (0.25 units); lane 6, no
additions; lane 7, nigericin (4 µM).
View larger version (22K):
[in a new window]
Fig. 6.
Analysis of integration of radioactively
labeled FCP protein from Odontella into thylakoid
membranes from pea. Radiolabeled mFCP protein was incubated with
isolated thylakoids from pea as described under "Experimental
Procedures." After incubation the thylakoids were washed with 2 M KSCN as described before separation by SDS-gel
electrophoresis. The amount of radioactively labeled proteins within
the bands (shown in window on top) was analyzed
by a PhosphorImager. Values show percentage of radioactivity compared
with the residual activity of the control reaction without washing
steps. The following reagents were present during incubation:
lanes 1 and 3, stromal extract and GTP (8 mM); lanes 2 and 4, stromal
extract.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pH-dependent system
allows the translocation of more tightly folded proteins (twin arginine
translocase; see Ref. 34). For both systems an N-terminal targeting
domain is required for transport, which is found between the transit
peptide for chloroplast import and the mature protein. Some proteins
that need to be inserted into the thylakoid membrane use different
mechanisms. Insertion of CAB proteins is dependent on the presence of
GTP and different stromal proteins, which are homologous to the SRP
transport system at ER membranes. Initial reports demonstrated that
integration into thylakoid membranes required ATP (35), but later
experiments demonstrated that GTP promotes membrane insertion 10 times
more effectively (10). Other proteins that are related to CAB proteins like early light-inducible proteins (ELIPs), PsbB, and PsbW are suggested to integrate spontaneously into the membrane without the aid
of proteins or energetic components (24). It is still unclear whether
these different integration processes are due to structural differences
between CAB and ELIP proteins. The mode of thylakoid membrane insertion
of LHC proteins so far has been analyzed for CAB proteins of higher
plants only. In this study we therefore have addressed the question
whether FCP proteins follow the same insertion pathway as CAB proteins
and whether this process might be a general feature for LHC proteins
from all groups of photoautotrophic eukaryotes.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Colin Robinson, Alexandra Mant, and Peter Hynds (University of Warwick, Great Britain) for discussion and support during a short visit by M. Lang in Warwick, Great Britain. We are also grateful to Kenneth Cline (University of Florida) and Bernhard Grimm (IPK Gatersleben, Germany) for genes of higher plant LHCs. We also thank Peter Jahns (University of Düsseldorf, Germany) for helpful suggestions regarding the manuscript.
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FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 189 Project B3 (to P. G. K.).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: Institut für
Biochemie der Pflanzen, Heinrich-Heine-Universität
Düsseldorf, Universitätsstraße 1, D-40225
Düsseldorf, Germany. Tel.: 49-211-811-2343; Fax: 49-211-811-3706;
E-mail: Peter.Kroth@uni-duesseldorf.de.
Published, JBC Papers in Press, December 18, 2000, DOI 10.1074/jbc.M006417200
2 K. E. Apt, L. Zaslavskaia, J. C. Lippmeier, M. Lang, O. Kilian, R. Wetherbee, A. R. Grossman, P. G. Kroth, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: LHC, light-harvesting complex; CAB, chlorophyll a/b-binding protein; ELIP, early light-inducible protein; ER, endoplasmic reticulum; FCP, fucoxanthin chlorophyll a/c-binding protein, Fcp, gene encoding FCP; PCP, peridinin chlorophyll a-binding protein; PAGE, denaturing polyacrylamide gel electrophoresis; SRP, signal recognition particle; DTT, dithiothreitol.
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