From the Wolfson Laboratories, Department of Biological Sciences, South Kensington Campus, Imperial College London, London SW7 2AZ, United Kingdom
Received for publication, January 9, 2003
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ABSTRACT |
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A supercomplex containing the photosystem I (PSI)
and chlorophyll a/b light-harvesting complex I
(LHCI) has been isolated using a His-tagged mutant of
Chlamydomonas reinhardtii. This LHCI-PSI supercomplex
contained ~215 chlorophyll molecules of which 175 were estimated to
be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b
ratio measurements. Its room temperature long wavelength
absorption peak was at 680 nm, and it emitted chlorophyll fluorescence
maximally at 715 nm (77 K). The LHCI was composed of four or more
different types of Lhca polypeptides including Lhca3. No LHCII proteins
or other phosphoproteins were detected in the LHCI-PSI supercomplexes
suggesting that the cells from which they were isolated were in State
1. Electron microscopy of negatively stained samples followed by image
analysis revealed the LHCI-PSI supercomplex to have maximal dimensions
of 220 Å by 180 Å and to be ~105 Å thick. An averaged top view was
used to model in x-ray and electron crystallographic data for PSI and
Lhca proteins respectively. We conclude that the supercomplex consists
of a PSI reaction center monomer with 11 Lhca proteins arranged along
the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are
located. The estimated molecular mass for the complex is 700 kDa
including the bound chlorophyll molecules. The assignment of 11 Lhca
proteins is consistent with a total chlorophyll level of 215 assuming
that the PSI reaction center core binds ~100 chlorophylls and that
each Lhca subunit binds 10 chlorophylls. There was no evidence for
oligomerization of Chlamydomonas PSI in contrast to the
trimerization of PSI in cyanobacteria.
Photosystem I
(PSI),1 a membrane-bound
multisubunit protein complex with a molecular mass of around 356 kDa,
is located in the thylakoids of plants, algae, and cyanobacteria (1).
It utilizes light energy to oxidize plastocyanin or cytochrome
c and to reduce ferredoxin or flavodoxin. The monomeric PSI
reaction center complex is composed of more than 11 different proteins, of which the PsaA and PsaB are functionally the most important since
they bind the majority of the chlorophyll (Chl) and redox-active cofactors involved in energy conversion and charge separation. Recently
a high resolution structure of PSI isolated from the thermophilic
cyanobacterium Synechococcus elongatus has been obtained at
2.5 Å resolution (2). This work revealed the precise organization of
the transmembrane helices of PsaA and PsaB proteins together with the
redox active cofactors and Chls they bind. Surrounding these two
proteins are other, smaller transmembrane PSI subunits of which some,
namely PsaJ, PsaK, PsaL, PsaM, and PsaX, were also shown to directly or
indirectly bind Chl a in the x-ray structure of
cyanobacterial PSI and are likely to aid or regulate excitation energy
transfer within the PSI complex. In addition, three stromally bound
extrinsic proteins, PsaC, PsaD, and PsaE, optimize the binding of the
mobile electron carriers ferredoxin or flavodoxin prior to reduction of
NADP+ with PsaC containing the iron-sulfur centers
FA and FB (1).
In higher plants, green algae, and red algae, the outer
light-harvesting system associated with PSI is made up of Lhca proteins that are encoded by the cab genes and known collectively as
LHCI (light-harvesting complex I) (3). The oligomeric state of Lhca proteins in vivo is as yet not well understood but is often
assumed to be dimeric, with each Lhca monomer having a structure
similar to that of the related Lhcb proteins of the LHCII complex of
photosystem II (PSII), which has been determined to 3.4 Å (4). Note
that in cyanobacteria another type of outer light-harvesting system is
present, composed of phycobiliproteins encoded by apc and
cpc genes (5), which may, under some circumstances, attach
to the stromal surface of the PSI core complex, although this has yet to be shown conclusively. Under conditions of iron deficiency, however,
cyanobacteria form an additional outer light-harvesting system,
composed of an 18-subunit membrane-intrinsic antenna "ring" around
a PSI trimeric reaction center core (6, 7). The subunit is the Chl
a-binding protein encoded by the isiA gene, which
is homologous to the CP43 Chl a-binding protein of PSII. A
similar protein, known as Pcb, which binds both Chl a and
Chl b has also been found to form an 18-subunit
light-harvesting antenna ring around trimeric PSI of the prochlorophyte
Prochlorococcus marinus SS120 (8).
To date all studies of the structure of PSI have concluded that unlike
cyanobacteria, PSI is monomeric in higher plants and green algae (9).
Some studies have explored the structural basis by which LHCI acts as
the outer light-harvesting system of PSI. Indeed, Boekema et
al. (10) concluded that in spinach the Lhca proteins were bound to
one side of the PSI monomer. The increase in antenna size by 100 Chl
a would correspond to the association of eight subunits of
Lhca proteins with the PSI reaction center assuming that each subunit
binds 12 Chl molecules as does the Lhcb monomer (4). However, unlike
LHCII, the Chl a/b ratio is higher for LHCI,
often assumed to be 3.3 or more (11, 12) compared with 1.4 for LHCII
(4). Also it has been found that higher plant Lhca proteins bind 10 Chls (11). Boekema et al. (10) also noted that according to
their interpretation of images derived from electron microscopy and
single particle analysis, the positioning of LHCI on the outer side of
the PSI reaction center would not preclude the possibility that under
some conditions PSI of higher plants and green algae could form trimers
similar to those found in cyanobacteria. Indeed PSI from these
eukaryotic organisms contains the PsaL subunit, which in cyanobacteria
is required for the stabilization of trimeric PSI (13).
Using Chlamydomonas reinhardtii we set out to examine the
oligomeric state of PSI in this green alga and establish structural details of the arrangement of LHCI with the reaction center core. A
LHCI-PSI supercomplex preparation has been isolated using a His-tagged
mutant. Projection maps of PSI with and without LHCI components bound
have been obtained by electron microscopy and single particle image
processing of negatively stained preparations. By difference mapping
and by modeling in higher resolution structures of the underlying
intrinsic proteins derived from x-ray diffraction studies (2) and
electron crystallography (4), these maps have provided information
about the number and positioning of the Lhca proteins within the supercomplex.
Isolation of PSI Complexes from C. reinhardtii--
The C. reinhardtii mutant psbD-His containing
C-terminal fusion of a His6 tag with the D2 protein (14)
was kindly donated by J. Minagawa (Hokkaido University, Sapporo,
Japan). Cells were grown in Tris acetate-phosphate medium (15)
to exponential growth phase. Thylakoid membranes were isolated from 20 liters of cultures by a procedure of Diner and Wollman (16) and
resuspended in 400 mM sorbitol/5 mM Hepes-OH,
pH 7.5/15 mM NaCl/10 mM MgCl2. LHCI-PSI supercomplexes were purified from the thylakoids by a modified
method of Fischer et al. (17). Thylakoid membranes were
harvested at 149,000 × g for 30 min, washed with 5 mM Tris-HCl, pH 7.5/15 mM NaCl/10
mM MgCl2, centrifuged as above, and resuspended in 5 mM Tris-HCl, pH 7.5 at a chlorophyll concentration of
0.8 mg/ml. The membranes were solubilized with 0.9% (w/v)
Characterization of Purified PSI Complexes--
SDS-PAGE was
carried out using the Tris-Tricine system described by Schägger
and von Jagow (20). Protein bands were resolved on 10% polyacrylamide
gels in the presence of 6 M urea and visualized with
Coomassie Brilliant Blue R-250 using standard procedures. For Western
blotting, proteins separated on SDS-PAGE were transferred onto
polyvinylidene difluoride membranes (BioRad) in 10 mM CAPS, pH 11/10% methanol using Mini-Trans-Blot apparatus (BioRad), according to the manufacturer's instructions. Blots were probed with polyclonal antibodies raised against maize LHCI complex and Chlamydomonas psa gene products, kindly donated by R. Bassi (University of
Verona, Verona, Italy) and J.-D. Rochaix (University of Geneva, Geneva, Switzerland), respectively. C. reinhardtii Lhca3- and
Lhcb1-specific polyclonal antisera were kindly provided by M. Hippler
(University of Jena, Jena, Germany). Anti-Lhcb2 and
anti-phosphothreonine polyclonal antisera were purchased from AgriSera
and Zymed Laboratories Inc., respectively. Blots were
developed with anti-rabbit IgG-horseradish peroxidase conjugate
(Amersham Biosciences). Immunocomplexes were visualized by the
ECL method using a SuperSignal® West Pico Chemiluminescent
Substrate kit (Pierce). Optical absorption spectra were measured at
room temperature using a Shimadzu UV-1601 spectrophotometer with a 2-nm
slit size. Steady-state fluorescence spectra were obtained using a
Perkin-Elmer luminescence spectrometer LS 50 at 77 K and an excitation
wavelength of 435 nm. For the P700+ measurements, PSI
complexes were diluted to 20 µg/ml Chl in 50 mM Mes-KOH,
pH 6.5/10 mM CaCl2/0.05% DDM.
P700+ was quantified by measuring absorbance changes at 700 nm in the presence of 0.2 mM
dichlorophenolindophenol, 2 mM ascorbate as a
reductant, and 0.5 mM ferricyanide as an oxidant as
described in Marsho and Kok (21). Reverse phase HPLC pigment analyses to determine Chl a/b ratios were performed
according to Zheleva et al. (22).
Electron Microscopy and Single Particle Analyses--
The
samples were negatively stained with 2% uranyl acetate by the droplet
method and imaged using a Philips CM100 electron microscope (EM) at 80 kV. Phase contrast was introduced by defocusing the microscope whereby
the first minima of the contrast transfer function (23) was
subsequently calculated to fall consistently in the range of 18-22 Å.
Subsequent image analyses were conducted using the best 23 micrographs,
imaged at 51,500× and displaying minimal or no discernible astigmatism
and drift, of the C. reinhardtii LHCI-PSI supercomplex
fraction. In addition, 11 micrographs for the PSI monomer/PSII dimer
fraction were selected through similar criteria. The micrographs were
digitized using a Leafscan 45 densitometer at a step-size of 10 µm
(1.94 Å per pixel on the specimen scale). All image processing was
performed within the Imagic-5 software environment (24, 25), running
under the Linux operating system on dual processor PC computers. For
the LHCI-PSI supercomplex a data set of 8,950 single particle images
was obtained by interactively picking all discernible particles that
were not overlapping or in close contact with other particles, and each
image was floated into a 96 × 96 pixel box. A similar data set of
3300 images was picked from micrographs of the C. reinhardtii PSI/PSII fraction. Images of the particles were
coarsened by a factor of 2-3.88 Å per pixel to aid the speed of
subsequent processing. An analysis of these two data sets was made
starting with the reference-free alignment-by-classification procedure
(26). This identified several particle subpopulations of differing size
and shape in each preparation. These subpopulations were in turn
analyzed independently, with the reference-free alignment giving the
initial class averages necessary for multireference alignments,
eventually leading to averages with enhanced signal to noise ratios
after iterative refinement (23, 25). Relative orientations were
determined for the class averages by the angular reconstitution
technique (27), resulting in initial three-dimensional reconstructions gained from implementation of the exact back projection technique (28).
Reprojections were taken from each three-dimensional model and used to
identify additional atypical views and to further refine the class
averages within each subpopulation data set. The resolution was
estimated by Fourier shell correlation between two independent
three-dimensional reconstructions (23). PDB coordinate data sets
derived for a monomer of LHCII, a kind gift of W. Kühlbrandt,
Frankfurt, Germany, calculated at 3.4 Å resolution by electron
crystallography (4) and the 2.5 Å resolution PSI monomer (PDB
accession number 1JB0; http://www.rcsb.org) from the cyanobacterium
S. elongatus (2) were modeled into the reconstructions using
the "O" modeling software package (29).
Separation of Fractions--
After solubilization with DDM, PSII
was partially removed by nickel affinity chromatography and the
remaining fraction was subjected to sucrose density centrifugation.
Three Chl-containing bands were obtained (F1, F2, and F3), and
absorption spectroscopy identified the top fraction (F1) as LHC
proteins having an absorption maximum at 673 nm and a shoulder at 653 nm due to Chl b. Fig. 1a shows strong absorption in
the 450-500 nm range, in part due to Chl b (peaking at 476 nm) and in part due to the presence of carotenoids in the F1 fraction.
F2 (Fig. 1a) is characterized by the lack of Chl
b absorption and a red shift of the long wavelength absorption peak to 676 nm compared with F1 (Fig. 1a). In
contrast the densest fraction (F3) showed some Chl b
absorption at 653 nm and 467 nm and had maximum long wavelength
absorption at 680 nm. Low temperature fluorescence was measured at 77 K
for each band. The emission profile peaking at 681 nm for the top band is consistent with it containing free Lhc proteins. The middle fraction
(F2) showed a broad emission spectrum peaking at 686 nm but having
significant contribution beyond 700 nm. This broad, low temperature
fluorescence spectrum is indicative of a mixture of PSII (emission
<700 nm) and PSI (emission >700 nm). However, the fluorescence
spectrum of F3 peaks at 715 nm, which implies that it is highly
enriched in PSI. Reverse phase HPLC pigment analyses of the F3 sample
gave a Chl a/b ratio of ~4.5.
Protein Characterization of the Isolated Fractions--
To
characterize further each fraction of the sucrose density gradient
SDS-PAGE and immunoblotting analyses were conducted. As shown in Fig.
2a, the top fraction (F1) was
enriched in polypeptides, mainly LHCII but also LHCI as confirmed by
immunoblotting (see Fig. 2b). On the other hand, none of the
PSI antibodies raised to the proteins of the reaction center core of
PSI cross-reacted with proteins in this fraction. According to SDS-PAGE
and immunoblotting, F2 contained a range of proteins consistent with it
being a mixture of PSI and PSII core proteins, while the LHC protein
level was generally lower in this fraction compared with F1.
F3 was distinguished by having no cross-reactions with antibodies
raised to PSII (D1-antibody) or to LHCII antibodies raised against
Lhcb1 and Lhcb2 (see Fig. 2b). On the other hand the
SDS-PAGE profile and immunoblotting showed that F3 contains the PSI
core subunits, PsaA/B, PsaC, PsaD, PsaE, and PsaF. Moreover
polypeptides having apparent molecular masses in the range
22-30 kDa were present in F3. Immunoblotting showed these to be
proteins of LHCI, and in particular Lhca3 was identified by a specific
antibody response.
The protein analysis shown in Fig. 2 seems to indicate that the dense
fraction (F3) separated by sucrose density gradient centrifugation
consists of a supercomplex composed of PSI and its associated light
harvesting Lhca proteins. To dissociate the Lhca proteins from the PSI
reaction center core we treated the F3 fraction with 0.6% zwittergent
16 plus 1% DDM, and separated the solubilized products by sucrose
density centrifugation. Despite the relatively strong solubilizing
conditions used, we were unable to remove all the Lhca subunits from
the PSI reaction center core. Those that were removed formed a band at
the top of the sucrose gradient, and SDS-PAGE analyses coupled with
immunoblotting showed that several different types of Lhca proteins
were present (see Fig. 3a
inset), including Lhca3 (Western blot, not shown). The absorption spectrum of this LHCI fraction is shown in Fig.
3a, and its emission in Fig. 3b. Reverse phase
HPLC analyses gave a Chl a/b ratio of 2 for this
LHCI fraction. Both spectra contrast with the corresponding spectra
obtained with the F1 fraction shown in Fig. 1a in that they
have long wavelength maxima that are blue-shifted. Moreover, the lower
level of Chl b is evident in the absorption spectrum of the
Lhca proteins in comparison to F1 (compare Figs. 1a and
3a). The differences between this LHCI fraction and fraction F1 are consistent with the presence of a significant level of LHCII
proteins in the latter.
Electron Microscopy--
To investigate further the nature of the
LHCI-PSI complex in F3, the contents of this fraction were viewed by EM
after negative staining and the images processed by single particle
analysis. Fig. 4a shows an
averaged top view obtained by processing 8950 particles, which has
maximum dimensions of 220 × 180 Å. To interpret this projection
map the EM structure of the PSI complex of Chlamydomonas, free of LHCI protein, was investigated. To this end, images of fraction
F2 were recorded and subjected to single particle analysis. After
multistatistical analysis of the data set it was clear that the F2
fraction contained two subpopulations based on their surface area and
differences in domain organization. The subpopulation with a two-domain
character were assigned to the PSII dimeric core present in F2, which
is known to have dimensions of 220 × 150 Å in negative stain
(not shown) (30, 31), while the other major population contains
particles having a single domain and dimensions of 150 × 100 Å,
indicative of a PSI core monomer. Fig. 4c shows the average
top view of the Chlamydomonas PSI monomer obtained by
processing 680 particles. Of note is that neither in fractions F2 nor
F3 did we observe larger particles that would suggest oligomerization
of the PSI monomer and, in particular, the existence of a trimer
similar to that found for PSI in cyanobacteria (2). The LHCI-PSI
supercomplex projection shown in Fig. 4a has strong density
features along one side. We assign these features to the Lhca proteins
present. To focus on and improve the quality of these features, we
overlaid the PSI monomer projection onto the projection of the
supercomplex and used this as a mask to conduct further rounds of image
analyses utilizing the whole supercomplex data set. The resulting image
is shown in Fig. 4b. The overlaying of the PSI monomer
projection onto that of the supercomplex was aided by reference to the
x-ray structure of PSI derived by Jordan et al. (2). In the
x-ray map the PsaL subunit, with its three transmembrane helices, forms
a distinctive bulge at one end of the monomer. We therefore assumed
that a similar feature in the projection maps of the supercomplex and
monomer (starred in Fig. 4, a and c)
are due to PsaL and used this for an approximate alignment of the maps
(Fig. 4d). This comparison further suggests that the top
views of the supercomplex and monomer (shown in Fig. 4, a and c) are being viewed from the stromal side since the
feature assigned to PsaL lies left of center in the stromal top view of the x-ray-derived PSI asymmetric monomer (see red ring; Fig.
4d).
As our native EM data set contained images of the LHCI-PSI supercomplex
in different orientations we have obtained a range of class averages
and calculated a low resolution, three-dimensional model. Fig.
5 shows typical class averages taken from
the total pool of 76 class averages and shows a range of orientations.
All 76 class averages were used to construct the three-dimensional model presented in Fig. 5c as surface-rendered views and at
the same orientation as the class averages given in Fig. 5a.
The calculated projections derived from the three-dimensional model are
shown in Fig. 5b and, according to Fourier shell correlation
analysis, have a resolution of about 30 Å.
First, we conclude that using mild isolation conditions we have
not identified a trimeric form of PSI in Chlamydomonas as typically found in cyanobacteria (1, 2) and other types of
oxyphotobacteria (6-8). The monomeric nature of PSI in higher plants
and green algae has also been concluded by others (9, 10, 32). On the
other hand Chlamydomonas has a dimeric PSII reaction center
core similar to that found in higher plants and cyanobacteria (30,
33-35).
Despite the apparent absence of a trimeric PSI complex in
Chlamydomonas we have isolated a LHCI-PSI supercomplex
suggesting that our isolation procedures are sufficiently mild to
maintain oligomeric organization of PSI. The LHCI-PSI supercomplex was present in F3 of the sucrose density gradient, and its emission spectrum indicates that chlorophylls bound to the Lhca proteins are
functionally coupled since there was no significant fluorescence at 674 nm as found with the detergent-solubilized, isolated Lhca proteins
(Fig. 3b). However, it has been suggested previously in
Refs. 9, 11, and 12 that when the Lhca proteins associate with the PSI
reaction center core their long wavelength absorption and emission
spectra undergo a significant red shift. In the case of the long
wavelength absorption maxima the overall shift is from 671 nm (Fig.
3a) to beyond 677 nm since the LHCI-PSI supercomplex absorbs
maximally at 680 nm (Fig. 1a). The Chlamydomonas
PSI reaction center core without LHCI present has a red absorption peak
at 677 nm as reported by Bassi et al. (12) and confirmed
here (data not shown). According to Bassi et al. (12)
aggregated forms of Chlamydomonas Lhca proteins can be
isolated as two populations, consisting of the same Lhca proteins but
absorbing at 673 nm and 680 nm and having 77 K fluorescence peaks at
685 nm and 705 nm, respectively. They also isolated a Lhca fraction
having an absorption maximum at 670 nm and low temperature emission at
about 675 nm, which probably consisted of non-aggregated protein, in
agreement with our findings. Similar aggregations of Lhca proteins
leading to red shifts in absorption and emission have been found for
higher plants (11).
In Chlamydomonas there seems to be at least 10 Lhca proteins
(12, 36) encoded by different genes, but just how many gene products
are contained in the F3 fraction is difficult to assess. Immunoblotting
with a polyclonal LHCI antibody indicates the presence of four or more
different forms of Lhca proteins (Fig. 2b). Importantly we
did not detect LHCII proteins in the F3 fraction by immunoblotting with
Lhcb1 and Lhcb2 although the antibodies were effective in detecting
LHCII in the F1 and F2 fractions (Fig. 2b). The absence of LHCII proteins in the F3 fraction suggests that
Chlamydomonas cells were in State 1 when the thylakoid
membranes were isolated. It is generally believed that the State 1 to
State 2 transition involves transfer of Lhcb subunits from PSII to PSI
in response to N-terminal phosphorylation of these Chl
a/b-binding proteins (37). To check this further
we conducted an immunological analysis using antibody specific for
phosphorylated threonine (purchased from Zymed Laboratories
Inc.). As can be seen in Fig. 6 we
did not detect LHCII phosphorylation in F1 or F3. In F2 clear signals were identified for CP29 (Lhcb4) and PsbH proteins that are known to
dephosphorylate significantly more slowly than the LHCII proteins involved in State transitions (38).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-dodecyl-maltoside (DDM) on ice in the dark for 20 min
and then centrifuged at 48,400 × g for 25 min to
remove insoluble material. To remove His-tagged PSII complexes,
solubilized membranes were incubated with equilibrated nickel-nitrilotriacetic acid-agarose (Qiagen) at a 4:1 (v/v) ratio for
1 h in the dark. Partially PSII-depleted samples (unbound fraction) were subjected to fractionation on continuous sucrose density
gradients prepared by the modified "freeze-thaw" method (18) with
the interim buffer composed of 0.5 M sucrose/5
mM Tricine-NaOH, pH 8/0.05% DDM/0.5 M betaine.
The LHCI-PSI supercomplexes were observed in the lowest, most dense
green fraction of the gradient. LHCI complexes were purified as
described in Croce et al. (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Absorbance and fluorescence properties of
C. reinhardtii sucrose gradient fractions. Spectra
corresponding to F1 (LHCs), F2 (PSI/PSII), and F3 (LHCI-PSI) fractions
are presented in gray, fine black and bold
black, respectively. a, room temperature absorbance
spectra. b, fluorescence emission spectra at 77 K excited at
435 nm. Peaks have been normalized to facilitate their
comparison.
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Fig. 2.
Protein composition of sucrose gradient
fractions obtained from solubilized C. reinhardtii
thylakoids. a, SDS-PAGE of sucrose gradient
fractions. b, Western detection of PSI core and LHCI
subunits. M, prestained protein markers with their apparent
molecular mass (in kDa) indicated on the left;
T, thylakoids; T, DDM-solubilized thylakoids following
partial PSII depletion; F1-F3, see Fig. 1 legend. Proteins were loaded
at 5 and 2 µg Chl in a and b,
respectively.
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Fig. 3.
Optical absorbance and fluorescence
properties of the C. reinhardtii LHCI complex
following its dissociation from LHCI-PSI. a, room
temperature absorbance of LHCI. Inset represents detached
LHCI components resolved on SDS-PAGE. b, fluorescence
emission spectra of dissociated LHCI antenna (black) and
LHCI-PSI holocomplex (gray) at 77 K excited at 435 nm. Peaks
have been normalized to facilitate their comparison.
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Fig. 4.
Structural comparison between the
LHCI-PSI supercomplex and PSI monomer. a, typical top
view class average projection of the LHCI-PSI supercomplex.
b, detailed top view of the LHCI antenna domain with the
density attributed to the PSI core overlaid with a surface-rendered
spacefill representation of the amino acids from the 2.5 Å resolved
cyanobacterial PSI monomer structure (2). c, typical top
view projection of the C. reinhardtii PSI monomer obtained
from the F2 fraction. d, the green
surface-rendered cyanobacterial PSI monomer view overlaid onto the
C. reinhardtii PSI monomer from c. Circled in
red is the PsaL subunit region of 1JB0.pdb. Bar
represents 10 nm.
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Fig. 5.
Single particle image analysis of the
C. reinhardtii LHCI-PSI supercomplex.
a, selection of four typical class averages used for the
three-dimensional reconstruction. b, reprojections of the
three-dimensional map in identical orientation as the class averages
presented in a. c, surface-rendered views of the
final three-dimensional map viewed in the same orientation. Fourier
shell correlation analysis (3 criterion) calculated the preliminary
LHCI-PSI three-dimensional map to be at 30 Å resolution.
Bar corresponds to 10 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Phosphorylation status of C. reinhardtii thylakoids used for purification of the LHCI-PSI
supercomplex. Molecular mass of protein markers (in kDa) is
indicated on the left. T, thylakoids;
T, DDM-solubilized thylakoids following partial PSII
depletion; F1-F3, sucrose gradient fractions as described in Fig. 1
legend. Proteins were loaded on SDS-PAGE at 2 µg of Chl.
Immunodetection of phosphorylated proteins was performed using
anti-phosphothreonine antiserum (see "Materials and
Methods").
Overlay of the PSI monomer projection from the 2.5 Å x-ray coordinates of cyanobacterial PSI (2) onto the Chlamydomonas LHCI-PSI supercomplex shows that there are additional densities (Fig. 4b). We assign the majority of this density to Lhca proteins. With the reaction center core, it has been found that higher plant PSI does not contain the PsaM and PsaX proteins (9) as does cyanobacterial PSI (2). However it does contain the additional low molecular weight subunits PsaG, PsaH, PsaN and possibly PsaO (9, 39). It has been suggested that PsaH is located close to PsaL, and in Arabidopsis it was shown that inactivation of the psaH gene inhibited the State 1 to State 2 transition suggesting that phosphorylated LHCII proteins bind to PsaH (40). The PsaN subunit is extrinsically located on the lumenal surface, while PsaG, like PsaH, is an intrinsic protein possibly located on the opposite side of the complex to the PsaH, PsaL, and PsaI proteins (9). Studies by Boekema et al. using spinach (10) concluded that Lhca proteins bind to one side of the PSI monomer in the membrane where the PsaF, PsaJ, PsaG, and PsaK are thought to be located (9). This suggestion is supported by deletion studies using Arabidopsis, which have shown that in the absence of the PsaK and PsaG proteins the Lhca proteins associate less strongly with the PSI reaction center core (41, 42). Similarly, in Chlamydomonas PsaK has been implicated in the association of LHCI with the PSI reaction center core (44). The idea that the LHCI subunits are located on the outer PsaF, PsaJ, PsaG, PsaK-containing side of PSI is in line with the recent discovery that iron stress-induced IsiA or CP43 proteins of cyanobacteria (6, 7, 43) or the Chl a/Chl b-binding Pcb proteins of Prochlorococcus SS120 (8) associate with this side of the PSI reaction center complex.
More recently Germano et al. have reported a top view projection map of the LHCI-PSI supercomplex of Chlamydomonas derived from single particle analysis (32). The size and shape of the PSI particle they studied is similar to that reported here. Based on an overlay of the x-ray structure of the cyanobacterial PSI and comparison with the LHCI-PSI supercomplex isolated from spinach, they have concluded that 14 Lhca proteins are bound to the PSI reaction center core. If each Lhca subunit binds 10 Chls, as reported for higher plant Lhca (11) then this would increase the antenna size of the PSI reaction center by an additional 140 Chls thus giving a total of 240 Chls per P700, assuming that the PSI reaction center binds about 100 Chl. This, they argued, contrasts with the 200 Chl per P700 of the LHCI-PSI supercomplex isolated from spinach (10) and accounts for the smaller size of the latter. In coming to their conclusion they not only placed Lhca proteins along the outer side, as in the case of spinach, but also identified possible locations of Lhc proteins closer to the region assigned to the PsaL, PsaH, and PsaI cluster (9). They noted in a small number of their class averages that some of the extra density in this region, amounting to a surface area of 40 nm2, was missing. They argued that this missing density seemed to be close to PsaH and because PsaH has been implicated in the formation of State II (40) considered the possibility that these Lhc subunits could in part be Lhcb of LHCII.
The projection map reported here for the LHCI-PSI supercomplex of
Chlamydomonas (Fig. 4a) does not seem to have
extra density close to the region assigned to PsaH noted by Germano
et al. (32). Its absence would be consistent with our
conclusion that the PSI structure we report is for State 1 and concurs
with the proposal of Germano et al. (32) that this density
could be due to the presence of additional Lhc proteins, possibly those
of LHCII. By using coordinates for the structure of Lhcb monomer
derived from electron crystallography (4) we can model 11 Lhca proteins into the density we assign to the LHCI as shown in Fig.
7. We propose that eight form dimers and
correspond to four clear densities identified in Fig. 4b.
The remaining densities can accommodate three monomers located toward
the end of the crescent composed of the four Lhca dimers. If each
monomer binds 10 chlorophylls then the LHCI more than doubles the
light-harvesting size of PSI. Indeed we found that there are about 215 Chls per reaction center in the Chlamydomonas supercomplex
by measuring the Chl/P700 ratio using chemical reduction and oxidation
and comparing the same measurements conducted on the isolated PSI
reaction center of S. elongatus, which is known to bind
~100 Chl molecules (2). This stoichiometry is also consistent with
the Chl a/b ratios measured. We found that the
LHCI fraction removed from the supercomplex (Fig. 3) had a Chl
a/b ratio of 2, which is in agreement with an
overall Chl a/b ratio of ~4.5 measured for the
LHCI-PSI supercomplex. This means that the Chlamydomonas
LHCI-PSI supercomplex contains ~40 Chl b and 175 Chl
a. Moreover with 11 Lhca subunits, the estimated molecular
mass of the supercomplex, including the bound Chls is in the region of
700 kDa.
|
Our analysis of the Chl content of the LHCI-PSI supercomplex of Chlamydomonas should be compared with the interpretations of Germano et al. (32). Given that we do not detect as much density in the region of the PsaH subunit, which Germano et al. (32) suggest is sufficient to accommodate three Lhc subunits, then our conclusion that there are 11 Lhc subunits in our supercomplex agrees with the assignment of Germano et al. for the remaining density in their projection map. We also agree with the general conclusion of these authors that the majority of the Lhca subunits are positioned along one side of the PSI monomer where the PsaK, PsaJ, PsaF, and PsaG subunits are localized, a picture that has emerged from studies with higher plants, including the use of reverse genetics (9). The consistency between our analyses and those of Germano et al. (32) does not explain the discrepancy between chlorophyll levels measured by us and the size differences between the LHCI-PSI supercomplexes of Chlamydomonas and spinach (10). The significantly smaller size of the LHCI-PSI supercomplex isolated from spinach, studied by Boekema et al. (10), suggests that perhaps it does not bind 200 Chls per P700.
In conclusion we show a surface-rendered oblique view of the LHCI-PSI
supercomplex in Fig. 8, the first
reported three-dimensional model for this type of supercomplex. To
improve its resolution beyond 30 Å will require either the application
of cryo-EM in the absence of electron-dense stain or elucidation of its
structure by x-ray crystallography.
|
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ACKNOWLEDGEMENTS |
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We thank the Biotechnology and Biological Sciences Research Council for financial support. We are grateful to Alison Telfer (Imperial College, London, UK) for helpful suggestions on P700 measurement.
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FOOTNOTES |
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* This work was supported by a grant from the Biotechnology and Biological Sciences Research Council.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.
Currently holds a Royal Society University Research Fellowship.
§ To whom correspondence should be addressed. Tel.: 44-207-594-5266; Fax: 44-207-594-5267; E-mail: j.barber@ic.ac.uk.
Published, JBC Papers in Press, February 14, 2003, DOI 10.1074/jbc.M300262200
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ABBREVIATIONS |
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The abbreviations used are:
PSI/II, photosystem
I/II;
Chl, chlorophyll;
F, fraction;
DDM, -D-dodecyl
maltoside;
LHCI/II, light-harvesting complex I/II;
EM, electron
microscopy;
CAPS, 3-(cyclohexylamino)propanesulfonic acid;
MES, 4-morpholineethanesulfonic acid;
HPLC, high performance liquid
chromatography.
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