Three-dimensional Reconstruction of a Light-harvesting Complex I- Photosystem I (LHCI-PSI) Supercomplex from the Green Alga Chlamydomonas reinhardtii

INSIGHTS INTO LIGHT HARVESTING FOR PSI*

Joanna Kargul, Jon NieldDagger, and James Barber§

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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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) beta -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).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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; beta 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.

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.


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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.

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).


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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.

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.


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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 (3sigma 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

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).


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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; beta 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.


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Fig. 7.   Modeling study using the top view difference projection map of the LHCI-PSI supercomplex (see Fig. 4b) viewed from the stromal side. Overlaid onto this projection are: (i) in red, coordinates containing the carbon atom positions for LHCII at a resolution of 3.4 Å, a kind gift from Prof. W. Kühlbrandt, Max Planck Institute for Structural Biology, Frankfurt, Germany, and (ii) the 2.5 Å resolved structure for the PSI monomer (2), Protein Data Bank accession number 1JB0. Carbon atoms and amino acid side chains are in green spacefill representation, except for subunits PsaJ (yellow), PsaK (pink), PsaL (cyan), and PsaX (purple). Heteroatoms are not shown. Bar represents 5 nm.

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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Fig. 8.   Surface representation of the three-dimensional reconstruction of the Chlamydomonas LHCI-PSI supercomplex at 30 Å resolution as viewed from an oblique angle of ~30 degrees to aid visualization of the LHCI complex and PSI core monomer. Note that the maximum dimensions are inclusive of a DDM detergent "shell" present in the membrane plane, which is also encompassed by the negative stain used.


    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

The abbreviations used are: PSI/II, photosystem I/II; Chl, chlorophyll; F, fraction; DDM, beta -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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