Purification Properties and Biogenesis of Chlamydomonas reinhardii Photosystem I Reaction Center*

A photosystem I reaction center was isolated from Chlamydomonas reinhardii chloroplasts. It consists of four different polypeptides with M, =: 70,000 (subunit I), 19,000 (subunit 11), 10,000 (subunit 111), and 8,000 (sub- unit IV). In the presence of salts, the purified reaction center was active in cytochrome 552 photooxidation. Short term labeling experiments with [36S]sulfate revealed that subunit I11 contains no cysteine or methio- nine. Subunits I and IV were shown to be chloroplast translation products, while subunit I1 appears to be synthesized on cytoplasmic ribosomes. The site of synthesis of the subunits to the proton-ATPase complex was studied. A differential effect of cycloheximide on the assembly of photosystem I reaction center and the proton-ATPase complex was indicated. The green alga Chlamydomonas reinhardii has been exten-sively used in genetic and biogenesis studies of photosynthesis (1-4). Ohad and his co-workers (2, 5) conducted a detailed study of the development of the photosynthetic membranes during greening of the cells. The onset, upon illumination of the activity of the two photosystems, was worked out. More-over, the presence of polypeptides of cytoplasmic and chloro-plastic origin in partially purified preparations of photosys-tems I and I1 was indicated (6). A photosystem I reaction center was isolated from higher plants (7-10). The purified reaction center was shown to consist of six different pc!ypeptides designated as subunits I to

The green alga Chlamydomonas reinhardii has been extensively used in genetic and biogenesis studies of photosynthesis (1-4). Ohad and his co-workers (2, 5) conducted a detailed study of the development of the photosynthetic membranes during greening of the cells. The onset, upon illumination of the activity of the two photosystems, was worked out. Moreover, the presence of polypeptides of cytoplasmic and chloroplastic origin in partially purified preparations of photosystems I and I1 was indicated (6).
A photosystem I reaction center was isolated from higher plants (7)(8)(9)(10). The purified reaction center was shown to consist of six different pc!ypeptides designated as subunits I to VI in order of decreasing M , = 70,000 (I), 25,000 (II), 20,000 (111), 18,000 (IV), 16,000 (V), and 8,000 (VI). Subunits I11 and IV switch their positions in Tris/glycine/sodium dodecyl sulfate slab gels and subunit VI splits into two apparent protein bands (10). The role of subunits I and 111 in the photobiochemical activity of the photosystem I reaction center have been worked out, and the subunit structure within the chloroplast membrane has been proposed (8,9). In this article, we report on the purification of the photosystem I reaction center from Chlamydomonas chloroplasts.
The subunit structure and some biochemical properties of this reaction center and the photosystem I reaction center from higher plants are compared. The sites of synthesis of individual subunits are studied by labeling with [35S]sulfate in the presence of protein synthesis inhibitors. The effect of polypeptide(s) originating in the cytoplasm on the assembly of the reaction center is indicated.

EXPERIMENTAL PROCEDURES
Materials-Most of the chemicals were purchased from Sigma.
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
["%]Sulfate was obtained from the Nuclear Research Centre, Negev, Israel.
Analytical Methods-Published procedures were used for chlorophyll (11) and protein (12) determinations. Reconstitution of photophosphorylation was performed according to Hauska et at. (13). was determined as previously described (7,8). Gel electrophoresis in slabs containing an exponential gradient of 10 to 15% acrylamide was performed according to Douglas and Butow (14).
Preparations-Proton ATPase complexes from lettuce and Chlamydomonas chloroplasts were prepared as before (15). Cytochrome 552 was purified from Euglena cells according to Perini et al. (16).
Growing and Labeling of Cells-Chlamydomonas reinhardii (strain Y-1) was grown as previously described (17). For labeling with [35S]sulfate, -6 liters of cell suspension containing 10 to 20 pg of chlorophyll/ml were harvested by centrifugation at 1500 X g for 3 min and washed twice with growth medium in which the MgSO, was replaced by an equal weight of MgC12. The cells were suspended in 2 liters of the same medium and incubated overnight at room temperature under fluorescent light (5000 ergs cm-*s"). Usually the suspension was then divided into four equal parts and protein synthesis inhibitors (cycloheximide at final concentration of 100 pg/ml or chloramphenicol at final concentration of 2 mM) were added when specified. To each flask, 0.5 mCi of ["Slsulfate was added, and after incubation for 30 min under fluorescent light, over 80% of the 35S was incorporated in the control experiments. The cells were then harvested by centrifugation, broken in a French pressure cell, and the protein complexes were isolated.
For labeling with [I4C]carbonate, cells were grown in 1 liter of medium in which the amounts of citrate and acetate were decreased to 10% of that in the original growth medium. Five ml of cells and 5 mCi of ['4C]Na*COa were added and the 2-liter flask was sealed with Parafilm. After -10 days of growth, over 70% of the I4C was incorporated into trichloroacetic acid-insoluble material. The cells were then harvested, washed, and broken as described above and under "Results."

RESULTS
Purification of Photosystem I Reaction Center-About 6 liters of cell suspension a t chloraphyll concentrations of 10 to 20 pg/ml were harvested by centrifugation at 1,500 X g for 3 min. The cells were washed twice with -200 ml of solution containing 0.4 M sucrose, 0.01 M NaC1, and 0.01 M Tricine-NaOH (pH 8). The cells were suspended in 30 m l of the same solution and were broken twice at -4 "C in a French pressure cell at 4000 p.s.i. The suspension was centrifuged a t 1,500 X g for 3 min and NaCl was added to the supernatant from a stock solution of 4 M to give a final concentration of 0.25 M. The suspension was then centrifuged at 20,000 x g for 15 min and the pellet was homogenized in -40 ml of 10 mM Tricine-NaOH (pH 8). After centrifugation at 60,000 X g for 20 min, the pellet was homogenized in a solution containing 10 mM Tricine (pH 8 ) and 0.15 M NaCl. After similar centrifugation, the pellet was further washed in 10 mM Tricine (pH 8) and suspended in a solution containing 250 mM sucrose and 50 mM Tricine at 0 "C, the suspension was centrifuged at 200,000 X g for 60 min. The supernatant was used for the preparation of the proton-ATPase complex as previously described (15). The pellet was homogenized in a solution containing 25 mM Tricine (pH 8) and 2% Triton X-100 to give a final chlorophyll concentration of 0.5 mg/ml. It was then centrifuged at 10,000 X g for 10 min and the supernatant was kept at -20 "C until used. A sample of -10 ml was thawed and centrifuged at 10,000 X g for 10 min and the supernatant was applied to a DEAE-cellulose column (1 X 12 cm) equilibrated with a solution containing 20 mM Tris-C1 (pH 8) and 2% Triton X-100. The reaction center was eluted with a linear NaCl gradient of 0 to 300 mM (20 d in each chamber) in a solution containing 20 mM Tris-C1 (pH 8) and 2% Triton X-100. The tubes of the fist green peak (containing more than 0.2 mg of chlorophyll/ml) were pooled and fractions of 1 ml were applied to linear sucrose gradients of 5 to 30% sucrose in a solution containing 20 mM Tris-C1 (pH 8) and 0.2% Triton X-100. The gradients were centrifuged in an SW-41 rotor a t 35,000 rpm for 15 h a t 2 "C. The lower green band was collected and used promptly or stored frozen at -70 "C. When desired it could be concentrated on a small DEAE-cellulose column as previously described (9). The purification of the photosystem I reaction center is summarized in Table I. The purified preparation contained -75 chlorophyll a molecules per/l Piw, and it enriched -25fold over the chloroplast membranes. The absorbance spectrum of the isolated reaction center is nearly identical to the one isolated from higher plants. Light-induced P~O O oxidation was readily observed in the purified preparation, but NADP photoreduction could not be detected either in the purified reaction center or in the chloroplast membranes. The regularity of the donor site of the photosystem I reaction center was examined by following the light-induced cytochrome 552 oxidation. Fig. 1 shows that under conditions in which high rates of cytochrome 552 photooxidation were recorded with the photosystem I reaction center from higher plants (8), the chlamydomonas reaction center was not active. However, the inclusion of 200 mM NaCl or 8 mM MgC12 or lowering the pH to 5 induced the light-dependent cytochrome 552 photooxidation activity of the ChZamydomonas photosystem I reaction center. Recent studies in our laboratory have shown that the Swiss chard photosystem I reaction center that was depleted of subunit I11 is analogous to the Chlamydomonas reaction center.' The regularity of the acceptor site of the reaction center is currently being studied by low temperature EPR spectroscopy.
The Chlamydomonas photosystem I reaction center was reconstituted into phospholipid vesicles together with purified proton-ATPase complex from lettuce chloroplasts (13). Upon illumination in the presence of N-methylphenazonium methosulfate, phosphorylation rates above 10 pmol of ATP/mg of chlorophyll/h were recorded.
On sodium dodecyl sulfate gels, the reaction center dissociated into four major bands that were designated as subunits I, 11, 111, and IV with M, =: 70,000, 19,000, 10,000, and 8,000, respectively (Fig. 2). The stoichiometry between subunits I, 11, 111, and IV was found to be 2:l:l:l in three different experiments. This was deduced from the amount of I4C that Stained gel and autoradiography of "C-labeled Chldnydomorum photosystem I reaction center. Lane 1, photosystem I reaction center, containing -30 pg of protein, was electrophoresed on exponential 10 to 15% acrylamide gel as described under "Experimental Procedures." Lane 2, autoradiography of purified photosystem I reaction center that was prepared from cells that were grown in the presence of ["C]Na2C03 as was described in the text. The reaction centers were prepared in the presence of 1 mM each of p-aminobenzamidine, phenylmethylsulfonyl fluoride, N-tosyl-L-phenylalanyl chloromethane, and N-tosyl-L-lysyl chloromethane. was incorporated into the protein bands and the above mentioned molecular weights of the polypeptides.
The fact that in all the preparations of the reaction center, the same subunit composition was obtained strongly suggests that we are dealing with a tight protein complex which is typical for a functional unit in the chloroplast membrane. The designation of its various subunits was done according to the order of decreasing molecular weights and their relation to the subunits of photosystem I reaction center from higher plants will be pointed out in the discussion. Subunit I was dissociated into two apparent protein bands (Fig. 2). This phenomenon was observed previously in photosystem I preparations from Chlamydomonas chloroplasts (3, 4, 6), and in purified photosystem I reaction center from higher plants (8,9). Fig. 3   imide abolished the synthesis of subunit I1 of the photosystem I reaction center and the y, 6, and 11 subunits of the proton-ATPase complex. While preincubation with cycloheximide for 1 h completely prevented the assembly of labeled subunits into the photosystem I reaction center (Fig. 5, lunes 4), labeled (Y and p subunits in the amount of up to 40% of the control were incorporated into the proton-ATPase complex.

DISCUSSION
A photosystem I reaction center containing four different subunits has been isolated from Chlamydomonas chloroplasts. Subunit I resembles that of higher plants in M, = 70,000, its stoichiometry with the other subunits (2:1), and even its appearance on sodium dodecyl sulfate gels as a diffuse band or even resolved into two distinct bands (7-10). A question has been raised as to whether these two bands are two different polypeptides, or whether they are related to each other (3,6,9). The one-dimensional fingerprinting experiment (Fig. 3) clearly indicated a resemblance between the two polypeptides; it is quite likely that their different mobilities on the gel are due to posttranslational modification of the polypeptide.
Attempts to detect sugar moieties covalently bound to subunit I failed, and therefore, the nature of the modification is not known.
of the secondary electron acceptors is missing from the preparation. Since subunit III lacks cysteine, it cannot serve as across the chloroplast membranes.' It is also possible that one "bound ferredoxin;" only subunits II and IV are candidated for this function.
Short term labeling experiments with [""Slsulfate were employed for the study of subcellular sites of synthesis of chloroplast proteins. In uiuo labeling of Chlamydomonas cells in the presence of specific inhibitors revealed that subunits (Y, p, l , and III (the chloroplast proteolipid) (see Refs. 15 and 19) of the proton-ATPase complex are chloroplast products while the y, 6, and subunit II of CFo have been synthesized in a cycloheximide sensitive system. These findings are in line with previous in uiuo studies in higher plants (10,20). The biological significance of synchronizing the synthesis of the proton-ATPase subunits has been discussed elsewhere (10,15). In the photosystem I reaction center, the synthesis of subunits I and The number of chlorophyll a molecules and P700 pigment are comparable to the higher plant reaction center (7,8). However, the Chlamydomonas reaction center is inactive in NADP photoreduction. This might be due to the lack of a polypeptide analog to subunit III of the photosystem I reaction center from higher plants (8,9). We do not yet know whether such a subunit was dissociated from the complex during its preparation or whether the Chlamydomonas photosynthetic system replaces its function by a proton gradient IV was inhibited by chloramphenicol, while the synthesis of subunit I1 was inhibited by cycloheximide. Therefore, subunit I1 appears to be synthesized on cytoplasmic ribosomes, whereas subunits I and IV appear to be chloroplast translation products. It was previously observed that a polypeptide corresponding to subunit I from photosystem I reaction center is made inside the chloroplasts of Chlamydomonas and higher plants (21, 22).
The effect of preincubation with cycloheximide on the incorporation of "S into the various protein complexes is of special interest. The preincubation completely prevented the incorporation of "S into assembled photosystem I reaction center while appreciable amounts of "S were observed in the chloroplast translation products of the assembled proton-ATPase complex. This must be due to the existence of pools of cytoplasmic products as precursors outside the chloroplasts, or as mature subunits inside the organelle (15). The assembly of photosystem I reaction center was abolished by blocking the protein synthesis on cytoplasmic ribosomes. It is tempting to suggest that subunit I1 is responsible for this property and that this subunit serves as a template for the assembly of photosystem I reaction center. However, it was recently shown by Girard et al. (23) that several nuclear genes are responsible for the functional assembly of photosystem I in the chloroplast membrane.
It is quite clear that photosystem I reaction centers from higher plants and Chlamydomonas resemble each other in several respects. It is also likely that one or more of the missing three subunits of the Chlamydomonas reaction center might be loosely bound polypeptides that were lost during its preparation.