Photosystem I1 Particles from Chlamydomonas reinhardtii PURIFICATION, MOLECULAR WEIGHT, SMALL SUBUNIT COMPOSITION, AND PROTEIN PHOSPHORYLATION*

particles from Chlamydomonas reinhardtii by They contained thepsbA,psbB,psbC, and psbD gene products in a 1/1/1/1 stoichiometry, cytochrome and several small polypeptides, and exhibited electron transfer from donor Z to acceptor chlorophylls/reducible Upon ultracen- trifugation and molecular sieving in the presence of as monomers of a small of a partially intercon- nuclear-encoded

from higher plants, cyanobacteria, and algae have been extensively studied. They comprise numerous prosthetic groups and a large number of subunits encoded either by chloroplast or nuclear genes. The function of many of these polypeptides remains unknown (reviewed by Ghanotakis and Yocum, 1990).
The simplest photoactive PSII reaction centers yet obtained in higher plants contain five intrinsic subunits encoded in the chloroplast by psbA, psbD, psbE, psbF, andpsbl genes (Nanba and Satoh, 1987;Webber et al., 1989). psbA and psbD encode the two larger subunits, respectively D l and D2, which cooperate in the binding of the primary reactants. These subunits show sequence homologies with subunits L and M of the purple bacteria reaction centers (Michel et al., 1986;Trebst, 1986). The three other subunits are small, and each probably forms a single transmembrane a-helix: psbE and psbF encode subunits a and p of cytochrome b559, respectively (Herrmann et al., 1984), whereas the product of thepsbl gene is not known to bind any cofactor.
Larger PSII complexes contain additional subunits, many of which are involved in energy collection or oxygen evolution. The "core antenna" is comprised of two intrinsic subunits encoded in the chloroplast by the psbB and psbC genes (Bricker, 1990). A major light-harvesting complex (LHCII) and minor chlorophyll-protein complexes (CPmin) are present in PSII membranes but essentially absent in PSII particles (Ghanotakis and Yocum, 1990;Bassi and Dainese, 1990); they are comprised of nuclear-encoded polypeptides that bind chlorophylls a and b. Three extrinsic subunits (OEEl,OEE2, and OEE3), encoded by nuclear genes, are part of the oxygen evolution center; the smaller oxygen-evolving PSII particles contain OEEl but neither OEE2 nor OEE3 (e.g. see Ghanotakis et al. (1987) and Haag et al. (1990)). Several other subunits (such as those encoded in chloroplasts by the psbH, psbJ, psbK, psbL, psbM, andpsbN genes) have been identified (generally by N-terminal sequencing) in PSII particles from various species (Ikeuchi et al. 1989a(Ikeuchi et al. , 1989b(Ikeuchi et al. , 1989cKoike et al., 1989;Webber et al., 1989).
The unicellular green alga Chlamydomonas reinhardtii is a good model system for studying PSII because of the possibility of growing the cells heterotrophically, of obtaining photosynthesis mutants, of transforming cells with foreign DNA, and of studying nucleus-organelle interactions. Several C. reinhardtii polypeptides, some of them phosphorylated, have been proposed to be PSII subunits on the basis of their presence in PSII particles and their absence in PSII-deficient mutants (Delepelaire, 1984;Delepelaire and Wollman, 1985;de Vitry et al., 1987). Although the genes encoding the large subunits of PSII have been identified and sequenced, little is known about the small subunits. One aim of the present study is to further characterize PSII subunits in C. reinhardtii and to establish to what extent these subunits are common to higher plants and cyanobacteria. We describe the isolation of PSII particles from C. reinhardtii, further purified with respect to the earlier procedure (Diner and Wollman, 1980). We have analyzed the particle composition (components and stoichiometry). We show that the molecular weight of the complex in two detergents (lauryl maltoside and CI2EB) corresponds to that of a monomer. We have characterized several small PSII subunits by N-terminal sequencing, immunoblotting, pulselabeling in the presence of translation inhibitors, and 32Plabeling of phosphopolypeptides.

DISCUSSION
Purification and Molecular Weight of PSII Particles-Further purification of photoactive PSII particles from C. reinhardtii was achieved by following the previous procedure (Diner and Wollman, 1980) with an ion-exchange chromatography step. This step removes the extrinsic proteins and most of the peripheral antenna without inactivating the reaction center. The monodispersity of the preparations was established by HPLC gel molecular sieving. The particles are similar in photoactivity (electron transfer from secondary donor Z (a tyrosine residue of subunit Dl) to primary quinone acceptor QA, with a stoichiometry of [40][41][42][43][44][45][46][47][48][49][50] chlorophylls/ reducible QA), in subunit composition, and in size (without detergent) to previously described preparations of non-02evolving PSII particles from cyanobacteria (Rogner et al., 1990;Dekker et al., 1988) and higher plants (Akabori et al., 1988). Such particles are sometimes referred to as PSII cores.
Taking into account the contribution of two small subunits whose presence is highly probable but which remained undetected, presumably because of blocked N termini (psbF and psbl gene products), the total M , of identified proteins and cofactors in C. reinhardtii PSII particles (see below) is -274,000 (Table 1A). This value is slightly higher than that of 255,000 (without detergent) estimated for Synechococcus particles on the basis of gel filtration (Rogner et al., 1990). When the contributions of bound lipids and detergent (including hydration water) are included, the total calculated value (417-430 kDa, depending on the detergent) is close to that determined from the particles' hydrodynamic properties (440-510 kDa), indicating that they are monomers (Table  1B).
Polypeptide Composition-The purified particles contained psbA, psbB, psbC, and psbD in a 1/1/1/1 stoichiometry and several small polypeptides. Establishing the exact complement of low M , subunits is not straightforward because they generally stain poorly and some of them comigrate. Four of them, namely a subunit of 6.1 kDa encoded in the nucleus and the products of chloroplast genes psbE, psbM, and psbK could be identified by sequencing their N terminus. This is the first report of their presence in PSII particles from a green alga. A homologous nuclear subunit of 6.1 kDa has been previously described in higher plants (Ikeuchi et al., 1989a;Schroder et al., 1988), but has not been hitherto observed in cyanobacteria. The a subunit of cytochrome bSs9 (product of the psbE gene) is present, as it is in all PSII reaction centers preparations hitherto described (Ghanotakis and Yocum, 1990). The very low yield of PTH-derivatives from the subunit Portions of this paper (including "Materials and Methods," "Results," Figs. 1-6, and Tables 1-5) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press. presenting some homology with the psbM gene product suggests a blocked N terminus, as in cyanobacteria (Ikeuchi et al., 1989b). The psbM protein has not yet been observed in higher plant particles. Finally, the sequence data show that the psbK gene, whose sequence in C. reinhardtii has recently been established (Silk et al., 1990), is expressed and its product is part of PSII particles, as in higher plants (Murata et al., 1988) and cyanobacteria (Koike et al., 1989).
Three putative subunits whose presence was suspected could not be identified, presumably because of blocked N termini: 1) the product of the psbl gene, whose N terminus is blocked in all species hitherto studied (Ikeuchi et al., 1989c;Webber et al., 1989), 2 ) that of the psbN gene, hitherto detected only in cyanobacteria where its N terminus is blocked (Ikeuchi et al., 1989b), and 3) that of the psbF gene (cytochrome bSS9 p subunit), which is blocked in higher plants (Webber et al., 1989) but not in cyanobacteria (Ikeuchi et al., 1989a). We attempted to establish by immunoreaction the presence of the / 3 subunit, but the antiserum we used, raised against spinach cytochrome b559, recognized only the a subunit in C. reinhardtii.
The composition in low molecular weight subunits of C. reinhardtii PSII particles therefore appears consistent with that observed in other organisms. Except for cytochrome bnns, none of these small polypeptides has been shown to bind cofactors, and their role is still unknown. Given the small size of the corresponding genes, the probability of nondirected mutations is low. The possibility to disrupt or modify these genes by homologous recombination should give clues as to how essential they are for the function and assembly of the PSII complex.
Many small subunits present in PSII particles are predicted to form a single transmembrane a-helix with short extramembrane segments (e.g. psbM in Table 3). Altogether, 10 or 11 different subunits of this type have been characterized (see Table 2). This process of "microassembly" (de Vitry and Popot, 1989;Popot and de Vitry, 1990) is exemplified by the proposed structure of cytochrome bss9, in which two independent subunits, each forming a single transmembrane helix, contribute 1 histidine residue each toward ligation of the heme (Herrmann et al., 1984). The existence of such subunits reflects the strong conformational constraints to which transmembrane polypeptides are subjected. As the larger folding domains encountered in soluble proteins, single transmembrane helices can fold autonomously, providing specific surfaces for association with the rest of the complex (Popot and Engelman, 1990).
PSII Phosphorylation-At least three PSII proteins can be phosphorylated, namely P6 (product of the psbC gene, which yields P6'), D2.2 (psbD product, yielding D2.1), and L7 (presumed psbH product, yielding L5 and L6). The apparent Mr in SDS-urea-PAGE gels of the nonphosphorylated forms was lower than that of the phosphorylated ones. Their amount increased with dephosphorylation by phosphatase, paralleling the decrease of the phosphorylated forms. They were still present in PSII mutants devoid of PSII phosphorylation, whereas the phosphorylated forms were absent.
The existence of slow posttranslational phosphorylations leading to the appearance of P6', D2.1, L5, and L6 was first established by Delepelaire (1984). In his 14C-labeled pulsechase experiments, appearance of the phosphorylated forms paralleled a decrease in the nonphosphorylated proteins P6, D2.2, L7, and L3. Our data suggest that L7 is the nonphosphorylated form and that L5 and L6 are two different phosphorylated forms of the psbH gene product. However, we cannot exclude that L5, L6, and L7 correspond to more than one protein.
The N-terminal sequence of psbH product in C. reinhurdtii is aligned with that in other species in Table 4. The second residue (Thr-2) has been shown to be phosphorylated both in C. reinhurdtii (Dedner et al., 1988) and in spinach (Michel and Bennet, 1987). The existence of two distinct phosphorylated forms, L5 and L6, suggests that in C. reinhurdtii other residue(s) could also be phosphorylated. Thr-17 is a possible candidate. Phosphorylation of Thr-17 would be consistent with the low yield of PTH-derivative observed for this residue, similar to that for Thr-2, in the N-terminal sequence analysis of Dedner et al. (1988). Distinct forms of phosphorylation of the psbH gene product have not been detected previously in other organisms, possibly because of limited resolution of the gels.
The existence of one (or more) phosphopolypeptide(s) migrating with an apparent M , of about 5,000 has already been reported in C. reinhurdtii (de Vitry et al., 1987) and wheat (Webber et al., 1989). In the latter case, it was proposed to originate from phosphorylation of the psbL gene product on Thr-2. The sequence of C. reinhardtii psbL, aligned with that of other species in Table 5, does not include Thr-2. All of its threonine and serine residues are located very close to, if not within, the putative transmembrane segment, and their accessibility to kinases may be limited. It seems possible that the psbL protein may not be phosphorylated in C. reinhardtii, but rather that the psbI and/or psbF proteins, which are expected to migrate in the same region of the gels, are.
Phosphorylation of D l in C. reinhardtii could not be detected in our hands even though the sequence (Erickson et al., 1984) includes a threonine residue that is phosphorylated in higher plants (Michel et al., 1988). Barring this exception, the PSII phosphopolypeptides appear to be similar in C. reinhardtii to those observed in higher plants.
Monomers or Dimers?-The molecular weight of the PSII/ LM and PSII/CI2Es particles indicates they are monomers, with one RC/particle. In LM, monomers were the only detectable form. In CI2E8, however, a small proportion of dimers was detected by rate zonal centrifugation. Monomers and dimers had the same absorption spectrum, the same polypeptide composition, and the same photoactivity and they bound the same amount of ClzEs per RC. Fluorescence induction curves measured on the two forms were similar, indicating the absence of energy transfer between the two centers in the dimer. This suggests that the centers interact by their extramembrane regions, keeping their core antennae distant one from another, rather than by their transmembrane regions. If dimers of the latter type exist and cooperate in uiuo, they did not resist our isolation protocol.
Dimeric PSII particles interacting by their transmembrane regions have been observed by freeze fracture EM and isolated in a thermophilic cyanobacterium, Synechococcus sp. ( Morschel and Schatz, 1987;Dekker et al., 1988); whether the two centers in these dimers cooperate is not known. Spectroscopic measurements on whole cells of purple bacteria (Joliot et al., 1989(Joliot et al., , 1990 showed PSII reactions centers to be dimeric in uiuo. Existence of PSII dimers in higher plants has been suggested on the basis of EM observations of Tris-washed membranes (Seibert et al., 1987;Bassi et al., 1989) and in order to account for the noninteger stoichiometry of antenna subunits (Bassi and Dainese, 1990). On the other hand, a recent EM examination of isolated PSII complexes from spinach has shown most dimers to be comprised of RCs aggregated by their stromal faces (Haag et al., 1990). In intact cells of higher plants, the size and appearance of EFs particles observed by freeze fracture is difficult to reconcile with their representing dimers; size variability was attributed to a variable content of antennae (Staehelin, 1986). In C. reinhurdtii, data are presently limited, but EFs particles observed in a mutant devoid of antennae (LHCII and CPmin) had a diameter of only 10 nm (Olive et al., 1981), which is similar to that of the PSII monomer in Synechococcus. The existence of PSII dimers in uiuo in higher plants and algae and the possibility for two centers in a dimer to cooperate therefore remain open questions.
In conclusion, we have isolated purified PSII particles from C. reinhurdtii. Their polypeptide and pigment composition is very similar to that of higher plant PSII particles, as well as their complement of phosphopolypeptides. Depending on the detergent, the particles are either totally or mainly monomeric. Indirect evidence suggests that dimers may be an artefact of isolation and not naturally occurring. The similarity of C. reinhurdtii and higher plant PSII complexes confirms the usefulness of this unicellular green alga as an organism in which to study the structure, assembly, and regulation of PSII, using combined biophysical, biochemical, and genetic approaches.
Supplemental Yaterial to: Photosystem I1 particles from Chlamydomonas reinhardtil: purlfrcatron, molec~lac weight. small aubunlt COmpOsition. Protcrn phosphorylation. Of the peripheral antenna. The PSI1 particles were then eluted with a buffer contalnrnq 0.2 W NaCl. AI1 steps were done In the presence Of 0.03% LH. The particles wero collected by centrifugstlan after dllutIOD below the CmC Of LW and frozen. For analysxs. the particles were thawed and resuspended Into elther 0.03\ LW or 0.02% C,,E, aolutlon, as needed (see Mat8rI612 6 Nethodr).

O C C U~D~C O of s dlmcrIC form
In the presence Of C,,E,, a rlnsll proportion of a heavler form vas detected by sedlnentatmn velocltycantrlfugatlon (hesvy/heevy*llght -17 t 43; ponded closely to that expected for a dlmer of the llght form (Table IO).

"
As PSI1 SUbUnlt names and W. dlffer from one organlsm to the next. nomenclature tends to be Confusing. The denonlnatlon and prlnclpal characteuhth Informathon gathered in the present vork about their presence or absence rlstlcs Of PSII Subunits ~n C. relnhardtll are summarlred in Table 2, together rn purlfled partlcles and the occ~rence Of phosphorylated forms (see below).   l n h a r d t i i &,a submit (Pig. 4 8 ) . conflrmlng that Its apparent molecular welght (6.000) Indeed is smaller than observed 1" hxgher plants (9.400). The I3 Subunlt of C . r e l n h a r d t l l , which had antiserum. not been xdentlfxed by N-termlnal sequencing, was not detected elther by the C. r. Z. m.

E L g w E e _ l :
DenSltomEtrlC scanning of an autoradlagram of "C-labelled PSII partlcler.
As shown below, thls band includes the D subunit of cytochrome b,,,, encoded the Chloroplast by the pSbE gene, and a nuclear- therefore could be detected on autoradiograms. They mlgrated wlth an apparent Polypeptides 15 and 16 stained poorly In CBB but were labelled by "P and