Role of sulfoquinovosyl diacylglycerol for the maintenance of photosystem II in Chlamydomonas reinhardtii Ayumi Minoda 1 , Norihiro Sato 1 , Hisayoshi Nozaki 2 , Katsuhiko Okada 1 , Haruko Takahashi 1 , Kintake Sonoike 3 and Mikio Tsuzuki 1 1 School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, Japan; 2 Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Hongo, Tokyo, Japan; 3 Department of Integrated Biosciences, Graduate School of Frontier Sciences, the University of Tokyo, Kashiwa, Chiba, Japan The physiological role of sulfoquinovosyl diacylglycerol (SQDG) in photosynthesis was investigated with a SQDG defective mutant (hf-2) of Chlamydomonas reinhardtii that did not have any detectable amount of SQDG. The mutant showed a lower rate of photosystem II (PSII) activity by  40% and also a lower growth rate than those of the wild- type. Results of genetical analysis of hf-2 strongly suggest that the SQDG defect and the lowered PSII activity are due to a single gene mutation. The supplementation of SQDG to hf-2 cells restored the lowered PSII activity to the same level as thatof wild-type cells, and also enabled the mutant to grow even in the presence of 135 n M 3-(3,4-dichlorophenyl)-1,1- dimethylurea. Moreover, the incubation of isolated thylakoid membranes of hf-2withSQDGraisedthelowered PSII activity. Chemical modifications of SQDG impaired the recovery of PSII activity. The results suggest that SQDG is indispensable for PSII activity in Chlamydomonas by main- taining PSII complexes in their proper state. Keywords: sulfoquinovosyl diacylglycerol; photosystem II; Chlamydomonas; thylakoid membrane; glycolipid. The primary process in photosynthesis occurs in thylakoid membranes. Protein complexes such as photosystem (PS) I and PSII play a role of energy conversion from excitation energy to redox potential. As autotrophic organisms obtain energy by photosynthesis, its efficiency is a matter of survival. Thylakoid membranes consist of four glyceroli- pids; monogalactosyl diacylglycerol (MGDG), digalactosyl diacylglycerol (DGDG), phosphatidylglycerol (PtdG) and sulfoquinovosyl diacylglycerol (SQDG). The lipophilic matrix may promote the efficiency of the reaction by organized pigment protein complexes [1–3]. Thylakoid membranes adapt to environmental conditions such as the unsaturation of lipids under low temperatures [4,5]. Thus, it is important to investigate the participation of thylakoid lipids in photosynthesis. Of all known thylakoid membrane lipids, SQDG is a unique acidic glycolipid due to an unusual head group, sulfoquinovose, and is likely to be present almost exclusively in thylakoid membranes [6]. In anoxygenic photosynthetic bacteria, it is localized in the Proteobacteria a-subgroup except for Rhodopseudomonas viridis whose photosystem resembles the PSII complex of higher plants [7]. Addition- ally, SQDG is present in organisms that perform oxygenic photosynthesis from cyanobacteria to higher plants, except for Gloeobacter violaceus PCC7421, which is considered as one of the ancient cyanobacteria and lacks thylakoid membranes [8,9]. SQDG-null mutants of Rhodobacter sphaeroides and Synechococcus sp. PCC7942 did not show any effect on the photosynthetic apparatus [10,11]. By separation of the PSII complex using detergents, it can be shown that SQDG is associated with the PSII complex in thylakoid membranes [1,12–15]. In contrast to the SQDG-null mutants of bacteria, the SQDG-defective mutant (hf-2) obtained by UV irradi- ation in Chlamydomonas reinhardtii showed a slightly slower growth rate and a 40% decrease in PSII activity as compared with that of the wild-type [16,17]. We will report here the genetic analysis of hf-2 in detail and the attempt to compensate the deficiency of SQDG in hf-2 by the culture in the presence of SQDG to elucidate that SQDG participate in PSII activity in C. reinhardtii. MATERIALS AND METHODS Algal culture SQDG-deficient mutant of C. reinhardtii,whichwasdesig- nated as hf-2, was originally obtained by Sato et al.[16]and was backcrossed with wild-type cells five times to segregate the deficiency of SQDG from other mutations. Most of the experiments were carried out with the F5 population of the mutant, except for segregation analysis and for the deter- mination of peptide composition in thylakoid membranes where the F3 population was used. Cells of C. reinhardtii CC125 (mt+) and hf-2 (mt–) were grown with 3/10HSM medium [17] in an rectangular glass vessel under continuous Correspondence to M. Tsuzuki, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Fax: + 81 426 76 6721, Tel.: + 81 426 76 6713, E-mail: mtsu@ls.toyaku.ac.jp Abbreviations: Chl, chlorophyll; SQDG, sulfoquinovosyl diacylglycerol; MGDG, monogalactosyl diacylglycerol; DGDG, digalactosyl diacylglycerol; PtdG, phosphatidylglycerol; PSI, photo- system I; PSII, photosystem II; LHC, light-harvesting chlorophyll a/b- protein complex; diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea. (Received 17 December 2001, revised 13 March 2002, accepted 20 March 2002) Eur. J. Biochem. 269, 2353–2358 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02896.x light (90 lEÆm )2 Æs )1 )at28°Cwith2%CO 2 bubbling. The trace elements of the 3/10HSM medium were replaced by Arnon’s A 5 solution [17]. Measurement of PSII activity Cells at mid-logarithmic phase were harvested by centrifu- gation, resuspended with 50 m M Tricine/KOH (pH 7.5), and continuously shaken in the light before measurement. Oxygen evolution was monitored with a Clark-type elec- trode (Rank Brothers Ltd, Cambridge, UK) at a light intensity of 500 lEÆm )2 Æs )1 and 25 °C. The reaction medium for the measurement of PSII activity was composed of 50 m M Tricine/KOH (pH 7.5), 3.3 m M ammonium chlor- ide, 0.5 m M p-benzoquinone as an electron acceptor and cells corresponding to 6.0 lg chlorophyll (Chl) per mL. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea (diuron) dissolved in ethanol was used and carefully diluted with 1 : 100 (v/v) of the reaction mixture. For the preparation of thylakoid membranes for PSII activity, 5 mL of cell suspension was sonicated for 9 s in 25 m M Hepes (pH 7.5), 0.4 M sucrose, 1 m M MgCl 2 ,1% BSA, and 5 m M bicarbonate at a power setting between three and four with the Sonifier 250D at Branson, CT, USA [18]. The suspension was centrifuged at 1200 g for 1 min at 4 °C to remove unbroken cells and cell debris. Its supernatant was recovered by centrifugation at 13 000 g for 10 min at 4 °C, and resuspended in 25 m M Hepes (pH 7.5) containing 0.4 M sucrose and 2 m M MgCl 2 . The same buffer was used for measurements of PSII activity in thylakoid membranes. PSII activity in thylakoid membranes was also determined with essentially the same method as that in cells. Either 0.5 m M p-benzoquinone in dimethylsulfoxide or 1 m M ferricyanide in H 2 O was used as an electron acceptor. Chlorophyll was determined as described previously [17]. Polypeptide analysis of thylakoid membranes Thylakoid membranes were isolated by a method described previously [19] with minor modifications. Cells grown in the 3/10HSM medium containing SQDG were washed three times with a first buffer in the procedure [19], and then were disrupted by sonication using the Sonifier 250D at power setting three for 15 s with cooling. This process was repeated several times with cooling intervals of 1 min until 80–90% of the cells was disrupted as observed under the light micros- copy. An ultracentrifugation over a sucrose gradient was carried out at 150 000 g for 90 min at 4 °C with the SW40Ti rotor in the Ultracentrifuge L8-T from Beckman (Palo Alto, CA, USA). Other ultracentrifugations were carried out at 24 000 g for 15 min at 4 °C. Thylakoid membranes corres- ponding to 0.5 lg Chl per mL were denatured by the incubation with 5% lithium dodecyl sulfate, 60 m M dithio- threitol and 30% sucrose for 2 h at room temperature. The polypeptides in thylakoid membranes corresponding to 10 lg Chl were separated by SDS/PAGE in a 16–22% gra- dient containing 0.1% SDS and 22.5% urea [20]. Polypeptide bands were stained with Coomassie Brilliant Blue. Lipid analysis Lipids of whole cells and isolated thylakoid membranes were extracted as described previously [21]. Lipids were separated on Silica gel 60 plate (Merck & Co. Inc., Rahway, NJ, USA; 0.25 mm) by developing with solvent A (chloro- form/methanol/25% ammonia solution, 65 : 35 : 5; v/v/v) or solvent B (chloroform/methanol/H 2 O, 65 : 25 : 4; v/v/v). Separated lipids were detected by spraying with primuline (Tokyo Kasei, Tokyo, Japan) in 80% acetone and visual- ized with a UV illuminator. For determination of the quantity of lipids, they were transformed to methylesters with 5% methanolic HCl and then analyzed by gas-liquid chromatography (Shimadzu, Kyoto, Japan; GC-14B) equipped with a hydrogen flame- ionization detector. Arachidic acid was used as an internal standard. The fatty acid methyl esters were separated on a capillary column HR-Thermon-3000B from Shinwa Chem- ical Industries Ltd, Kyoto, Japan (0.25 mm; inside diameter 25 m). Temperatures of the column and flame-ionization detector were 180 °Cand250°C, respectively. The analysis of chromatographic data was performed with a data processor (Shimadzu, Chromatopack CR-7 plus). Lipids were dissolved in chloroform/methanol (2 : 1, v/v) and stored at )20 °C. Supplementation of algal cells and thylakoid membranes with SQDG SQDG for supplementation to hf-2 cells and their thylakoid membranes was extracted and separated with solvent A from total lipids of Synechocystis PCC6803 and C. rein- hardtii. The isolated SQDG was suspended in 3/10HSM medium after removing organic solvents. The lipid in 3 mL medium was transformed to liposomes by a sonication for 30 min with an ultrasonic cleaner (Branson, CT, USA), and then was filtered with a disposable mixed cellulose ester filter (0.02 mm, Toyo Roshi Kaisha, Ltd, Tokyo, Japan) before it was supplied to the culture. The cells were cultured for 2 days in 3/10HSM medium containing the lipid at 8.5 l M . For the analysis of the diuron effect, various concentrations (0–100 l M ) of SQDG were added to the cells on agar plates containing 135 n M diuron. For lipid analysis of the cells cultured in the presence of SQDG, the cells were washed three times with fresh medium before the lipid extraction. The lipids were analyzed using silica gel plates with solvent A. Thylakoid membrane lipids corresponding to 2 lmol of fatty acid were developed with solvent B and a spot containing SQDG and PtdG was scraped out. The mixture of SQDG and PtdG extracted from the silica powder was developed with solvent A. Sugar-oxidized and methylated SQDG for supplementa- tion were prepared as described below. The sugar-modified SQDG and PtdG were incorporated into liposomes prior to their addition to the culture, while methylated SQDG was dissolved in ethanol. Modifications of SQDG The sulfonic residue of SQDG was methylated by incuba- tion with diazomethane in ether after being protonated by the addition of 0.1 M HCl [22]. For oxidation of the sulfoquinovose of SQDG, SQDG in H 2 O was transformed to liposomes by sonication. The suspension was shaken with 5% sodium periodate for at least 1 h at room temperature [22]. Modified SQDGs were purified by separation on silica gel plates. 2354 A. Minoda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 RESULTS Co-segregation of the SQDG defect and decrease in PSII activity To investigate phenotypes derived from SQDG deficiency, we obtained 36 tetrads in the F3 population by repeated crossings of hf-2 with wild-type cells. Lipid compositions of all tetrads revealed that two progenies were similar in phenotype to the wild-type and the others to hf-2ineach tetrad (Fig. 1). None of the hf-2 mutants had detectable amounts of SQDG on the thin layer chromatography plate. The amount of SQDG in hf-2 was quantified to be under 2.8% of that in the wild-type by 35 S-labeling experiments (data not shown), suggesting that the average number of SQDG molecules bound to each PSII complex was less than one [17]. The average of PSII activities in SQDG defective progenies was 225 ± 20 lmol O 2 Æmg Chl )1 Æh )1 , which cor- responds to 67% of the activity in wild-type progenies (338 ± 24 lmol O 2 Æmg Chl )1 Æh )1 ) (Fig. 1). These results strongly suggest that a single gene mutation caused the SQDG defect and the decrease of PSII activity in hf-2. There was no difference between the wild-type and backcrossed hf-2 in peptide composition of thylakoid membranes (Fig. 2). The backcrossed hf-2 mutants did not show any abnormal structure of thylakoid membranes with transmission electron microscopy (data not shown), though the original hf-2 showed extremely curved thylakoid membranes [19]. The decrease of PSII activity in hf-2 compared with that of the wild-type is therefore neither due to the change in peptide composition of the PSII complex nor to the ultrastructure of thylakoid membranes. The growth rate of hf-2 was slightly slower than that of the wild-type when cells were grown photoautotrophically, as described previously [17], while it was equal to that of the wild-type when cells were grown photomixotrophically (data not shown). Cells of hf-2 tended to suffer from photo- inhibition, if light intensities higher than 600 lEÆm )2 Æs )1 were used for 30 min [23]. These results suggest that SQDG is important for the maintenance of a high activity of PSII in Chlamydomonas. Incorporation of SQDG to hf -2 Although the hf-2 mutant could grow photoautotrophically on agar plates, it could not grow in the presence of 135 n M diuron, but the wild-type could grow under this condition. The supplementation of SQDG in the medium enabled the mutant to grow even in the presence of diuron (Fig. 3). The exogenously applied SQDG is incorporated into the mutant cells in the form of liposomes [24], probably in the thylakoid membrane in the region of PSII. The effect was observed at over 2.4 l M of SQDG, but not at 0.48 l M . When hf-2 was cultured in the medium containing 8.5 l M SQDG for 1 day, SQDG was detected in the whole cell (data not shown). An exact amount of SQDG was detected in isolated thylakoid membranes when hf-2 was cultured under the same conditions for 2 days (Fig. 4). The exogen- ous SQDG did not have a significant effect on the remaining lipid composition, neither in the whole cell nor in thylakoid membranes. No effect was observed on the peptide composition in thylakoid membranes by the supply of SQDG (data not shown). Fig. 1. Segregation pattern of phenotypes in SQDG-defective mutant of C. reinhardtii hf-2. Thirty-six tetrads in the F3 population were obtained by crossing hf-2withwild-typecells.Fourcirclesoneachver- tical line means a single tetrad from the same zygote; white circles show wild-type progenies and black ones SQDG-defective progenies on lipid composition. All tetrads examined were numbered horizontally. PSII activity was measured with p-benzoquinone as an electron acceptor and the values were the average of two independent experiments. Fig. 2. Polypeptide composition in thylakoid membranes of the CC125 (lane a) and hf-2 (lane b) in C. reinhardtii. Thylakoid membranes of the wild-type and hf-2 corresponding to 10 lgChlÆml )1 were separated with SDS/PAGE. Bands indicate as follows: 1, apoproteins of the PSI complex; 2, ATP synthase ab subunits; 3, apoproteins of the PSII complex; 4, apoproteins of LHCII. Ó FEBS 2002 Sulfolipid maintains PSII in Chlamydomonas (Eur. J. Biochem. 269) 2355 We then measured the PSII activity in hf-2culturedinthe presence of SQDG (Fig. 5). Inhibition of PSII activity by diuron was more pronounced in hf-2thanthatinthewild- type. Exogenous SQDG raised the lowered PSII activity to the same level as in wild-type cells in the absence of diuron, and decreased the sensitivity to diuron in hf-2. There was no difference in the effect of SQDG isolated from either Synechocystis PCC6803 or C. reinhardtii (data not shown). Measurements of PSII activities in isolated thylakoid membranes showed a recovery of the lowered PSII activity when hf-2 was cultured in the presence of SQDG (Table 1). A similar result was obtained when ferricyanide was used as an electron acceptor (data not shown). No effect was found by the addition of SQDG to the medium, neither in wild- type cells nor in the isolated thylakoid membranes of the wild-type. Moreover, when isolated thylakoid membranes from hf-2wereincubatedwith100 l M SQDG for 10 min on ice at 0.75 mg ChlÆmL )1 , the PSII activity of the membranes increased from 210 ± 7 to 256 ± 4 lmol O 2 Æmg Chl )1 Æh )1 , which is almost the same rate as in wild-type cells. This result suggests that the de novo protein synthesis is not required for the restoration. Exogenous SQDG incorporated in thylakoid membranes may directly affect the PSII activity in thylakoid mem- branes. We investigated PSII activity of hf-2culturedinthe presence of PtdG and two modified SQDGs; methylated- SQDG, in which a sulfonic residue of sulfoquinovose was methylated, and sugar-oxidized SQDG, in which the sugar part of sulfoquinovose was cleaved by a periodate oxidation. Neither methylated SQDG nor PtdG affected the PSII activity of hf-2 (Table 1). Sugar-oxidized SQDG raised ABC Fig. 3. Effect of exogenous SQDG on photo- autotrophic growth of C. reinhardtii CC125 (right part) and hf-2 mutant (left part) on agar plates in the presence of 135 n M diuron. Medium: (A) 3/10HSM (B) 3/10HSM containing diuron, and (C) 3/10HSM containing both diuron and 100 l M SQDG. Fig. 5. Effect of exogenous SQDG addition on PSII activity of hf-2 and the wild-type in C. reinhardtii in the presence of various concen- trations of diuron. The values of PSII activities are the means of three independent experiments and SD. PSII activities were measured with 0.5 m M p-benzoquinone in CC125 (circles) and hf-2 (squares) fed with 8.5 l M SQDG (closed symbols) or without SQDG (open symbols) for 2days. Fig. 4. Incorporation of exogenous SQDG into thylakoid membranes of C. reinhardtii hf-2 cultured in the presence of SQDG. The wild-type (CC125 strain) (lane 1) and hf-2 (lanes 2,3) grown in the medium with (lane 3) or without (lanes 1,2) 8.5 l M SQDG for 2 days. Spots on the silica gel plate were visualized with an UV illuminator after being sprayed with primulin. Table 1. Photosystem II activity of C. reinhardtii hf-2 in which deriva- tives of SQDG and PtdG were incorporated by incubation for 48 h. The values obtained in the presence of p-benzoquinone are given as means of three independent experiments ± SD. Addition Photosystem II activity (lmol O 2 Æmg Chl )1 Æh )1 )in Cells Isolated thylakoid membranes Control 189 ± 21 210 ± 7 SQDG 261 ± 19 256 ± 4 Methylated SQDG a 207 ± 14 198 ± 17 Sugar-oxidized SQDG b 223 ± 21 231 ± 7 PtdG 174 ± 22 206 ± 16 a Sulfonate group of sulfoquinovose in SQDG was methylated. b The sugar part of SQDG was broken with sugar oxidization. 2356 A. Minoda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the PSII activity of hf-2 only a little (Table 1). The sulfonic residue of SQDG may be more important than the sugar part for the maintenance of PSII activity, although both the sulfonic residue and the sugar part of SQDG may be required for the appearance of full PSII activity. DISCUSSION Genetic analyses strongly suggest that the deficiency of SQDG and the lowered PSII activity are due to a single gene mutation in the SQDG-deficient mutant, C. rein- hardtii hf-2. The mutant is more sensitive to diuron than the wild-type (Figs 4 and 5). These phenomena in hf-2are due to the missing SQDG. The activity of PSII was restored by incorporation of exogenous SQDG from the medium (Figs 3–5), but the recovery greatly reduced when chemically modified SQDGs and PtdG were supplied (Table 1). We therefore concluded that the PSII complex is affected by SQDG in thylakoid membranes of Chlamydo- monas. As the incubation of thylakoid membranes of hf-2with SQDG significantly raised the PSII activity, SQDG may interact with the PSII complex directly. Specific binding sites could be implicated, as the restoration of PSII activity is achieved by addition of a very small amount of SQDG to thylakoid membranes. The lowered PSII activity in hf-2 could be due to an impairment of the reaction center in the PSII complex, rather than to a decrease of the antenna size in PSII, or a decrease in the efficiency of energy transfer from LHCII to the reaction center [17]. Therefore, a conformational change of the PSII complex may cause the decrease of PSII activity in hf-2bythelackofspecific binding of SQDG to the PSII complex. Alternatively, the change of the lipophilic surrounding at Q B site of the PSII complex might cause the decrease of PSII activity even without the conformational change in protein complex. Diuron inhibits PSII activity by binding to the relatively hydrophobic Q B pocket, which is the exit of the electron flow in the PSII complex. According to the increase in the sensitivity to diuron in hf-2, the limitation of the electron transfer at the exit of the PSII complex might lower the whole PSII activity as a result of the change in the lipophilic environment at the Q B pocket. The electron flow in PSII might be commonly affected by the negative charge of SQDG and/or PtdG which are both located in the region of the PSII complex. A PtdG- null mutant of Synechocystis PCC6803 showed an impaired rate of PSII activity [25,26], suggesting a change in the conformation of PSII complexes or in the lipophilic environment at Q B site. Immunological experiments in the filamentous cyanobacterium Oscillatoria chalybea showed that PtdG with its negatively charged surface increased the hydrophobicity of D1 peptides [27]. The rate of electron flow from Q A – was intensified by the treatment of thylakoid membranes with the appropriate concentra- tion of phospholipase C which removed the head-group of phospholipid molecules [28]. Acidic lipids localized around protein complexes could affect electron flow, a finding which may be a key to realizing the role of acidic lipids in photosynthesis. The contribution of SQDG to the maintenance of maxi- mum PSII activity in C. reinhardtii was not observed with SQDG-null mutant in R. sphaeroides and Synechococcus PCC7942 [10,11]. The discrepancy between C. reinhardtii and these bacteria may not be due to structural differences of the lipids, which are almost the same [6]. In fact, the lowered PSII activity in hf-2 was restored by the incorpor- ation of SQDG prepared from both Synechocystis sp. PCC6803 and C. reinhardtii. The discrepancy may be due to the differences in PSII complexes of C. reinhardtii and the bacteria. The reaction center of PSII complex is highly conserved from photosynthetic bacteria to higher plants, whereas some differences have been found among Synecho- cystis PCC6803 and C. reinhardtii especially in the periph- eral polypeptides of 5–15 kDa such as psbK and psbH of the PSII complex [29,30]. These differences in PSII imply a variety of roles of peripheral components of the protein– lipid interactions. In Oscillatoria chalybea, the D1 polypep- tide reacted only with antibodies against PtdG, but not with those against galactolipids [27], in contrast to tobacco where the D1 peptide reacted also with antibodies to MGDG [12]. In this relation, a disruptant of Synechocystis sp. PCC6803, which has an ORF homologous to sqdB, was found to require SQDG supplementation for its photoautotrophic growth [31]. The localization of SQDG in thylakoid membranes of Chlamydomonas showed heterogeneity, and was associated with the PSII core complex and parts of LHCII [17]. Additionally, SQDG was found to be tightly bound to purified LHCII of C. reinhardtii [13]. In higher plants, SQDG was found in all fractions of Chl-binding proteins of PSII in very low amounts in maize mesophyll chloroplast [14] and in the outer surface of the heterodimer D1/D2 in the PSII complex of tobacco where it was accessible to antibodies to SQDG [12]. SQDG was also found in PSII membranes [32] and the PSII core dimers [1] of spinach. Therefore, SQDG is associated with the PSII complex in thylakoid membranes of chloroplasts regardless of the species, even though there may be variability in the amount among species. In higher plants, photosynthetic perform- ance was not affected by the antisense expression of SQD1 cDNA of Arabidopsis thaliana, while it caused a decrease in the amount of SQDG [33]. Acyl chains of SQDG in higher plants are more unsaturated than those of algal SQDG, like other glycerolipids in thylakoid membranes, although the lipid composition is almost equal between Chlamydomonas and higher plants [2,6]. In light of the result that a very small amount of SQDG is enough to recover the lowered PSII activity in Chlamydomonas, the SQDG-deficient mutant, if it was obtained, might show some different phenotypes from the wild-type in Arabidopsis. In conclusion, we have shown the requirement of SQDG for the maintenance of PSII activity in Chlamydomonas.The molecular mechanism of the role of SQDG in PSII should be further investigated. ACKNOWLEDGEMENTS The authors thank Drs A. Yamagishi and K. Iguchi in Tokyo University of Pharmacy and Life Science for their helpful discussion and technical support. They are also indebted to Dr T. Kuroiwa in the University of Tokyo and Drs M. Washizu and K. Okabe in Advance Co. Ltd. for their kind help during the research. This work was supported by grants from the Ministry of Education, Science, Sports and Culture, from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and CREST of JST (Japan). Ó FEBS 2002 Sulfolipid maintains PSII in Chlamydomonas (Eur. J. Biochem. 269) 2357 REFERENCES 1. Kruse, O., Hamkamer, B., Konczak, C., Gerle, C., Morris, E., Radunz, A., Schmid, G.H. & Barber, J. (2000) Phosphatidylgly- cerol is involved in the dimerization of photosystem II. J. 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