Role of sulfoquinovosyl diacylglycerol for the maintenance
of photosystem II in
, Norihiro Sato
, Hisayoshi Nozaki
, Katsuhiko Okada
, Haruko Takahashi
and Mikio Tsuzuki
School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, Japan;
Department of Biological Sciences, Graduate School of Science, the University of Tokyo, Hongo, Tokyo, Japan;
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
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 . 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 . 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
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
SQDG-deﬁcient mutant of C. reinhardtii,whichwasdesig-
nated as hf-2, was originally obtained by Sato et al.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  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,
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
trace elements of the 3/10HSM medium were replaced by
Measurement of PSII activity
Cells at mid-logarithmic phase were harvested by centrifu-
gation, resuspended with 50 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
and 25 °C. The reaction medium
for the measurement of PSII activity was composed of
Tricine/KOH (pH 7.5), 3.3 m
ide, 0.5 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
Hepes (pH 7.5), 0.4
sucrose, 1 m
BSA, and 5 m
bicarbonate at a power setting between three
and four with the Soniﬁer 250D at Branson, CT, USA .
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
Hepes (pH 7.5) containing 0.4
sucrose and 2 m
. 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
p-benzoquinone in dimethylsulfoxide or 1 m
O was used as an electron acceptor. Chlorophyll was
determined as described previously .
Polypeptide analysis of thylakoid membranes
Thylakoid membranes were isolated by a method described
previously  with minor modiﬁcations. Cells grown in the
3/10HSM medium containing SQDG were washed three
times with a ﬁrst buffer in the procedure , 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
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 . Polypeptide
bands were stained with Coomassie Brilliant Blue.
Lipids of whole cells and isolated thylakoid membranes
were extracted as described previously . 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
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
For the analysis of the diuron effect, various concentrations
) of SQDG were added to the cells on agar plates
containing 135 n
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
HCl . For oxidation of the
sulfoquinovose of SQDG, SQDG in H
O was transformed
to liposomes by sonication. The suspension was shaken with
5% sodium periodate for at least 1 h at room temperature
. Modiﬁed SQDGs were puriﬁed by separation on silica
2354 A. Minoda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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
(data not shown), suggesting that the average number of
SQDG molecules bound to each PSII complex was less than
one . The average of PSII activities in SQDG defective
progenies was 225 ± 20 lmol O
, which cor-
responds to 67% of the activity in wild-type progenies
(338 ± 24 lmol O
) (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 . 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 , 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
were used for 30 min . These results suggest that SQDG
is important for the maintenance of a high activity of PSII in
Incorporation of SQDG to
Although the hf-2 mutant could grow photoautotrophically
on agar plates, it could not grow in the presence of 135 n
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 , probably in the thylakoid
membrane in the region of PSII. The effect was observed at
over 2.4 l
of SQDG, but not at 0.48 l
When hf-2 was cultured in the medium containing 8.5 l
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
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
SQDG for 10 min on
ice at 0.75 mg ChlÆmL
, the PSII activity of the membranes
increased from 210 ± 7 to 256 ± 4 lmol O
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
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
Medium: (A) 3/10HSM (B) 3/10HSM
containing diuron, and (C) 3/10HSM
containing both diuron and 100 l
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
p-benzoquinone in CC125 (circles) and hf-2 (squares) fed with
SQDG (closed symbols) or without SQDG (open symbols) for
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
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.
Photosystem II activity
Control 189 ± 21 210 ± 7
SQDG 261 ± 19 256 ± 4
207 ± 14 198 ± 17
223 ± 21 231 ± 7
PtdG 174 ± 22 206 ± 16
Sulfonate group of sulfoquinovose in SQDG was methylated.
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.
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-
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 . 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
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
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
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
site. Immunological experiments in
the ﬁlamentous cyanobacterium Oscillatoria chalybea
showed that PtdG with its negatively charged surface
increased the hydrophobicity of D1 peptides . The rate
of electron ﬂow from Q
was intensiﬁed by the treatment
of thylakoid membranes with the appropriate concentra-
tion of phospholipase C which removed the head-group of
phospholipid molecules . 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
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 . 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 , in contrast to tobacco where
the D1 peptide reacted also with antibodies to MGDG .
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
The localization of SQDG in thylakoid membranes of
Chlamydomonas showed heterogeneity, and was associated
with the PSII core complex and parts of LHCII .
Additionally, SQDG was found to be tightly bound to
puriﬁed LHCII of C. reinhardtii . In higher plants,
SQDG was found in all fractions of Chl-binding proteins of
PSII in very low amounts in maize mesophyll chloroplast
 and in the outer surface of the heterodimer D1/D2 in
the PSII complex of tobacco where it was accessible to
antibodies to SQDG . SQDG was also found in PSII
membranes  and the PSII core dimers  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 . 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.
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
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. Biol.
Chem. 275, 6509–6514.
2. Siegenthaler, P.A. (1998) Molecular organization of acyl lipids in
photosynthetic membranes of higher plants. In Lipids in Photo-
synthesis: Structure, Function and Genetics (Siegenthaler, P.A. &
Murata, N., eds) pp. 119–144. Kluwer Academic Publishers,
Dordrecht, the Netherlands.
3. Garnier, J., Wu, B., Maroc, J., Guyon, D. & Tremolieres, A.
(1990) Restoration of both an oligomeric form of the light-har-
vesting antenna CPII and a ﬂuorescence state II-state I transition
-trans-hexadecenoic acid-containing phosphatidilglycerol, in
cells of a mutant of Chlamydomonas reinhardtii. Biochim. Biophy.
Acta 1020, 153–162.
4. Sato, N. & Murata, N. (1981) Studies on the temperature shift-
induced desaturation of fatty acid compositions in the blue-green
algae, Anabaena variabilis. Plant Cell Physiol. 22, 1043–1050.
5. Vogg, G., Heim, B., Gotschy, B., Beck, E. & Hansen, J. (1998)
Frost hardening and photosynthetic performance of Scots pine
(Pinus sylvestris L.). II. Seasonal changes in the ﬂuidity of thyla-
koid membranes. Planta 204, 201–206.
6. Harwood, J.L. (1998) Membrane lipids in algae. In Lipids in
Photosynthesis: Structure, Function and Genetics (Siegenthaler,
P.A. & Murata, N., eds) pp. 53–64. Kluwer Academic Publishers,
Dordrecht, the Netherlands.
7. Linscheid, M., Diehl, B.W.K., O
hle, M., Riedl, I. & Heinz,
E. (1997) Membrane lipids of Rhodopseudomonas viridis. Biochim.
Biophys. Acta 1347, 151–163.
8. Sestam, E. & Campbell, D. (1996) Membrane lipid composition of
the unusual cyanobacterium Gloeobacter violaceus sp. PCC 7421,
which lacks sulfoquinovosyl diacylglycerol. Arch. Microbiol. 166,
9. Benning, C. (1998) Biosynthesis and function of the sulfolipid
sulfoquinobosyl diacylglycerol. Annu. Rev. Plant Physiol. Plant
Mol. Biol. 49, 53–75.
10. Benning, C., Beatty, J.T., Prince, R.C. & Somervile, C.R. (1993)
The sulfolipid sulfoquinovosyl diacylglycerol is not required for
photosynthetic electron transport in Rhodobacter sphaeroides but
enhances growth under phosphate limitation. Proc. Natl Acad.
Sci. USA 90, 1561–1565.
ler, S., Seelinger, A., Ha
(1996) A null mutant of Synechococcus sp. PCC7942 deﬁcient in
the sulfolipid sulfoquinovosyl diacylglycerol. J. Biol. Chem. 271,
12. Voß, R., Radunz, A. & Schmid, G.H. (1992) Binding of lipids
onto polypeptides of the thylakoid membrane. I. Galacto-lipids
and sulfolipid as prosthetic groups of core peptides of the pho-
tosystem II complex. Z. Naturforsch. 47c, 406–415.
13. Sigrist, M., Zwilliengerg, C., Giroud, C.H., Eichenberger, W. &
Boschetti, A. (1988) Sulfolipid associated with the light-harvesting
complex associated with photosystem II apoproteins of Chlamy-
domonas reinhardtii. Plant Sci. 58, 15–23.
res, A., Dainese, P. & Bassi, R. (1994) Heterogeneous
lipid distribution among chloroplast-binding proteins of photo-
system II in maize mesophyll chloroplasts. Eur. J. Biochem. 221,
characterization of a photosystem II reaction center lipoprotein
complex. FEBS Lett. 188, 68–72.
Kawaguchi, A. (1995) Isolation and characterization of mutants
aﬀected in lipid metabolism of Chlamydomonas reinhardtii. Eur.
J. Biochem. 230, 987–993.
17. Sato, N., Sonoike, K., Tsuzuki, M. & Kawaguchi, A. (1995)
Impaired photosystem II in a mutant of Chlamydomonas
reinhardtii defective in sulfoquinovosyl diacylglycerol. Eur.
J. Biochem. 234, 16–23.
18. Roﬀey, R.A., Kramer, D.M., Govindjee & Sayre, R.T. (1994)
Lumenal side histidine mutations in the D1 protein of photo-
system II aﬀect donor side electron transfer in Chlamydomonas
reinhardtii. Biochim. Biophys. Acta 1185, 250–270.
19. Chua, N. & Bennon, P. (1975) Thylakoid membrane polypeptides
of Chlamydomonas reinhardtii: wild-type and mutant strains deﬁ-
cient in photosystem II reaction center. Proc. Natl Acad. Sci. USA
20. Ikeuchi, M. & Inoue, Y. (1988) A new 4.8-kDa polypeptide
intrinsic to the PSII reaction center, as revealed by modiﬁed
SDS-PAGE with improved resolution of low-molecular-weight
proteins. Plant Cell Physiol. 29, 1233–1239.
21. Bligh, E.G. & Dyer, W.J. (1959) A rapid method of total
lipid extraction and puriﬁcation. Can. J. Biochem. Physiol. 37,
22. McMurry, J. (1999) Organic Chemistry, 5th edn. Brooks/Cole
Thomson Learning, Belmont, CA.
23. Minoda, A., Sonoike, K., Nozaki, H., Okada, K., Sato, N. &
Tsuzuki, M. (2001) Contribution of SQDG in photosystem II of
Chlamydomonas reinhardtii. PS2001. Proceedings of the 12th
International Congress on Photosynthesis, S5–039.
A. (1991) Modiﬁcation of the membrane fatty acid composition of
Chlamydomonas reinhardtii cultured in the presence of liposomes.
Plant Physiol. 29, 429–440.
25. Sato, N., Hagio, M., Wada, H. & Tsuzuki, M. (2000)
Requirement of phosphatidylglycerol for photosynthetic function
in thylakoid membranes. Proc. Natl Acad. Sci. USA 97, 10655–
26. Hagio, M., Gombos, Z., Va
rkonyi, Z., Masamoto, K., Sato, N.,
of phosphatidylglycerol in photosystem II of photosynthesis. Plant
Physiol. 124, 795–804.
27. Kruse, O., Radunz, A. & Schmid, G.H. (1994) Phosphatidylgly-
cerol and b-carotene bound onto the D1-core peptide of photo-
system II-particles of the cyanobacterium Oscillatoria chalybea.
Z. Naturforsch. 49c, 380–390.
28. Droppa, M., Horva
th, G., Hideg, E
. & Farkas, T. (1995) The role
of phospholipids in regulating photosynthetic electron transport
activities: Treatment of thylakoids with phospholipase C. Photo-
synthesis Res. 46, 287–293.
29. Nield, J., Kruse, O., Ruprecht, J., Fonseca, P., Bu
chel, C. &
reinhardtii and Synechococcus elongatus photosystem II complexes
allows for comparison of their oxygen-evolving complex organi-
zation. J. Biol. Chem. 275, 27940–27946.
30. Ruﬄe, S.V. & Sayre, R.T. (1998) Functional analysis of photo-
system II. In The Molecular Biology of Chloroplasts and
Mitochondria in Chlamydomonas (Rochaix, J D.Goldschmidt-
demic Publishers, Dordrecht, the Netherlands.
31. Aoki, M., Sato, N. & Tsuzuki, M. (2001) Synechocystis sp.
PCC6803 required sulfoquinovosyl diacylglycerol for its growth.
Plant Cell Physiol. 42S, s194.
32. Murata, N., Higashi, S. & Fujimura, Y. (1990) Glycerolipids in
various preparations of photosystem II from spinach chloroplasts.
Biochim. Biophys. Acta 1019, 261–268.
33. Essigmann, B., Guler, S.A., Narang, R.A., Linke, D. & Benning, C.
(1998) Phosphate availability aﬀects the tylakoid lipid composi-
tion and the expression of SQD1, a gene required for sulfolipid
biosynthesis in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 95,
2358 A. Minoda et al. (Eur. J. Biochem. 269) Ó FEBS 2002