Protein assembly of photosystem II and accumulation of subcomplexes
in the absence of low molecular mass subunits PsbL and PsbJ
Marjaana Suorsa
1
, Ralph E. Regel
2
, Virpi Paakkarinen
1
, Natalia Battchikova
1
, Reinhold G. Herrmann
2
and Eva-Mari Aro
1
1
Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Finland;
2
Botanisches Institute der
Ludwig-Maximilians Universita
¨
t, Mu
¨
nchen, Germany
The protein assembly and stability of photosystem II (PSII)
(sub)complexes were studied in mature leaves of four plastid
mutants of tobacco (Nicotiana tabacum L), each having one
of the psbEFLJ operon genes inactivated. In the absence of
psbL, no PSII core dimers or PSII–light harvesting complex
(LHCII) supercomplexes were formed, and the assembly of
CP43 into PSII core monomers was extremely labile. The
assembly of CP43 into PSII core monomers was found to be

559
), the chloro-
phyll a-binding antenna proteins CP43 and CP47, and a
number of low molecular mass (LMM) proteins, the
functions and locations of which in PSII are still largely
unknown. They include both chloroplast-encoded (PsbH, I,
J, K, L, M, N, T and Z) and nucleus-encoded (PsbR, W and
X) proteins with generally only one membrane-spanning
helix [1]. During the past few years, enormous progress has
been made in determining the structure of PSII [2–4]. The
functional form of PSII is apparently a dimer [5]. The
oxygen-evolving complex (OEC) situated on the lumenal
side of PSII is composed of the PsbO (33 kDa), PsbP
(23 kDa) and PsbQ (17 kDa) proteins in higher plants. PSII
dimers further associate with the light-harvesting complex II
(LHCII) to form PSII–LHCII supercomplexes, the minor
antenna proteins CP24, CP26 and CP29 probably serving as
linker proteins [2,5,6]. It has been suggested that several
LMM proteins participate in PSII dimerization [7,8].
However, despite the available structure of PSII at 3.8
and 3.7 A
˚
resolution [3,4], the exact locations and roles of
most of the LMM proteins in the assembly and stability of
PSII remain to be determined.
Today it is a challenge to resolve the assembly steps of
PSII. Various approaches have been fruitful in analysing the
primary assembly steps of PSII [9]. The best-characterized
LMM proteins of PSII, the a and b subunits of Cyt b
559

accepted 4 November 2003)
Eur. J. Biochem. 271, 96–107 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03906.x
because of resolution problems and, furthermore, only some
of the LMM subunits of PSII incorporate [
35
S]methionine.
Another approach to understanding the assembly of
LMM subunits into PSII is to use specific PSII protein
deletion mutants and to analysethe ability of such mutants to
form various PSII subassemblies. This approach has only
seldom been taken because of technical problems, and, when
applied, the fractionation of PSII subcomplexes has been
based in sucrose-density centrifugation with limited resolu-
tion capacity [14]. Moreover, none of the numerous studies
with Synechocystis 6803 mutants of the LMM subunits of
PSII has addressed the PSII assembly process as such, but
instead the focus has been on functional properties of PSII
and the overall synthesis or composition of thylakoid
polypeptides. Furthermore, despite remarkable similarities
between cyanobacterial and chloroplast PSIIs [15], many of
the PSII LMM subunits, which are completely dispensable
for the assembly of PSII in Synechocystis, are necessary for
the formation of functional PSII in the respective LMM
mutants of Chlamydomonas reinhardtii.Representative
examples of differential effectson the formation of functional
PSII in Synechocystis and Chlamydomonas are the deletion
mutants of psbH [16,17], psbI [18,19] and psbK [20,21].
However, it is not known at which assembly step these
proteins are crucial for the formation of functional PSII in
Chlamydomonas. So far only a few studies have seriously

turnover of the reaction center D1 protein [30]. In
particular, the role of PsbL and PsbJ in the assembly and
stability of PSII was addressed. To maximize separation of
PSII subcomplexes, we applied 2D Blue-native (BN) gel
electrophoresis followed by protein identification with
immunoblotting and MS. In addition, comparative analysis
of both very young and mature leaves was performed to
examine the developmental aspects of PSII core and OEC
protein accumulation in the psbEFLJ operon mutants with
impaired PSII assembly.
Materials and methods
Transformation of tobacco chloroplasts
Tobacco (Nicotiana tabacum cv. Petit Havanna) psbEFLJ
operon mutants were constructed by replacing portions of
the four individual genes of the operon with a terminator-
less aadA gene cassette. A similar cassette with a terminator
wasalsoinsertedintoanEcoRV site, located in the 3¢ UTR
of the operon, to generate the RV control plants. The
plasmid construct and the transformation, selection and
culture of the transformants is described in detail elsewhere
[14,28,31]. Mutants and controls (wild-type and RV plants)
were aseptically grown in MS medium [32] supplemented
with 3% (w/v) sucrose under low light conditions
( 10 lmol photonsÆm
)2
Æs
)1
)at25°C. Mature, fully
expanded green leaves, but not senescing ones (hereafter
referred to as mature leaves), were used for all experiments

pellet was resuspended in 50 m
M
Hepes/KOH, pH 7.5,
containing 100 m
M
sorbitol, 10 m
M
MgCl
2
and 10 m
M
NaF, centrifuged at 2500 g for 3 min at 4 °C, and finally
resuspended in the same buffer. Chlorophyll was extracted
in 80% (v/v) buffered acetone (2.5 m
M
Hepes/NaOH,
pH 7.5) and quantitated as described [33].
BN-PAGE, SDS/PAGE and protein identification
Blue-native PAGE (BN-PAGE) was performed as des-
cribed previously [34] with slight modifications. Thylakoid
membrane suspensions containing 20 lg chlorophyll were
used as starting material. Thylakoids were washed with
50 m
M
BisTris/HCl, pH 7.0, containing 330 m
M
sorbitol
and 0.25 lgÆlL
)1
Pefabloc (Roche), sedimented at 3500 g

gels were silver-stained or electroblotted on to a poly(viny-
lidene difluoride) membrane. Western blotting with chemi-
luminescence detection was performed with standard
techniques using protein-specific antibodies (D1, D2, PsbE,
CP43, CP47, PsbO, PsbP, PsbQ, Cyt f, Lhcb1,2, CP26,
CP29) or an antibody raised against the PSI complex. The
AIS Analytical Imaging Station (version 3.0 rev 1.7;
Imaging Research Inc., Brock University, St Catharines,
Ontario, Canada) was used for quantitation of the Western
blots. For each quantitation, a minimum of three inde-
pendent Western blots was used.
Several protein components of PSII, OEC and LHCII
complexesaswellasCytb
6
f and PSI were also identified by
MS MALDI-TOF analysis. Protein in-gel digestion with
modified trypsin (Promega) and sample preparation for MS
analysis were performed manually [36]. Samples were
loaded on to the target plate by the dried droplet method
using a-cyano-4-hydroxycinnamic acid as a matrix.
MALDI-TOF analysis was performed in reflector mode
on a Voyager-DE PRO mass spectrometer (Applied Bio-
systems, Foster City, CA, USA). Internal mass calibration
of spectra was based on trypsin autodigestion products
(842.5094 and 2211.1046 m/z). Proteins were identified as
the highest ranking result by searching in the NCBI
database using Mascot ().
The search parameters allowed for carbamidomethylation
of cysteine, one miscleavage of trypsin, and 50 p.p.m. mass
accuracy. For positive identification, the score of the result

determined using 1D SDS/PAGE and immunoblotting with
protein-specific antibodies. DpsbE and DpsbF thylakoids
were practically devoid of all PSII core proteins tested
(including D1, D2, CP43, CP47, PsbE and PsbZ, Fig. 1A).
Similarly, all three OEC proteins, PsbO, PsbP and PsbQ,
were completely missing from thylakoids of these two
mutants (Fig. 1A). PsbW, on the other hand, represented a
PSII LMM protein that was present at reduced amounts in
the thylakoids of both DpsbE and DpsbF (33 ± 11 of that in
the control thylakoids). To investigate the apparent devel-
opmental control of the accumulation of PSII proteins, we
also isolated thylakoids from very young, rapidly expanding
leaves of DpsbE and DpsbF and analysed their protein
composition (Fig. 1B). In contrast with mature leaves, the
young leaves of both DpsbE and DpsbF accumulated all
Fig. 1. Immunoblots of thylakoid membrane proteins of the four tobacco
psbEFLJ operon mutants and the controls (wild-type and RV). Thyla-
koids were isolated from mature green leaves (A) and rapidly
expanding young leaves (B). Proteins were separated by SDS/PAGE,
electroblotted on to a poly(vinylidene difluoride) membrane and pro-
bed with antisera against different thylakoid membrane proteins.
Chlorophyll (1 lg) was loaded in each well, except for PsbW (0.3 lg)
and PsbO and PsbP (0.5 lg).
98 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003
OEC proteins and also traces of D2 and the CP43 protein
(Fig. 1B). Interestingly, traces of PsbE protein (the a
subunit of Cyt b
559
) could also be distinguished in young
leaves of DpsbF (Fig. 1B). Other PSII core proteins (D1,

considerable amounts. However, the DpsbJ mutant was
again the exception, accumulating only traces of PsbP
compared with the other mutants (Fig. 1B). Otherwise the
pattern of PSII proteins in young leaves of DpsbL and DpsbJ
resembled that of the mature leaves (Fig. 1B).
AswellasthePSIIcoreandOECproteins,we
investigated the amounts of the LHCII, CP26, Cyt f,LHCI
and PsaA/B proteins in mature leaves of the psbEFLJ
operon mutants. All mutants were capable of accumulating
these proteins and no clear differences were recorded
compared with thylakoids isolated from control plants
(Fig. 1A).
Assembly of thylakoid membrane protein complexes
in
psbEFLJ
operon mutants
Simple detection of thylakoid proteins by immunoblotting
does not reveal whether the proteins are assembled into
complexes or whether they exist as free proteins in the
membrane or lumen. The general assumption that good
quality control in chloroplasts results in rapid degradation
of unassembled proteins [41] does not always hold true. In
rapidly expanding young leaves in particular, some of the
PSII core proteins and all of the OEC proteins can
accumulate in thylakoids in the absence of any assembly
of PSII, as was evident for the DpsbE and DpsbF tobacco
mutants (Fig. 1B). Thus, to understand the role of various
LMM subunits in the stable assembly of PSII, it is necessary
to isolate various PSII assembly intermediates. For these
experiments we used only mature leaves to avoid accumu-

complexes on dodecyl maltoside solubilization and subse-
quent electrophoretic separation of thylakoid protein com-
plexes. Of the OEC proteins, the PsbO subunit was always
detected in association with the PSII–LHCII supercom-
plexes (Fig. 4A). The Cyt b
6
f complex was present in wild-
type thylakoids mainly as a dimer, and the PSI complex
Fig. 2. BN-PAGE of thylakoid protein complexes from mature leaves of
the four tobacco psbEFLJ operon mutants and the wild-type and RV
controls. Thylakoids (20 lg chlorophyll per well) were solubilized with
1% n-dodecyl maltoside before BN-PAGE. For identification of the
complexes, see Fig. 3.
Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271)99
Fig. 3. Two-dimensional gel analysis of the thylakoid protein complexes from mature leaves of wild-type and DpsbF, DpsbL and DpsbJ mutants of
tobacco. Thylakoids were solubilized and subjected to BN-PAGE separation of the protein complexes as described in Fig. 2. After the run, a lane of
BN-PAGE was cut out, solubilized with 5% (v/v) 2-mercaptoethanol, and placed horizontally on the top of the SDS/polyacrylamide gel. After
electrophoresis, the gel was silver-stained. Similar gels were also electroblotted on to poly(vinylidene difluoride) membranes and probed with
antisera against D1, D2, CP43, CP47 and PsbE (Cyt b
559
a subunit). Strips of such immunoblots are presented below the corresponding silver-
stained gels. Some of the immunoblots are overexposed and thus cannot be compared quantitatively. The D1, D2, CP43 and CP47 proteins from
the PSII complexes (PSII core monomers, CP43-less core monomers, PSII core dimers and PSII–LHCII supercomplexes) are circled. Positions of
PSI, Cyt b
6
f dimers and various LHCII subassemblies are circled in the silver-stained gel of the DpsbF mutant lacking all PSII complexes and were
identified by MALDI-TOF MS and immunoblotting (data not shown).
100 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003
Fig. 4. Presence of the 33-kDa PsbO protein of OEC in different PSII assemblies of the control and DpsbJ and Dpsb L mutant thylakoids isolated from
mature leaves. (A) Protein components of PSII (sub)complexes from wild-type and DpsbJ and DpsbL mutants of tobacco. The gels for the wild-type

synthetic complexes in the thylakoid membrane. This differs
from a recent study in which the amounts of some PSI
proteins were reduced in tobacco DpsbJ mutant [29].
Analysis of DpsbJ by 2D BN-PAGE revealed that both
PSII core monomers and dimers were correctly assembled
(Fig. 3). Considerable amounts of PSII monomers lacking
CP43 were, however, also present, although the relative
amount of free CP43 was much less than in DpsbL (see
below). It is noteworthy that not even traces of PSII–LHCII
supercomplexes were present in DpsbJ thylakoids. In the
absence of PSII–LHCII supercomplexes, the PsbO protein
of the OEC was found to be associated with the PSII core
dimers (Fig. 4A) in the thylakoid membranes of DpsbJ.
The DpsbL mutant was capable of partial assembly of the
PSII core monomers, whereas PSII core dimers and
supercomplexes were completely missing (Fig. 3). Small
amounts of both types of PSII core monomers, an intact
PSII monomer and a CP43-less monomer, were observed
(Fig. 3). It is noteworthy that, in DpsbL, the portion of free
CP43 compared with that assembled into the PSII core
monomer was extremely high (91 ± 5%). In wild-type
thylakoids, only a minor amount (2 ± 1%) of CP43 was
found free and unassembled into the PSII complexes under
similar experimental conditions. This indicates that, in the
absence of PsbL, the assembly of CP43 and thus the
formation of stable intact PSII core monomers is severely
impaired. None of the other PSII proteins were found free
after 2D BN-PAGE of DpsbL thylakoids (except for a tiny
amount of PsbE; Fig. 3), indicating no general disassembly
of PSII core complexes during electrophoretic separation.

peaks at 685 nm (CP43) and 695 nm (CP47) as well as the
PSI emission peak at 735 nm (Fig. 5) [42]. DpsbE, DpsbF
and DpsbL lacked the emission peaks at 685 and 695 nm
and instead had a prominent peak at 680 nm, characteristic
of free LHCII. The 730-nm PSI peak was shifted to a lower
wavelength. Interestingly, in DpsbJ, the 680-nm (LHCII),
685-nm (CP43) and 695-nm (CP47) 77 K fluorescence
emission peaks were all present, in addition to the promi-
nent PSI emission peak.
Fig. 5. 77 K fluorescence emission spectra of thylakoid membranes of
tobacco psbEFLJ operon mutants and controls (wild-type and RV).
Thylakoids were excited with visible light below 500 nm.
102 M. Suorsa et al.(Eur. J. Biochem. 271) Ó FEBS 2003
Discussion
PSII contains several chloroplast-encoded and nuclear-
encoded LMM subunits, the role of which in the assembly
and stability of the complex has remained poorly under-
stood. We have used a reverse genetics approach to
elucidate the role of proteins encoded by the psbEFLJ
operon, with special attention to PsbL and PsbJ, in the
stable assembly process of the PSII core subunits, the
LHCII antenna polypeptides, and the proteins of the OEC.
PsbJ is essential for correct association of LHCII
Although stable PSII core dimers were assembled in DpsbJ,
the PSII–LHCII supercomplexes were completely missing.
This indicates the importance of PsbJ in the steady-state
higher organization of the PSII complexes. This conclusion,
deduced from the 2D gel analysis (Fig. 3), was further
supported by the 77 K fluorescence emission spectrum of
DpsbJ revealing a distinct emission peak directly from

conceivable that PsbL is an essential protein component
of PSII for ensuring the stable assembly of CP43, and
therefore, in DpsbL, the CP43 protein readily becomes
detached from the PSII core monomer during the elec-
trophoretic run. On the basis of the crystal structure of PSII
[3], it was suggested that a transmembrane a-helix in the
vicinity of CP47 possibly represents PsbL. We are inclined,
however, to suggest that, rather than being located in the
vicinity of CP47, PsbL is one of the unassigned transmem-
brane a-helices in the vicinity of CP43 and D1 [3]. This
suggestion is also supported by the fact that CP47 stably
assembles with PSII core monomers even in the absence of
PsbL (Fig. 3).
Recently there has been a growing consensus in favour of
PSII dimers being the functional forms of PSII [2,5,6].
Whether PsbL has a direct role in PSII dimerization, as was
suggested by Barber and coworkers [7], is difficult to assess.
It is probable that problems in stable assembly of CP43
exert secondary effects on PSII dimerization, and thus the
role of PsbL in the dimerization process itself may be
indirect. The exact mechanism of PSII dimerization is not
known but it is conceivable that several small PSII subunits
collectively control the successful dimerization of PSII [7,8].
The presence of CP43 in PSII is a prerequisite for
association of PsbO whereas PsbL and PsbJ are needed
for correct association of PsbP and PsbQ
Three-dimensional OEC structures from spinach [2],
Chlamydomonas and Synechococcus elongatus [43] were
recently published. In all of these evolutionarily divergent
species, the PsbO protein was suggested to be located

assembly of the other OEC proteins, it does not seem to
provide any direct binding site for either PsbP or PsbQ,
which does not support the previous suggestion [49]. It is
evident that the presence of both PsbL and PsbJ is critical in
providing proper docking sites, either directly or indirectly,
by modifying the conformation of PSII on the lumenal side,
making efficient binding of PsbQ and PsbP of the OEC
possible.
A fundamental difference in the DpsbJ mutants between
cyanobacteria and the chloroplasts of higher plants was
Ó FEBS 2003 Low molecular mass proteins in the assembly of PSII (Eur. J. Biochem. 271) 103
recently described: only the cyanobacterial mutant is
capable of slow photoautotrophic growth [28]. This is
reflected in the capacity of the mutants to oxidize Q
A
–
.Itis,
however, likely that the water splitting and donation of
electrons to P680
+
also play a role in the better performance
of the cyanobacterial than the tobacco DpsbJ mutant.
Requirements for OEC proteins in cyanobacteria seem to be
less stringent than in eukaryotes. In cyanobacteria, the
presence of either PsbO or Cyt c
550
(PsbV) confers photo-
autotrophic growth [50,51], whereas three distinct proteins
form the OEC in eukaryotes [51]. Both PsbO and to a lesser
extent PsbQ were present in mature leaves of tobacco DpsbJ

assembly of D1 [53,54]. In addition, Cyt b
559
has been found
in barley etioplasts as a complex with D2 [11], emphasizing
the role of these two subunits as primary assembly partners
for construction of the PSII complexes. Indeed, the PsbE
protein was also present in tiny amounts in the thylakoid
membranes of young, developing leaves of DpsbF.Ofthe
internal antenna proteins of PSII, the assembly of CP47
possibly also occurs cotranslationally because no free
protein was found in the membrane, whereas the assembly
process of CP43 seems to be less stringent [9,54,55] and
some free CP43 was found in the thylakoid membrane of
young developing leaves (Fig. 1B).
Apparently a change in the developmental program upon
leaf maturation and cessation of chloroplast division leads
to down-regulation of both the chloroplast-encoded and
nucleus-encoded PSII proteins (Fig. 1A), avoiding the
wasteful synthesis of proteins when their assembly into
functional complexes is prohibited. The possible signaling
mechanisms leading to complete down-regulation of PSII
core and OEC proteins in the absence of PSII assembly,
manifested in DpsbE and DpsbF upon leaf maturation
(Fig. 1A,B), are not known. However, the notion of the
strict regulation of OEC protein synthesis in mature leaves
also is supported by the identification of PSII subcomplexes
that bind the PsbO protein in DpsbL and DpsbJ thylakoids
(Fig. 4). Demonstration of the association of PsbO with
PSII subcomplexes implies that free OEC proteins do not
accumulate in the thylakoid lumen of mature leaves, in

lacked PSII (DpsbE and DpsbF) or had a defectively
assembled PSII (DpsbL and DpsbJ), as was discussed in
the recent report on the DpsbJ tobacco mutant with
dramatically reduced photosynthetic performance [29].
Lessons from
psbEFLJ
operon mutants on the role
of PsbW and PsbZ subunits
PSII assembly studies on psbEFLJ operon mutants also
provided some information on the two other small PSII
proteins, PsbW and PsbZ. Nuclear-encoded PsbW has been
found to accumulate in the thylakoid membranes of both
mature (Fig. 1A) and young [14] leaves of psbEFLJ operon
mutants, even in the complete absence of PSII complexes
(DpsbE and DpsbF). Similarly, PsbW was present, but at
reduced amounts, in tobacco DpsbA mutant with no PSII
assembly and activity [56]. Less stringent mutual regulation
of the accumulation of PsbW and the other PSII core
proteins was also evident in psbW antisense mutants of
Arabidopsis [8]. All this suggests that PsbW is not under the
same strict regulation and/or quality control as the other
PSII core proteins and the OEC proteins in mature leaves.
Recently characterized chloroplast-encoded PsbZ [38–
40], on the other hand, accumulated in mature leaves of
DpsbL and DpsbJ, in comparable amounts to assembled
PSII complexes, while being absent from DpsbE and DpsbF
(Fig. 1A). The presence of PsbZ even in DpsbL may suggest
the location of PsbZ in a very central core of PSII. Such a
central location in PSII, however, seems to contradict the
fact that PsbZ is not required for correct assembly of the

clearly required for stable formation of PSII–LHCII
supercomplexes, thereby allowing greater organization of
PSII complexes in the thylakoid membrane.
Acknowledgements
Elena Baena-Gonzalez and Mika Kera
¨
nen are thanked for help with
the 77 K fluorescence measurements, and Drs Roberto Barbato, Toril
Hundal, Stefan Jansson, Wolfgang Schro
¨
der and Francis-Andre
Wollman for the gifts of antibodies. This work was supported by the
Academy of Finland, the Finnish Ministry of Agriculture and Forestry
(NKJ project), the German Research Foundation (SFB-TR1) and
Fonds der Chemischen Industrie.
References
1. Hankamer, B., Morris, E., Nield, J., Carne, A. & Barber, J. (2001)
Subunit positioning and transmembrane helix organisation in the
core dimer of photosystem II. FEBS Lett. 504, 142–152.
2. Nield, J., Orlova, E.V., Morris, E.P., Gowen, B., van Heel, M.
& Barber, J. (2000) 3D map of the plant photosystem II super-
comples obtained by cryoelectron microscopy and single particle
analysis. Nat. Struct. Biol. 1, 44–47.
3. Zouni, A., Witt, H T., Kern, J., Fromme, P., Krauss, N.,
Saenger, W. & Orth, P. (2001) Crystal structure of photosystem II
from Synechococcus elongatus at 3.8 A
˚
resolution. Nature 409,
739–743.
4. Kamiya, N. & Shen, J R. (2003) Crystal structure of oxygen-

ller, B. & Eichacker, L.A. (1999) Assembly of the D1 precursor
in monomeric photosystem II reaction center precomplexes pre-
cedes chlorophyll a-triggered accumulation of reaction center II in
barley etioplasts. Plant Cell 11, 2365–2377.
12. Zhang, L., Paakkarinen, V., van Wijk, K.J. & Aro, E M. (1999)
Co-translational assembly of the D1 protein into photosystem II.
J. Biol. Chem. 274, 16062–16067.
13. Zhang, L., Paakkarinen, V., van Wijk, K.J. & Aro, E M. (2000)
Biogenesis of the chloroplast-encoded D1 protein: regulation of
translational elongation, insertion and assembly into photosystem
II. Plant Cell 12, 1769–1781.
14. Swiatek, M., Regel, R.E., Meurer, J., Wanner, G., Pakrasi, H.B.,
Ohad, I. & Herrmann, R.G. (2003) Effects of selective inactivation
of individual genes for low molecular mass subunits on the pho-
tosystem II assembly by chloroplast transformation: the psbEFLJ
operon in Nicotiana tabacum. Mol. General Genomics 268,
699–710.
15. Barber, J. & Nield, J. (2002) Organization of transmembrane
helices in photosystem II: comparison of plants and cyano-
bacteria. Philos. Trans. R. Soc. Lond. B 357, 1329–1337.
16. Mayes,S.R.,Dubbs,J.M.,Vass,I.,Hideh,E.,Nagy,L.&Barber,
J. (1993) Further characterization of the psbH locus of Syne-
chocystis sp. PCC6803: inactivation of psbH impairs Q
A
to
Q
B
electron transport in photosystem II. Biochemistry 32, 1454–
1465.
17. Summer, E.J., Schmid, V.H., Bruns, B.U. & Schmidt, G.W. (1997)

24. Anbudurai, P.R. & Pakrasi, H.B. (1993) Mutational analysis of
the PsbL protein of photosystem II in the cyanobacterium
Synechocystis sp PCC 6803. Z. Naturforsch. 48, 267–274.
25. Kitamura, K., Ozawa, S., Shiina, T. & Toyoshima, Y. (1994)
L protein, encoded by psbL, restores normal functioning of the
primary quinone acceptor, Q
A
, in isolated D1/D2/CP47/Cytb-
559/I photosystem II reaction center core complex. FEBS Lett.
354, 113–116.
26. Ozawa, S., Kobayashi, T., Sugiyama, R., Hoshida, H., Shiina, T.
& Toyoshima, Y. (1997) Role of PSII-L protein (psbL gene
product) in the electron transfer in photosystem II complex 1.
Over-production of wild-type and mutant versions of PSII-L
protein and reconstitution into the PSII core complex. Plant Mol.
Biol. 34, 151–161.
27. Lind, L.K., Shukla, V.K., Nyhus, K.J. & Pakrasi, H.B. (1993)
Genetic and immunological analyses of the cyanobacterium
Synechocystis sp. PCC 6803 show that the protein encoded by the
psbJ gene regulates the number of photosystem II centers in thy-
lakoid membranes. J. Biol. Chem. 268, 1575–1579.
28. Regel, R.E., Ivleva, N.B., Zer, H., Meurer, J., Shestakov, S.V.,
Herrmann, R.G., Pakrasi, H.B. & Ohad, I. (2001) Deregulation of
electron flow within Photosystem II in the absence of the PsbJ
protein. J. Biol. Chem. 276, 41473–41478.
29.Hager,M.,Hermann,M.,Biehler,K.,Krieger-Liszkay,A.&
Bock, R. (2002) Lack of the small plastid-encoded PsbJ poly-
peptide results in a defective water-splitting apparatus of photo-
system II, reduced photosystem I levels, and hypersensitivity to
light. J. Biol. Chem. 277, 14031–14039.

37. Kera
¨
nen, M., Aro, E M. & Tyystja
¨
rvi, E. (1999) Excitation-
emission map as a tool in studies of photosynthetic pigment-
protein complexes. Photosynthetica 37, 225–237.
38. Ruf, S., Biehler, K. & Bock, R. (2000) A small chloroplast-
encoded protein as a novel architectural component of the light-
harvesting antenna. J. Cell Biol. 149, 369–378.
39. Baena-Gonzalez, E., Gray, J.C., Tyystja
¨
rvi, E., Aro, E M.
&Ma
¨
enpa
¨
a
¨
, P. (2001) Abnormal regulation of photosynthetic
electron transport in a chloroplast ycf9 inactivation mutant.
J. Biol. Chem. 276, 20795–20802.
40. Swiatek, M., Kuras, R., Sokolenko, A., Higgs, D., Olive, J.,
Cinque, G., Muller, B., Eichacker, L.A., Stern, D.B., Bassi, R.,
Herrmann, R.G. & Wollman, F A. (2001) The chloroplast gene
ycf9 encodes a Photosystem II (PSII) core subunit, PsbZ. that
participates in Photosystem II supramolecular architecture. Plant
Cell 13, 1347–1367.
41. Yamamoto, Y. (2001) Quality control of Photosystem II. Plant
Cell Phys. 42, 121–128.

49. Miyao, M. & Murata, N. (1989) The mode of binding of three
extrinsic proteins of 33 kDa, 23 kDa and 18 kDa in the Photo-
system II complex of spinach. Biochim. Biophys. Acta 977,
315–321.
50. Shen, J R., Burnap, R.L. & Inoue, Y. (1995) An independent role
of cytochrome c-550 in cyanobacterial photosystem II as revealed
by double-deletion mutagenesis of the psbO and psbV genes in
Synechocystis sp. PCC 6830. Biochemistry 34, 12661–12668.
51. Seidler, A. (1996) The extrinsic polypeptides of Photosystem II.
Biochim. Biophys. Acta 1277, 25–60.
52. Rova, M., Franzen, L G., Fredriksson, P O. & Styring, S.
(1994) Photosystem II in a mutant of Chlamydomonas reinhardtii
lacking the 23 kDa PsbP protein shows increased sensitivity to
photoinhibition in the absence of chloride. Photosynth. Res. 39,
75–83.
53. van Wijk, K.J., Roobol-Boza, M., Kettunen, R., Andersson, B. &
Aro, E M. (1997) Synthesis and assembly of the D1 protein into
photosystem II: processing of the C-terminus and identification of
the initial assembly partners and complexes during photosystem II
repair. Biochemistry 36, 6178–6186.
54. Wollman, F A., Minai, L. & Nechustai, R. (1999) The biogenesis
and assembly of photosynthetic proteins in thylakoid membranes.
Biochim. Biophys. Acta 1411, 21–85.
55. deVitry,C.,Olive,J.,Drapier,D.,Recouvreur,M.&Wollman,
F A. (1989) Posttranslational events leading to the assembly of
photosystem II protein complex: a study using photosynthesis
mutants of Chlamydomonas reinhardtii. J. Cell Biol. 109, 991–
1006.
56. Baena-Gonzalez, E., Allahverdiyeva, Y., Svab, Z., Maliga, P.,
Josse, E M., Kuntz, M., Ma