Báo cáo khoa học: Protein Extrinsic của Photosystem II - Thành viên trung gian của họ protein PsbQ trong tảo đỏ PS II

Extrinsic proteins of photosystem II

An intermediate member of the PsbQ protein family in red algal PS II

Hisataka Ohta

1,2

, Takehiro Suzuki

, Masaji Ueno

, Akinori Okumura

, Shizue Yoshihara

, Jian-Ren Shen

and Isao Enami

Department of Biology, Faculty of Science and

Tissue Engineering Research Center, Tokyo University of Science, Japan;

Department of Biology, Faculty of Science, Okayama University and PRESTO, JST, Japan

The oxygen-evolving photosystem II (PS II) complex of red

algae contains four extrinsic proteins of 12 kDa, 20 kDa,

33 kDa and cyt c-550, among which the 20 kDa protein is

unique in that it is not found in other organisms. We cloned

the gene for the 20-kDa protein from a red alga Cyanidium

caldarium. The gene consists of a leader sequence which can

be divided into two parts: one for transfer across the plastid

envelope and the other for transfer into thylakoid lumen,

indicating that the gene is encoded by the nuclear genome.

The sequence of the mature 20-kDa protein has low but

signiﬁcant homology with the extrinsic 17-kDa (PsbQ)

protein of PS II from green algae Volvox Carteri and

Chlamydomonas reinhardtii, as well as the PsbQ protein of

higher plants and PsbQ-like protein from cyanobacteria.

Cross-reconstitution experiments with combinations of the

extrinsic proteins and PS IIs from the red alga Cy. calda-

rium and green alga Ch. reinhardtii showed that the

extrinsic 20-kDa protein was functional in place of the

green algal 17-kDa protein on binding to the green algal

PS II and restoration of oxygen evolution. From these

results, we conclude that the 20-kDa protein is the ancestral

form of the extrinsic 17-kDa protein in green algal and

higher plant PS IIs. This provides an important clue to

the evolution of the oxygen-evolving complex from pro-

karyotic cyanobacteria to eukaryotic higher plants. The

gene coding for the extrinsic 20-kDa protein was named

psbQ¢(prime).

Keywords: photosystem II; oxygen evolution; extrinsic

protein; psbQ; red alga.

Oxidation of water by photosystem II (PS II) is the source

of molecular O

, electrons, and protons in higher plants,

algae, and cyanobacteria. PS II is a multisubunit pigment–

protein complex containing intrinsic and extrinsic compo-

nents located in thylakoid membranes. More than 10

intrinsic, membrane-spanning proteins including CP47,

CP43, D1, D2, aand bsubunits of cytochrome b-559,

and the psbI gene product form the transmembrane core of

PS II. The extrinsic components are known to maintain and

optimize the stability and activity of the water oxidation site,

which is composed of a cluster of four manganese atoms

located close to the luminal surface of the transmembrane

domain and coordinated mainly by amino acids of the D1

protein [1–3]. The extrinsic domain of the oxygen-evolving

complex is composed of three proteins of 33 kDa, 23 kDa

and 17 kDa encoded by psbO, psbP, psbQ genes, respect-

ively, in PS II of green algae and higher plants (reviewed in

[4]). Among these three extrinsic components, the 33-kDa

manganese stabilizing protein (PsbO) is highly conserved

from prokaryotic cyanobacteria to eukaryotic higher plants,

while the 23-kDa and 17-kDa proteins are absent in PS II

from cyanobacteria and red algae, although a PsbQ-like

protein was recently reported to be associated with PS II

from Synechocystis sp. PCC 6803 [5]. Instead, cyanobacte-

rial PS II contains two other extrinsic proteins, PsbU

(12 kDa) and PsbV (cyt c-550), which functions to replace

to some extent the role of PsbP and PsbQ found in green

algae and higher plants [6].

Among photosynthetic organisms, red algae are one of

the most primitive eukaryotic algae phylogenetically closely

related to the prokaryotic oxygenic cyanobacteria. We have

found that the oxygen-evolving PS II complex puriﬁed from

aredalga,Cyanidium caldarium contained three extrin-

sic proteins of cyanobacteria-type, i.e. the 33-kDa, 12-kDa

proteins and cyt c-550 [7]. In addition to these three

proteins, the red algal PS II contained a fourth extrin-

sic protein of 20 kDa [7,8]. N-terminal amino acid sequence

of more than 30 residues of the protein revealed that it has

no signiﬁcant homology with any known PS II polypeptides

[7], suggesting that it is a new extrinsic component of PS II.

Release-reconstitution experiments in red algal PS II

showedthatthe20-kDaproteincanbindtoPSIItoa

signiﬁcant extent by itself, whereas the effective binding of

cyt c-550 and the 12-kDa protein requires the presence of

both the 33-kDa and 20-kDa proteins [8]. This is in contrast

to the situation found in cyanobacterial PS II where cyt c-

550 could bind to PS II essentially independently of the

binding of the 33-kDa protein, and where the homologous

Correspondence to I. Enami, Department of Biology,

Faculty of Science, Tokyo University of Science,

1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan.

Fax: +81 471 24 2150, Tel.: +81 471 24 1501 ex5022,

E-mail: enami@rs.noda.tus.ac.jp

Abbreviation: PS II, photosystem II.

Note: The sequence reported in this paper has been deposited in the

DDBJ database (accession No. AB111526)

(Received 8 July 2003, revised 21 August 2003,

accepted 29 August 2003)

Eur. J. Biochem. 270, 4156–4163 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03810.x

20-kDa protein was not found. These results suggest a

gradual change of the oxygen-evolving complex from

prokaryotic cyanobacteria to eukaryotic red algae and

higher plants. The unique 20-kDa protein found in red algal

PS II may provide insights into such changes.

In this work, we cloned the gene for the 20-kDa protein

from the red alga, Cy. caldarium,andcomparedits

sequence with those of other PS II extrinsic proteins. It

was shown that the 20-kDa protein is homologous to the

PsbQ protein found in green algal and higher plant PS IIs.

The 20-kDa gene was successfully expressed in Escherichia

coli, and cross-reconstitution with the recombinant 20-kDa

protein showed that this protein is functional in place of the

PsbQ protein in green algal PS II. These results provided

important clues to the evolution of oxygen-evolving com-

plex from cyanobacteria to higher plants.

Materials and methods

Preparations

PS II membranes of spinach were prepared according to

Berthold et al. [9]. The extrinsic proteins of PS II were

extractedwith1

CaCl

as described by Enami et al. [10].

Oxygen-evolving PS II from the red alga Cy. caldarium was

prepared according to Enami et al. [7], and suspended in

40 m

Mes pH 6.5, 10 m

CaCl

, 25% glycerol. The four

extrinsic proteins were released with 1

CaCl

-wash and

puriﬁed as described by Enami et al. [8], and ﬁnally dialysed

extensively against 40 m

Mes pH 6.5 and concentrated.

Green algal oxygen-evolving PS II and its extrinsic proteins

were prepared from Chlamydomonas reinhardtii as described

by Suzuki et al. in [11].

Cloning and sequence analysis of the extrinsic

20-kDa protein

The N-terminal sequence of the 20-kDa protein was

determined as described by Enami et al.[12],andthe

sequence obtained was as follows: AGEPKMSFFGA

DAPSSPFTYNEREGEPVYK. Based on this sequence,

the gene coding for the 20-kDa protein from Cy. calda-

rium was cloned by a two-step PCR method. First, two

sets of degenerate oligonucleotide primers corresponding

to N-terminal sequence of AGEPKM and GEPVYK were

synthesized and used to amplify a 90-bp cDNA fragment

by RT/PCR from a Cy. caldarium cDNA library. The

cDNA fragment was sequenced to conﬁrm that it indeed

corresponded to the N-terminal sequence of the 20-kDa

protein. Based on this information, the second PCR step

was performed with the RACE procedure [13] using the

Marathon cDNA Ampliﬁcation Kit (Clontech) by which

DNA fragments including the 5¢-and3¢-ﬂanking regions

of the 20-kDa protein were ampliﬁed using primers newly

synthesized based on the N-terminal 90-bp cDNA frag-

ment. This second-step PCR resulted in 450-bp and

600-bp cDNA fragments from the 5¢-and3¢-RACE,

respectively. Sequencing of these cDNA fragments con-

ﬁrmed that they contained the cDNA for the 20-kDa

protein. These sequences were combined with the partial

sequence of the N-terminal part to yield the whole

sequence of the gene.

The PCR fragments obtained were inserted into the

plasmid pCRII (TA Cloning Kit, Invitrogen), and the DNA

sequences were determined by the method of Dye Deoxy

Terminator Cycle Sequencing with a DNA Sequencer

(Applied Biosystems, model 310).

Expression and purification of the recombinant

20-kDa protein

The whole gene encoding the mature 20-kDa protein was

cloned into the LIC site of plasmid pET-32Xa/LIC,

resulting in a fusion protein with thioredoxin and (His)

tag attached at its N-terminus [14,15]. The recombinant

protein was expressed with the host cell BL21 (Novagen)

and puriﬁed by His-bind afﬁnity chromatography according

to the manufacturer’s instructions. The fusion protein was

treated with Factor Xa to cleave off the thioredoxin and

His-tag and then puriﬁed again by afﬁnity column.

Reconstitution

Reconstitution experiments of CaCl

-washed PS II from

red and green algae with various combinations of extrinsic

proteins from different sources were performed according to

Enami et al. [8,10] and Suzuki et al. [11]. SDS/PAGE was

performed according to Ikeuchi and Inoue [16]. Oxygen

evolution was measured with a Clark-type oxygen electrode

at 25 Cwith0.4m

phenyl-p-benzoquinone (red alga)

or 2,6-dichloro-p-benzoquinone (green alga) as electron

acceptor.

Results

Cloning and sequence analysis of the 20-kDa protein

The DNA sequences obtained in the present study are

shown in Fig. 1. Two in-frame ATG codons were found

upstream of the N-terminal alanine residue of the mature

polypeptide, one at position 40 and the other at position 91.

The start codon for the 20-kDa protein gene was assigned at

the ﬁrst ATG codon of nucleotide number 40, because this

site (AAAAATGTT) has a better match with the consensus

sequence for plant translation initiation than the second site

(CTTGATGAT) [17]. According to this assignment, the

resulting gene encodes a polypeptide of 218 amino acid

residueswithatotalmolecularmassof24028Da.Asthe

N-terminal part of the mature 20-kDa protein corresponds

to sequence starting at residue number 73, residues 1–72

serve as leader sequences. Hydropathy analysis (data not

shown) revealed that there are two characteristic domains in

this leader sequence. The ﬁrst consists of residues 1–47 and

is enriched in basic, hydrophilic, as well as hydroxylated

residues; this is consistent with the characteristic features of

transit peptides for transport across the chloroplast envel-

ope [4] and suggests that this domain functions to direct the

transfer of the 20-kDa protein across the chloroplast

envelope. The second domain consists of residues 48–72

and has features characteristic of transit peptides for

transfer of proteins through the bacterial periplasmic

membranes and thylakoid membranes [4], because its

central part is enriched in hydrophobic residues and its C

terminus contains an alanine residue at position )1(thisis

FEBS 2003 Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4157

typically found in proteins transported across the periplas-

mic and thylakoid membranes). Thus, we conclude that the

20-kDa protein is encoded by the nuclear DNA in the red

alga. This is consistent with results of whole chloroplast

genome sequencing of the red algae Porphyra purpurea [18]

and Cy. caldarium RK1 [19], in which the gene coding for

the 20-kDa protein was not found in the plastid genome.

Cleavage of the transit peptides resulted in a mature

polypeptide of 146 amino acid residues with a calculated

molecular mass of 16 386 Da.

Blast analysis with the GenBank database showed a

signiﬁcant homology of the 20-kDa protein gene with a

cDNA clone, AV34507 from a marine red alga Porphyra

yezoensis [20]. Unexpectedly, this analysis also gave low but

signiﬁcant scores (53–64) with oxygen-evolving enhancer

(OEE) protein 3 (PsbQ) from green algae Volvox carteri [21]

and Ch. reinhardtii [22]. These results suggested that the

extrinsic 20-kDa protein in PS II from the red alga

Cy. caldarium is a homologue of one of the PS II extrinsic

proteins, PsbQ protein, in green algae. Recently, Kashino

et al. reported that the sll1638 gene product of cyanobac-

terium Synechocystis sp. PCC 6803 has a similarity to the

PsbQ protein and is associated with the cyanobacterial

PS II complex [5]. We thus aligned the red algal 20-kDa

protein sequence with the PsbQ-like protein from two

cyanobacteria, Synechocystis sp. PCC 6803 and Anabaena

sp. PCC7120, the PsbQ protein from green algae and higher

plants whose sequences are currently available, together

with the homologous 20-kDa protein from the red alga

P. yezoensis, using the global alignment algorithm

CLU-

STALW

[23] (Fig. 2A). Based on these sequences, a phylo-

genetic tree was constructed by the neighbour-joining

algorithm as shown in Fig. 2B [24]. Generally, in contrast

with the other PS II extrinsic proteins such as the PsbO

protein which has a relatively high homology from cyano-

bacteria to higher plants, the PsbQ protein has a low

homology even between green algae and higher plants. For

example, the similarities (number of identical residues out of

the total residues) of the PsbO protein between cyanobac-

teria and higher plants range from 42% to 53% (Blast

similarity score, > 200), whereas those of the PsbQ protein

between the green algae Ch. reinhardtii or V. carteri and

spinach are 23% and 25% (Blast similarity score, 51–55),

respectively. In particular, the homology between the red

algal 20-kDa protein and the cyanobacterial PsbQ-like

protein is not high; blast analysis gave rise to a similarity

score less than 28 (20% identity). This is reminiscent of the

similarity between the cyanobacterial PsbQ-like protein and

higher plant PsbQ protein (Blast similarity score, < 39).

Consequently, the

CLUSTALW

multiple sequence alignment

shows that only ﬁve residues are completely conserved in the

C-terminal half of all sequences (Fig. 2A). Examination of

individual sequences showed that the 20-kDa protein

among red algae, and the PsbQ protein within the same

category of organisms are rather conserved. The resulting

phylogenic tree indicated that the PsbQ protein family could

be classiﬁed into four groups: (a) cyanobacteria; (b) red

algae; (c) green algae; and (d) higher plants. If we assume

that all these proteins were arisen from a common ancestral

protein, the PsbQ proteins of higher plants and green algae

were diverged at a very early stage from those of prokaryotic

cyanobacteria, whereas the red algal 20-kDa protein

remains rather unchanged. As a result, the red algal

20-kDa protein has a relatively low similarity with PsbQ

proteins from green algae and higher plants.

Reconstitution using the recombinant 20-kDa protein

For reconstitution experiments, the 20-kDa protein of

Cy. caldarium was successfully expressed as a fusion protein

with a His-tag using the pET expression system. The

expressed protein was puriﬁed by His-bind afﬁnity chro-

matography, and the His-tag was proteolytically removed

by Factor Xa. This recombinant 20-kDa protein was used

for reconstitution experiments with the red algal PS II.

To compare the binding and functional properties of the

recombinant 20-kDa protein with those of the native

20-kDa protein, reconstitution experiments were ﬁrst car-

ried out with the native 20-kDa protein puriﬁed from the

red algal PS II. As described previously [8,10], four extrinsic

Fig. 1. Nucleotide sequence of the 20-kDa extrinsic protein of PS II

from the red alga, Cyanidium caldarium.The deduced amino acid

sequence is shown below the nucleotide sequence in the single-letter

code. The putative chloroplast envelope transit domain (solid line) and

thylakoid transfer domain (dashed line) are underlined. Arrowhead

indicates the cleavage site generating the mature 20-kDa protein, and

the asterisk indicates the stop codon.

4158 H. Ohta et al. (Eur. J. Biochem. 270)FEBS 2003

proteins were completely released by treatment with 1

CaCl

of the puriﬁed PS II particles from Cy. caldarium

(Fig. 3, lane 2). The 12-kDa protein and cyt c-550 rebound

to the CaCl

-washed PS II efﬁciently when they were recon-

stituted together with the 33-kDa protein (Fig. 3, lane 3),

but their rebinding was not complete. Reconstitution

Fig. 2. Phylogenetic analysis of the 20-kDa protein sequence. (A) Alignment of the mature part of the 20-kDa protein sequence of Cyanidium

caldarium with a homologous protein from a marine red alga Porphyra yezoensis [20], and those of the PsbQ related proteins from two cyano-

bacteria Anabaena sp. PCC7120 [27] and Synechocystis sp. PCC6803 [28], two green algae Volvox Carteri (U22330) [21] and Chlamydomonas

reinhardtii [22], and four species of higher plants Spinacia oleracea [29], Arabidopsis thaliana [30], Zea mays [31], Onobrychis viciifolia (GenBank

Accession: AAB81994). The alignment was made with the global alignment algorithm

CLUSTAL X

[23]. Asterisks indicate identical residues among

all the sequences compared; double dots indicate conserved replacement of the residue in some of the species, and single dots indicate a slightly less

conserved replacement of the residue in some of the species. (B) Phylogenetic tree of the PsbQ protein family constructed based on the alignment

shown above. See text for further discussions.

FEBS 2003 Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4159

of these three extrinsic proteins together with the native

20-kDa protein resulted in a complete rebinding of all of the

four extrinsic proteins (Fig. 3, lane 4). Similarly, reconsti-

tution of the recombinant 20-kDa protein together with the

other three proteins also resulted in the complete rebinding

of the four extrinsic proteins (Fig. 3, lane 5). This indicates

that the recombinant 20-kDa protein retained the same

binding ability as that of the native 20-kDa protein.

Table 1 shows the restoration of oxygen evolution of the

CaCl

-washed PS II upon reconstitution with the extrinsic

proteins. The native PS II of Cy. caldarium showed a high

activity of 2754 lmol O

Æmg chl

Æh

2in the absence of

NaCl in the assay medium; this activity did not increase

much upon supplemention by NaCl. Upon CaCl

-wash, no

activity was observed in the absence or presence of NaCl.

Reconstitution with all the four native proteins increased

the activity to 50% and 51% of that in the native PS II,

respectively, in the absence and presence of NaCl. Recon-

stitution with the recombinant 20-kDa protein together with

the other three native proteins restored the oxygen-evolving

activity to a similar level as that with the native 20-kDa

protein, indicating that the recombinant protein was

functional in the red algal PS II and was as effective as

the native protein.

Cross-reconstitution of the 20-kDa protein and green

algal extrinsic 17-kDa protein

As the 20-kDa protein from Cy. caldarium has a sequence

homology with higher plant PsbQ protein, we tried to cross-

reconstitute the 20-kDa protein to well characterized

spinach PS II in place of the 17-kDa protein. However,

the 20-kDa protein was neither able to bind to CaCl

washed spinach PS II in the presence of the spinach extrinsic

33-kDa and 23-kDa proteins nor contributed to increase of

the Cl

–

binding afﬁnity for oxygen evolution (data not

shown). Recently, we have puriﬁed oxygen-evolving PS II

complexes from a green alga, Ch. reinhardtii having His-

tagged CP47, and reported that the extrinsic 17-kDa protein

of Ch. reinhardtii directly bound to PS II independently of

the other extrinsic proteins [11], which is apparently in

contrast with the spinach 17-kDa protein which functionally

associates with PS II only through its interaction with both

the 33-kDa and 23-kDa proteins [25]. This binding property

of the 17-kDa protein in green algal PS II is similar to that

of the 20-kDa protein in the red algal PS II in that the latter

also binds to PS II by itself and promotes the complete

binding of the 12-kDa protein and cyt c-550totheredalgal

PS II [8]. Thus, we performed cross-reconstitution experi-

ments between the 20-kDa protein from the red alga and the

17-kDa protein from the green alga, with PS IIs from both

redandgreenalgae.

First, we examined whether the green algal 17-kDa

protein is exchangeable for the 20-kDa protein in binding

to the red algal PS II. The resulting PS II was analysed

by SDS/PAGE (Fig. 4A). In agreement with the results

obtained in Fig. 3, signiﬁcant amounts of the 12-kDa

protein and cyt c-550 bound to CaCl

-washed PS II from

the red alga in the presence of the 33-kDa protein, but the

20-kDa protein was essential for complete binding of the

12-kDa protein and cyt c-550 (Fig. 4A, lanes 1 and 2).

When the 20-kDa protein was replaced by the green algal

extrinsic 17-kDa protein, the 17-kDa protein was able to

bind to the red algal PS II to a moderate level, but this

binding scarcely enhanced the binding of 12-kDa protein

and cyt c-550 (Fig. 4A, lane 3). These results agree with the

restoration of oxygen evolution which showed a decreased

–

requirement upon reconstitution with the 20-kDa

Fig. 3. Reconstitution of CaCl

-treatedPSIIoftheredalgawitheither

the native 20-kDa protein or the recombinant 20-kDa protein, in com-

binations with other three native extrinsic proteins of 33 kDa, 12 kDa

and cyt c-550. Lane 1, control PS II; lane 2, CaCl

-treated PS II; lanes

3–5, CaCl

-treated PS II reconstituted with the three extrinsic proteins

of 33 kDa, 12 kDa and cyt c-550 (lane 3), with the three extrinsic

proteins plus the native 20 kDa protein (lane 4), and with the three

extrinsic proteins plus the recombinant 20 kDa protein (lane 5).

Table 1. Restoration of oxygen evolution of CaCl

-treated red algal PS II by reconstitution with native or recombinant extrinsic 20-kDa protein.

Oxygen evolving activity (lmol O

Æmg chl

Æh

)

–

ion (%) +10 m

NaCl (%)

Cyanidium PS II 2754 ± 21 (100) 2756 ± 31 (100)

CaCl

-treated PS II 0 (0) 0 (0)

+ 33 0 (0) 496 ± 22 (18)

+ 33 + cyt c-550 + 12 1157 ± 18 (42) 1350 ± 40 (49)

+ 33 + cyt c-550 + 12 + native 20 1378 ± 30 (50) 1402 ± 27 (51)

+ 33 + cyt c-550 + 12 + recombinant 20 1406 ± 16 (51) 1433 ± 38 (52)

4160 H. Ohta et al. (Eur. J. Biochem. 270)FEBS 2003

Báo cáo khoa học: Extrinsic proteins of photosystem II An intermediate member of the PsbQ protein family in red algal PS II

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