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Plant Cell Physiol. 43(4): 429–439 (2002)
JSPP © 2002
Comparison of the Structure of the Extrinsic 33 kDa Protein from Different
Organisms
Akihiko Tohri 1, 2, Takehiro Suzuki 1, Satoshi Okuyama 1, Kei Kamino 3, Akihiro Motoki 4, Masahiko Hirano 4,
Hisataka Ohta 1, Jian-Ren Shen 5, Yasushi Yamamoto 2 and Isao Enami 1, 6
1
Department of Biology, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601 Japan
The Graduate School of Natural Science and Technology, Okayama University, Tsushimanaka 3-1-1, Okayama, 700-8530 Japan
3
Shimizu Laboratories, Marine Biotechnology Institute, Sudeshi 1900, Shimizu, Shizuoka, 424-0037 Japan
4
Biological Science Department, Toray Research Center Incorporation, Tebiro 1111, Kamakura, Kanagawa, 248-8555 Japan
5
RIKEN Harima Institute, Sayo-gun, Mikazuki-cho, Hyogo, 679-5148 Japan
2
;
The psbO gene encoding the extrinsic 33 kDa protein
of oxygen-evolving photosystem II (PSII) complex was
cloned and sequenced from a red alga, Cyanidium caldarium. The gene encodes a polypeptide of 333 residues, of
which the first 76 residues served as transit peptides for
transfer across the chloroplast envelope and thylakoid
membrane. The mature protein consists of 257 amino acids
with a calculated molecular mass of 28,290 Da. The
sequence homology of the mature 33 kDa protein was
42.9–50.8% between the red alga and cyanobacteria, and
44.7–48.6% between the red alga and higher plants. The
cloned gene was expressed in Escherichia coli, and the
recombinant protein was purified, subjected to proteasetreatments. The cleavage sites of the 33 kDa protein by
chymotrypsin or V8 protease were determined and compared among a cyanobacterium (Synechococcus elongatus),
a euglena (Euglena gracilis), a green alga (Chlamydomonas
reinhardtii) and two higher plants (Spinacia oleracea and
Oryza sativa). The cleavage sites by chymotrypsin were at
156F and 190F for the cyanobacterium, 159M, 160F and
192L for red alga, 11Y and 151F for euglena, 10Yand 150F
for green alga, and 16Y for spinach, respectively. The cleavage sites by V8 protease were at 181E (cyanobacterium),
182E and 195E (red alga), 13E, 67E, 69E, 153D and 181E
(euglena), 176E and 180E (green alga), and 18E or 19E
(higher plants). Since most of the residues at these cleavage
sites were conserved among the six organisms, the results
indicate that the structure of the 33 kDa protein, at least the
structure based on the accessibility by proteases, is different among these organisms. In terms of the cleavage sites,
the structure of the 33 kDa protein can be divided into
three major groups: cyanobacterial and red algal-type has
cleavage sites at residues around 156–195, higher planttype at residues 16–19, and euglena and green algal-type at
residues of both cyanobacterial and higher plant-types.
Introduction
Photosystem II (PSII) is a pigment–protein complex
which drives the light-induced electron transfer from water to
plastoquinone with the concomitant production of molecular
oxygen. The PSII complex contains a number of intrinsic membrane protein components (for review, see Bricker and Ghanotakis 1996). In addition, there are 3–4 extrinsic proteins associated with PSII, which play important roles in maintaining the
function and stability of the oxygen-evolving complex (Shen et
al. 1992, Shen and Inoue 1993, Enami et al. 1995, Enami et al.
1998b, Seidler 1996). Among the extrinsic proteins, only the
33 kDa protein is common to all of the organisms (Koike and
Inoue 1985, Stewart et al. 1985, Enami et al. 2000). The other
extrinsic proteins are different among different organisms.
Higher plant PSII binds the extrinsic 23 and 17 kDa protein,
while cyanobacterial PSII binds cytochrome c550 and a 12 kDa
protein instead of the 23, 17 kDa proteins (Shen et al. 1992,
Shen and Inoue 1993), and red algal PSII has a 20 kDa protein
in addition to three cyanobacterial proteins (Enami et al. 1995,
Enami et al. 1998b). The 33 kDa protein plays an important
role in maintaining the stability and functional binding of the
Mn cluster that directly catalyzes the H2O-splitting reaction
(for review, see Seidler 1996). Removal of the 33 kDa protein
from PSII leads to a destabilization of the manganese cluster at low physiological chloride concentrations (Miyao and
Murata 1984, Ono and Inoue 1984). The other extrinsic proteins play a role in regulating the PSII affinity for calcium and
chloride (Åkerlund et al. 1982, Kuwabara and Murata 1982,
Ghanotakis et al. 1984, Miyao and Murata 1984, Andersson et
al. 1984, Miyao and Murata 1986, Shen et al. 1997, Shen et al.
1998, Enami et al. 1998b).
The amino acid sequences of the 33 kDa protein have
been reported from cyanobacteria (Kuwabara et al. 1987,
Philbrick and Zilinskas 1988, Borthakur and Haselkorn 1989,
Miura et al. 1993, Tucker et al. 2001), euglena (Shigemori et al.
1994), green alga (Mayfield et al. 1991), and higher plants
(Tyagi et al. 1987, Wales et al. 1989, Ko et al. 1990, Meadows
et al. 1991, Uchiyama and Tsugita 1991, Van Spanje et al.
1991, Görlach et al. 1993). The sequence of the 33 kDa protein
Key words: Evolution — Extrinsic 33 kDa protein — Limited
proteolysis — psbO gene — Red alga.
Abbreviation: CP, chlorophyll-binding protein; LIC, ligation
independent cloning; MALDI-TOF, matrix-assisted laser desorption/
ionization, time of flight; TFA, trifluoroacetic acid.
6
Corresponding author: E-mail, [email protected]; Fax, +81-471-24-2150.
429
430
Comparison of the structure of the 33 kDa protein
showed a relatively high homology around 40–50% from
cyanobacteria to higher plants. The 33 kDa protein has also
been reported to be exchangeable in binding to PSII and supporting oxygen evolution from various different organisms
(Koike and Inoue 1985, Enami et al. 2000). Thus, the structure
of the 33 kDa protein has been considered to be largely conserved during evolution from cyanobacteria to higher plants.
There are, however, some reports suggesting that the
structure of the 33 kDa protein may be different, at least in its
free form, among different plant species. The cleavage sites of
the 33 kDa protein by protease were reported to be different
between higher plant and cyanobacterium. The higher plant
33 kDa protein was cleaved at 16Y by chymotrypsin and at
18E by V8 protease (Eaton-Rye and Murata 1989), while the
cyanobacterial 33 kDa protein was cleaved primarily at 156F
and 190F by chymotrypsin (Motoki et al. 1998). Another line
of evidence suggesting a possible difference in the structure of
the 33 kDa protein is that oxygen evolution of cyanobacterial
PSII was restored to a lager extent with its own 33 kDa protein
than with the 33 kDa protein from other sources in crossreconstitution experiments (Enami et al. 2000).
Red algae are one of the most primitive eukaryotic algae
phylogenetically closely related to the most primitive oxygenic
organism, the prokaryotic cyanobacteria. The psbO gene coding for the extrinsic 33 kDa protein has not been cloned from
red algae so far, in spite of a number of reports for the
sequences of the protein from other species (Seidler 1996, De
Las Rivas and Heredia 1999). In the present study, we first
cloned and sequenced the psbO gene encoding the 33 kDa protein from a red alga, Cyanidium caldarium, and compared this
sequence with those from various organisms. Next, we determined and compared the cleavage sites by chymotrypsin or
Staphylococcus aureus V8 protease of the 33 kDa protein from
various species, i.e. a cyanobacterium (Synechococcus elongatus), a red alga (C. caldarium), a euglena (Euglena gracilis), a
green alga (Chlamydomonas reinhardtii), and higher plants
(Spinacia oleracea and Oryza sativa). The results showed that
the structure of the 33 kDa protein is different among different
organisms, and can be divided into three major groups of
higher plant-type, cyanobacterial-type (red algae and cyanobacteria) and their intermediate-type (green algae and euglena)
based on their protease-cleavage sites.
Results
Cloning and sequence analysis of the psbO gene coding for the
red algal (C. caldarium) 33 kDa protein
The psbO gene from the red alga C. caldarium was cloned
using the two-step PCR method. First, PCR was conducted to
amplify the DNA fragment corresponding to a region from 11Y
to 167G of the protein. This resulted in a 469-bp cDNA fragment from a C. caldarium cDNA library. Subsequent cloning
and sequencing of the amplified fragment revealed that the
deduced amino acid sequence from the amplified fragment was
identical to the N-terminal sequence of the protein. The second
PCR step involved the RACE procedure (Frohman et al. 1988)
by which the DNA fragments of 5¢- and 3¢-flanking regions of
the 33 kDa protein cDNA were amplified using primers newly
synthesized based on the 469-bp cDNA fragment, which
yielded a 565-bp cDNA fragment and a 640-bp cDNA fragment from 5¢- and 3¢-RACE, respectively. The nucleotide
sequences of these cDNA fragments were confirmed to contain the cDNA for the 33 kDa protein. These sequences were
combined to yield the whole sequence of the gene, which is
shown in Fig. 1. This sequence contained two in-frame ATG
codons upstream of the N-terminal leucine residue of the
mature protein; they are located at positions 100 and 193,
respectively. Among them, the ATG codon at nucleotide
number 100 was assigned as the start codon, because this
codon is similar to the start codon (AnnATGGC) for translation initiation in higher plants (Lutcke et al. 1987) (see also
below). According to this assignment, the gene encodes a
polypeptide of 333 amino acid residues with a total molecular
mass of 35,992 Da. The N-terminal sequence determined for
the mature 33 kDa protein corresponds to the sequence starting
from residue number 77 of the gene-derived amino acid
sequence. This indicates the cleavage of the first 76 residues
after synthesis of the protein, which gave rise to a mature
33 kDa protein of 257 residues with a calculated molecular
mass of 28,290 Da. Thus, the first 76 residues served as a transit peptide for transport of the protein across membranes.
In the 33 kDa presequence, the first 48 residues were
enriched in basic (K and R) and hydroxylated (S and T) residues which are characteristic features for the transit peptide of
chloroplast envelope (Keegstra et al. 1989). In contrast, the residues from 49 to 76 of the psbO gene presequence have typical
features for thylakoid transfer peptide (Keegstra et al. 1989).
Its N-terminal part is hydrophilic and its central part is hydrophobic which is predicted to constitute an a-helical structure,
and its C-terminal part contains alanine residue at position –1
and valine at position –3 upstream from N-terminus of the
mature protein which is consistent with the consensus sequence
(A)-X-A for recognition site of the thylakoidal processing
peptidase of higher plants (Fig. 1). Based on these features, the
residues from 49 to 76 were assigned as a thylakoid transfer
signal. Thus, the gene-derived amino acid sequence contained
two characteristic transit peptides, one for chloroplast envelope and the other for thylakoid membrane transfer signal. This
indicates that the extrinsic 33 kDa protein in the red algal PSII
is encoded in the nuclear genome, which implies that the psbO
gene had moved to nucleus during evolution from prokaryotic
cyanobacteria to the most primitive eukaryotic red alga, C.
caldarium. This is consistent with the fact that the psbO gene
was not found in the plastid genome of a red alga Porphyra
purpurea (Reith and Munholland 1996) whose complete plastid
sequences have been determined, and also suggests that our
assignment of the start codon was correct.
Comparison of the structure of the 33 kDa protein
431
Fig. 1 Nucleotide sequence of the psbO gene encoding the extrinsic 33 kDa protein 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 is underlined by a solid line, and the thylakoid transfer domain is underlined by a wavy line. Arrowhead indicates the cleavage site generating the mature
33 kDa protein, and the asterisk indicates the stop codon.
Fig. 2 Polypeptide analysis of the 33 kDa protein treated with a-chymotrypsin. The 33 kDa proteins from various species indicated in the figure were treated with chymotrypsin at 30°C, and then applied to a 16–22% gradient polyacrylamide gel containing 0.1% SDS and 7.5 M urea.
Lanes 1, 4, 7, 10, 13 and 16, control 33 kDa protein. Other lanes are treated with chymotrypsin at enzyme-protein ratio (mg mg–1) of 0.33 for 2 h
(lanes 2 and 5), of 1.0 for 2 h (lanes 3, 6, 8, 11 and 17), of 3.3 for 2 h (lanes 9, 12 and 18), of 1.0 for 30 min (lane 14), and of 3.3 for 30 min (lane
15).
Comparison of the cleavage sites of the 33 kDa protein from
various species by chymotrypsin and V8 protease
In order to determine the primary cleavage sites of the
33 kDa protein from different species, the 33 kDa protein was
treated with various enzyme-protein ratios of a-chymotrypsin.
The ratio of chymotrypsin to extrinsic proteins and the treat-
ment time were chosen to yield a typical digestion pattern;
excess treatments led to a total breakdown of the proteins
whose products were too small to be detected by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), while treatments at lower concentrations of chymotrypsin or shorter time are not enough to yield a constant pat-
432
Comparison of the structure of the 33 kDa protein
Table 1 Assignments for peptide fragments from a-chymotrypsin digests of the extrinsic
33 kDa proteins from various species*
Species
S. elongatus
Cyanidium
Euglena
Chlamydomonas
Spinach
Peptide bands in
SDS-PAGE
C1
C2
C3
C4
R1
R2
R2
R3
R3
R4
E1
E2
E3
E4
G1
G2
G3
S1
Observed
mass
Da
Predicted
mass
Da
Deviation
in mass
%
Peptide
assignment
20860.2
17216.4
9632.8
5975.9
21111.4
17844.3
17695.0
10606.7
10454.6
7202.5
24523.4
16472.5
15119.4
9421.6
24404.2
15092.5
9325.3
1187.9
24665.7
20867.1
17213.0
9628.6
5974.5
21098.8
17845.1
17697.9
10609.9
10462.7
7209.0
24541.1
16453.3
15139.9
9419.2
24394.2
15087.7
9324.5
1186.3
24663.1
–0.03
0.02
0.04
0.02
0.06
–0.00
–0.02
–0.03
–0.08
–0.09
–0.07
0.12
–0.14
0.03
0.04
0.03
0.01
0.13
0.01
1A-190F
1A-156F
157L-246A
191S-246A
1L-192L
1L-160F
1L-159M
160F-257D
161L-257D
193R-257D
12L-243K
1L-151F
12L-151F
152L-243K
11L-239K
11L-150F
151L-239K
1L-10Y
17L-247Q
* Rice was not listed in this Table since its 33 kDa protein was not cleaved by a-chymotrypsin under the
present conditions (see text for further details).
tern. The typical polypeptide patterns of the digested protein
were compared among six organisms, a cyanobacterium (S.
elongatus), a red alga (C. caldarium), a euglena (E. gracilis), a
green alga (C. reinhardtii), and two higher plants (S. oleracea
and O. sativa). As shown in lanes 1–3 of Fig. 2, chymotrypsin
treatments of the cyanobacterial 33 kDa protein produced four
peptide fragments of apparent molecular masses of 21, 19, 10
and 8 kDa; these four fragments were named as C1, C2, C3 and
C4, respectively. The resulting peptide mixture was analyzed
directly by a MALDI-TOF mass spectrometer in order to determine their cleavage sites. Since whether a peptide fragment can
be detected by the MALDI-TOF mass spectrometer depends on
the matrix employed and the molecular mass of that fragment,
two different matrices were used. One was a mixture of sinapic
acid and 2,5-dihydroxybenzoic acid (4 : 1), suitable for measurements of peptide fragments with molecular masses above
2,400 Da, and the other one was a-cyano-4-hydroxyciannamic
acid, suitable for peptide fragments below 2,400 Da. The protease-digested peptide fragments were assigned to fragments of
known amino acid sequences, if the measured mass of that
fragment is in the range of 0.14% mass error compared to the
calculated mass of the fragment from its known sequence
(Table 1). The results clearly showed that the cyanobacterial
33 kDa protein was primarily cleaved at 156F and 190F by
chymotrypsin, which is consistent with the results previously
reported by Motoki et al. (1998). When the red algal 33 kDa
protein was treated with chymotrypsin, four peptide fragments
appeared at approximately 21 (R1), 18 (R2), 12 (R3) and 7
(R4) kDa (Fig. 2, lanes 4–6). The molecular masses of these
fragments analyzed by MALDI-TOF mass spectrometer indicated that the red algal 33 kDa protein was cleaved at 159M,
160F and 192L, which were consistent with our results estimated by measurements of amino acid sequences of the
digested peptides (Tohri et al. 2001). Chymotrypsin-treatment
of the euglena 33 kDa protein also yielded four peptide fragments at apparent masses of 24 (E1), 17 (E2), 15 (E3) and 10
(E4) kDa on SDS-PAGE (Fig. 2, lanes 7–9). Measurements of
the molecular masses of these fragments, however, indicated
that the euglena 33 kDa protein was cleaved at 11Y and 151F
(Table 1). The green algal 33 kDa protein digested with chymotrypsin produced three peptide fragments at 24 (G1), 15 (G2)
and 7 (G3) kDa (Fig. 2, lanes 10–12), and mass-analysis of
these fragments (Table 1) indicated that the green algal 33 kDa
protein was cleaved at 10Y and 150F. When the spinach
33 kDa protein was treated with chymotrypsin, only one peptide band was obtained that had an apparent molecular mass of
26 (S1) kDa (Fig. 2, lanes 13–15). MALDI-TOF mass-analysis
(Table 1) indicated that the spinach 33 kDa protein was cleaved
at 16Y by chymotrypsin, which is consistent with the results
previously reported by Eaton-Rye and Murata (1989). On the
Comparison of the structure of the 33 kDa protein
433
Table 2 Assignments for peptide fragments from S. aureus V8 protease digests of the extrinsic
33 kDa proteins from various species
Species
S. elongatus
Peptide bands in
SDS-PAGE
Spinach
C5
C6
R5
R6
R7
R8
E5
E6
E6
E7
E8
E9
E9
G4
G5
G6
S2
Oryza
O1
Cyanidium
Euglena
Chlamydomonas
Observed
mass
Da
Predicted
mass
Da
Deviation
in mass
%
Peptide
assignment
19809.1
7028.3
21507.1
20138.7
8153.5
6788.3
24290.4
18555.8
18279.3
12142.6
9109.0
6030.3
5759.0
19365.1
18879.6
6228.4
24416.6
2132.0
24311.8
2297.1
19810.8
7030.8
21512.2
20146.7
8161.1
6795.6
24298.8
18552.4
18276.1
12173.3
9103.1
6040.8
5764.5
19352.3
18868.7
6228.1
24420.8
2130.3
24352.8
2299.5
–0.01
–0.04
–0.02
–0.04
–0.09
–0.11
–0.03
0.02
0.02
–0.25
0.06
–0.17
–0.10
0.07
0.06
0.00
–0.02
0.08
–0.17
–0.10
1A-181E
182L-246A
1L-195E
1L-182E
183A-257D
196N-257D
14V-243K
68F-243K
70R-243K
68F-181E
70R-153D
14V-69E
14V-67E
1T-180E
1T-176E
181N-239K
19V-247Q
1E-18E
20V-247E
1E-19E
other hand, the 33 kDa protein of rice was very resistant to
digestion with chymotrypsin: At the conditions shown in Fig.
2, the rice 33 kDa protein was not cleaved by chymotrypsin at
all (Fig. 2, lanes 16–18), although harsher treatments led to a
total breakdown of the rice 33 kDa protein (data not shown).
Cleavage sites of the extrinsic 33 kDa protein by S. aureus
V8 protease were also determined by the same method
described above. When the cyanobacterial 33 kDa protein was
treated with V8 protease, two peptide fragments appeared at
approximately 22 (C5) and 8 (C6) kDa on SDS-PAGE (Fig. 3,
lanes 1–3). The mass measurement of the fragments indicated
that the cyanobacterial 33 kDa protein was primarily cleaved at
181E by V8 protease (Table 2). The red algal 33 kDa protein
treated with V8 protease produced four fragments of 22 (R5),
20 (R6), 8 (R7) and 6 (R8) kDa (Fig. 3, lanes 4–6). The
MALDI-TOF mass analysis (Table 2) indicated that the red
algal 33 kDa protein was cleaved at 182E and 195E by V8 protease, which were consistent with our results estimated by
measurements of amino acid sequences of the digested peptides
(Tohri et al. 2001). V8 protease treatment of the euglena
33 kDa protein produced five fragments of 24 (E5), 19 (E6), 14
(E7), 10 (E8) and 6 (E9) kDa (Fig. 3, lanes 7–9); mass analysis
of these fragments (Table 2) showed that the euglena 33 kDa
protein was cleaved at 13E, 67E, 69E, 153D and 181E by V8
protease. Three fragments of 20 (G4), 18 (G5), and 6 (G6) kDa
were obtained by V8 protease-treatment of the green algal
33 kDa protein (Fig. 3, lanes 10–12). The MALDI-TOF mass
analysis of these fragments (Table 2) indicated that the green
algal 33 kDa protein was cleaved at 176E and 180E. The V8
protease-treatment of spinach 33 kDa protein primarily produced a fragment of 26 kDa (S2) (Fig. 3, lanes 13–15); its
molecular mass measurement (Table 2) showed that it was
cleaved at 18E. This is consistent with the previous results of
Eaton-Rye and Murata (1989). V8 protease cleaved the rice
33 kDa protein at 19E, which is similar with that of the spinach 33 kDa protein (Fig. 3, lanes 16–18, Table 2), although in
this case also, the rice 33 kDa protein is more resistant to the
V8 protease-digestion than the spinach 33 kDa protein so that
harsher treatments were needed for the rice protein (legends of
Fig. 3).
In the above experiments, the recombinant 33 kDa protein were used for the cyanobacterium, red alga, euglena, green
alga, while the native 33 kDa protein was used for the two species of higher plants. It is possible that the different pattern of
protease-digestion among different organisms observed may be
due to, at least partly, possible differences between the structure of the recombinant 33 kDa protein and that of the native
protein. In order to examine this possibility, we compared the
cleavage patterns between the recombinant and native 33 kDa
protein of both S. elongatus and C. caldarium. As shown in
Fig. 4, the cleavage patterns of the recombinant, cyanobacterial 33 kDa protein at different enzyme-protein ratios of either
434
Comparison of the structure of the 33 kDa protein
Fig. 3 Polypeptide analysis of the 33 kDa protein treated with S. aureus V8 protease. The 33 kDa proteins from various species indicated in the
figure were treated with V8 protease at 30°C, and then applied to a 16–22% gradient polyacrylamide gel containing 0.1% SDS and 7.5 M urea.
Lanes 1, 4, 7, 10, 13 and 16, control 33 kDa protein. Other lanes are treated with V8 protease at enzyme-protein ratio (mg mg–1) of 0.33 for 2 h
(lanes 2 and 5), of 1.0 for 2 h (lanes 3, 6 and 11), of 1.7 for 2 h (lane 8), of 10 for 2 h (lanes 9, 12 and 18), of 0.33 for 30 min (lane 14), of 10 for
30 min (lane 15), and of 3.33 for 2 h (lane 17).
chymotrypsin or V8 protease were completely the same as
those of the native protein treated in the same conditions. Also,
the cleavage patterns of the recombinant, red algal 33 kDa protein were the same as those of the native protein under variously different conditions. These results excluded the possibility that the different cleavage patterns observed are due to the
differences in the structure between recombinant proteins and
native ones, and suggest that they are indicative of differences
in the structure of the 33 kDa protein from various organisms.
Discussion
So far, the psbO gene has not been cloned from red algae,
although this gene has been cloned and sequenced from various organisms such as cyanobacteria, euglena, green algae, and
higher plants (Seidler 1996, De Las Rivas and Heredia 1999).
In this study, we cloned and determined the full sequence of the
psbO gene from an acidophilic red alga, C. caldarium. Our
results showed that the psbO gene from the red alga contained
two characteristic domains in its leader sequence, one has the
feature for transfer across chloroplast envelope and the other
one has typical feature for transit peptide across thylakoid
membranes. This indicates that the psbO gene is encoded by
the nuclear genome of the red algae, which confirms the results
from whole plastid genome sequencing showing that the psbO
gene is not located in the red algal plastid (Reith and Munholland 1996). This indicates that this gene had moved to the
nucleus during evolution from the prokaryotic cyanobacteria to
the most primitive eukaryotic red algae.
Table 3 compared the sequences of the mature 33 kDa
protein from C. caldarium with those from 12 different species
for which gene-derived amino acid sequence are available. The
sequence homology, that is, the ratio of identical residues out
of the total residues, was 50.0–50.8% between the red alga and
cyanobacteria (except the homology between the red alga and
Synechocystis which is 42.9%); this is slightly higher than the
homology between the red alga and euglena (47.3%), between
the red alga and green alga (47.6%), and between the red alga
and higher plants (44.7–48.6%). This is consistent with the fact
that red algae are the most primitive eukaryotic algae closely
related to the prokaryotic cyanobacteria. The homologies
between the red algal psbO gene with those of other species,
however, are apparently lower than the homology between different species of cyanobacteria, or between euglena and green
algae, between euglena and higher plants, or between green
algae and higher plants. The highest homology was observed
among different species of higher plants. This may suggest that
the sequence of the psbO gene changed frequently in lower
organisms such as cyanobacteria and red algae, but became
more rigid and conserved after it evolved into higher plants.
Fig. 5 shows the alignment of amino acid sequences of the
33 kDa protein from six typical organisms. Three major characteristic features can be identified. First, the red algal 33 kDa
protein has an additional insertion sequence between 185G and
193R that is not present in the sequence of other species. Sec-
Fig. 4 Comparison of the cleavage pattern between native and
recombinant 33 kDa proteins. Lanes 1 and 4, native and recombinant
33 kDa protein without protease-treatment; lanes 2 and 5, native and
recombinant 33 kDa protein treated with a-chymotrypsin at an
enzyme-protein ratio of 0.33 for 2 h at 30°C; lanes 3 and 6, native and
recombinant 33 kDa protein treated with V8 protease at an enzymeprotein ratio of 0.33 for 2 h at 30°C. Panel A, Synechococcus elongatus. Panel B, Cyanidium caldarium.
Table 3
Comparison of the extrinsic 33 kDa protein sequence among different species
Cyanobacteria
S.
Synecho- A.
elongatus cystis
nidulans
Anabaena
Euglena
Euglena
C. caldarium
S. elongatus
–
–
50.0
–
42.9
56.0
50.2
57.8
50.8
63.9
47.3
47.8
Synechocystis
–
–
–
59.6
57.0
A. nidulans
–
–
–
–
Anabaena
–
–
–
Euglena
–
–
–
Green algae
Chlamydomonas
Higher plants
Spinach
Arabidopsis Solanum Wheat
Pea
Oryza
47.6
47.1
46.3
46.9
44.7
48.2
45.7
46.7
45.5
47.6
46.5
48.8
48.6
49.8
42.9
47.9
44.3
45.5
45.3
41.8
44.9
45.9
60.1
43.3
45.1
44.9
44.9
44.8
43.5
45.2
46.2
–
–
50.4
52.5
50.0
48.3
51.2
49.2
51.7
52.9
–
–
–
63.9
60.2
61.4
59.3
56.1
60.5
61.4
Chlamydomonas
–
–
–
–
–
–
–
62.5
64.2
64.6
60.8
63.3
63.8
Spinach
–
–
–
–
–
–
–
–
83.1
88.9
82.3
87.7
88.5
Arabidopsis
–
–
–
–
–
–
–
–
–
83.0
79.8
83.4
83.8
Solanum
–
–
–
–
–
–
–
–
–
–
84.3
87.5
89.1
Wheat
–
–
–
–
–
–
–
–
–
–
–
82.7
89.1
Pea
–
–
–
–
–
–
–
–
–
–
–
–
86.6
Oryza
–
–
–
–
–
–
–
–
–
–
–
–
–
Comparison of the structure of the 33 kDa protein
Red algae
C.
caldarium
This table shows the ratio of identical residues out of the total residues of the mature 33 kDa protein from Synechococcus elongatus (Miura et al. 1993), Synechocystis 6803
(Philbrick and Zilinskas 1988), Anacystis nidulans R2 (Kuwabara et al. 1987), Anabaena PCC 7120 (Borthakur and Haselkorn 1989), Euglena gracilis (Shigemori et al. 1994),
Chlamydomonas reinhardtii (Mayfield et al. 1987), Spinacia oleracea (Tyagi et al. 1987), Arabidopsis (Ko et al. 1990), Solanum tuberosum (potato) (Van Spanje et al. 1991), wheat
(Meadows et al. 1991), pea (Wales et al. 1989), Oryza sativa (rice) (Uchiyama and Tsugita 1991) and Cyanidium caldarium (this study).
435
436
Comparison of the structure of the 33 kDa protein
Fig. 5 Alignment of the mature 33 kDa protein sequences from Synechococcus elongatus (Miura et al. 1993), Cyanidium caldarium (this study),
Euglena gracilis (Shigemori et al. 1994), Chlamydomonas reinhardtii (Mayfield et al. 1989), Spinacia oleracea (Tyagi et al. 1987), Oryza sativa
(Uchiyama and Tsugita 1991), and comparison of their cleavage sites by chymotrypsin (black triangles) and V8 protease (arrowheads). Residues
in boxes are not cleaved in that organism but are conserved and cleaved in at least some of the other organisms. Asterisks indicated identical residues among all of the six organisms, and dots indicate identical residues among majority of the six organisms.
ond, the psbO gene is shortest in its N-terminal region in
cyanobacteria, longer in the red algae, euglena and green algae,
and becomes the longest in higher plants. Finally, cyanobacterial and red algal proteins have an insertion around 135–145
residues that are absent in the other four species. The latter two
features are consistent with the fact that the red algal psbO
gene is most closely related to the cyanobacterial gene, but the
first feature also revealed a unique position of the red algal
gene.
In order to investigate the structural differences of the
33 kDa protein among different species, we determined the
cleavage sites of the 33 kDa protein by chymotrypsin and V8
protease. Variously different cleavage sites were observed for
the 33 kDa protein from different organisms. In the course of
these experiments, recombinant and native 33 kDa proteins
were used for different organisms. We confirmed that the
resulting cleavage patterns are the same for both of the recombinant and native proteins from the same organism. This is consistent with results from reconstitution experiments that
showed that recombinant 33 kDa protein can support oxygen
evolution as effectively as the native protein does (Seidler
1996, Motoki et al. 1998, Enami et al. unpublished results), and
suggests that the different cleavage patterns observed are due to
structural differences of the 33 kDa protein from different
organisms.
The cleavage sites of the 33 kDa proteins from different
species (S. elongatus, C. caldarium, E. gracilis, C. reinhardtii,
S. oleracea, O. sativa) are summarized in Fig. 5. The cleavage
sites of the cyanobacterial and red alga 33 kDa protein by chymotrypsin and V8 proteases were located in the region between
residues 156 and 195 in their sequences. In contrast, the higher
plant 33 kDa protein showed no cleavage sites at all in this
Comparison of the structure of the 33 kDa protein
region, although most of the amino acids that were cleaved
with these proteases in this region are conserved in the higher
plant 33 kDa protein (residues in boxes in Fig. 5, e.g. 156F and
181E in the cyanobacterial protein). Instead, the spinach
33 kDa protein was cleaved at 16Y by chymotrypsin, and at
18E by V8 protease. The rice 33 kDa protein was not cleaved
by chymotrypsin at 17Y (corresponding to 16Y of spinach
33 kDa protein). This may be due to the difference in the residue immediately following: the residue after 17Y is Met in rice
whereas that after 16Y in spinach is Leu. On the other hand,
the cleavage site by V8 protease was well conserved between
the spinach and rice 33 kDa protein. Cleavage in the region
corresponding to 16Y-18E of spinach, however, did not occur
in the cyanobacterial and red algal 33 kDa protein, in spite of
the fact that Y and E are all conserved in this region. The
euglena and green algal 33 kDa protein had cleavage sites
involving both of the cyanobacterial and higher plant 33 kDa
proteins. Since the amino acid corresponding to 18E of the
higher plant protein had changed to Q in the green algal protein, no cleavage at this region was detected in the green algal
protein. In the 33 kDa protein from euglena, additional cleavage sites were observed at 67E and 69E. No cleavage at the
position corresponding to 67E of euglena was detected in other
species, in spite of the well conservation of the acidic residue
among six species.
The occurrence of a cleavage site by protease digestion
depends on, in principle, two factors, one is the amino acid
sequence of that site and the other one is the accessibility of the
corresponding region (site) to the protease in solution. If different cleavage sites were observed with their sequences at the
particular cleavage sites conserved between two proteins, this
difference should be attributed to the accessibility of that
region to the protease, in other words, the structure of the
region in question is different between the two proteins. The
results obtained in this study indicated that the structure of the
33 kDa protein in solution is clearly different among different
species and can be divided into three major groups. They are
cyanobacterial-type (cyanobacteria and red algae), higher
plant-type, and an intermediate-type including euglena and
green algae. Since the cleavage sites are considered to be
exposed to the surface of the protein to be prone to protease
attack, in the cyanobacterial-type the region around 156–195
residues of the 33 kDa protein must be exposed to the protein
surface but the N-terminal region does not. In the higher planttype, the N-terminal region containing 16Y and 18E exposes to
the protein surface but the region around 156–195 residues
does not. In the intermediate-type, both the regions of the Nterminal and around 156–195 residues are exposed to the protein surface, though in the euglena protein additional region
around 67E-69E may also be exposed to the surface. This may
imply a gradual change of the structure of the 33 kDa protein
during evolution of oxygen-evolving system from cyanobacteria to higher plants. This change may correlate with the change
that occurred in the other extrinsic proteins, i.e. from cyto-
437
chrome c550 and a 12 kDa protein in cyanobacteria and red
algae to the 23 and 17 kDa proteins in euglena, green algae and
higher plants. Various studies concerning the structure of the
33 kDa protein and its binding sites to PSII have been published mainly using the spinach 33 kDa protein (for review, see
Bricker and Frankel 1998). Our present results suggested that
such studies should also be conducted for the 33 kDa protein
from various species, in order to better understand the structural difference of the protein among different species.
It was reported recently that conformational changes
occur in the 33 kDa protein upon its binding to PSII (Hutchison
et al. 1998, Enami et al. 1998a). It is interesting to compare the
structure of the 33 kDa protein in solution with that when it is
bound to PSII by means of limited proteolysis. We tried the
limited proteolysis of the 33 kDa protein when it is bound to
PSII. However, PSII intrinsic proteins such as CP47, CP43, D1
and D2 were cleaved concurrently with the cleavage of the
33 kDa protein, which interfered significantly with the identification of cleavage sites of the 33 kDa protein. Therefore, at
present we are unable to identify the cleavage sites of the
33 kDa protein when it is bound to PSII.
Materials and Methods
Cloning and sequence analysis of the red algal psbO gene
The 33 kDa protein was purified from the red alga C. caldarium
according to Enami et al. (Enami et al. 1998b, Enami et al. 2000). The
N-terminal 28 amino acid sequence was determined with an Applied
Biosystems 477A protein sequencer after the polypeptide was resolved
by SDS-PAGE and semi-dry-blotted at a constant current of 1.0 mA
cm–2 for 1.5 h onto a polyvinylidene difluoride membrane (Immobilon™-P, Millipore, MA, U.S.A.). Based on the N-terminal sequence
and the conserved regions of the 33 kDa protein among cyanobacteria,
green algae and higher plants, two regions of the amino acid
sequences, 11Tyr-16Gly (YEQVKG) and 160Asp-167Gly (DPKGRG), were chosen to synthesize degenerate oligonucleotide primers.
These primers were used to amplify a cDNA fragment by the polymerase chain reaction (PCR), with double-stranded cDNA as template,
constructed as described previously (Ohta et al. 1999). To prepare
cDNA fragments of the 5¢- and 3¢-flanking regions of the psbO gene,
the method of RACE-PCR (rapid amplification of cDNA ends) was
used with the Marathon cDNA Amplification Kit (Clontech, CA,
U.S.A.). DNA sequence was determined by Dye Deoxy Terminator
Cycle Sequencing (Applied Biosystems, CA, U.S.A.) with a DNA
Sequencing System (model 310) after the PCR fragment was inserted
into the plasmid pCR II (TA Cloning Kit, Invitrogen, CA, U.S.A.). The
flanking regions obtained were combined with the internal fragments
obtained by the first-step PCR to yield the full sequence of the psbO
gene.
Expression and purification of recombinant and native 33 kDa proteins
from various organisms
The red algal (C. caldarium) psbO gene was sub-cloned into an
expression plasmid pET-3d (STRATAGENE, CA, U.S.A.). Both of the
red algal and cyanobacterial (S. elongatus) psbO genes were expressed
in Escherichia coli and purified according to Motoki et al. (1995). The
psbO genes of a euglena and a green alga were amplified on the basis
of the nucleotide sequences coding for the 33 kDa protein from E. gracilis (Shigemori et al. 1994) and C. reinhardtii (Mayfield et al. 1989).
438
Comparison of the structure of the 33 kDa protein
These psbO genes were cloned into the LIC site of plasmid pET-32´a/
LIC, resulting in a fusion protein with thioredoxin and 6´ His-tag
attached at its N-terminus (Ohta et al. 1999, Okumura et al. 2001).
These recombinant plasmids were transformed into E. coli. (strain
BL21 (DE3)) (Novagen, WI, U.S.A.). The E. coli was grown at 37°C
with vigorous aeration in Luria-Bertani medium containing 100 mg ml–1
ampicilin. The fusion proteins were expressed efficiently by induction
with 1 mM isopropyl-b-D-thiogalactopyranoside after 4 h. The cell pellets were collected by centrifugation (1,500´g, 10 min) and suspended
in 20 mM Tris-HCl (pH 7.9) containing 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100, 0.1 mg ml–1 lysozyme, and the cells were
lysed by incubation at 30°C for 15 min followed by sonication. The
lysate was centrifuged at 15,000´g for 20 min and the supernatant was
applied to a 1 ml column packed with His-Bind resin (Novagen, WI,
U.S.A.). The fusion proteins were eluted with 1 M imidazole, dialyzed
against 20 mM Tris-HCl (pH 7.4), 0.1 M NaCl, and then treated with
Factor Xa (Novagen, WI, U.S.A.), followed by purification with the
His-Binding column in order to remove thioredoxin and 6´ His-tag.
All the purification steps of the recombinant proteins were carried out
on ice or at 4°C.
The native 33 kDa protein from S. elongatus and C. caldarium
was purified from isolated PSII complexes as described in Shen and
Inoue (1993) and Enami et al. (1998b).
Rice PSII membranes were prepared as described in Shen et al.
(1988). The higher plant 33 kDa protein was released and purified
from rice and spinach PSII membranes with the method described in
Enami et al. (2000).
All of the 33 kDa proteins were dialyzed against 50 mM MESNaOH (pH 6.5) and stored at –80°C, before being subjected to limited
proteolysis.
Limited proteolysis
The 33 kDa protein from various organisms was treated with achymotrypsin (VII from bovine pancreas, SIGMA, MO, U.S.A.) or S.
aureus V8 protease (XVII, SIGMA, MO, U.S.A.) in 50 mM MESNaOH (pH 6.5) for 30 min or 2 h at 30°C. The reaction was stopped
by addition of 3 mM phenylmethylsulfonyl fluoride for chymotrypsin
and 2 mM diisopropyl fluorophosphate for V8 protease.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SDS-PAGE was performed with a gradient gel containing 16–
22% acrylamide and 7.5 M urea according to Ikeuchi and Inoue
(1988). The samples were solubilized with 5% lithium lauryl sulfate
and 75 mM dithiothreitol for 30 min prior to electrophoresis.
Mass spectroscopic analysis
For determination of molecular masses of the digested peptide
fragments, the protease-digested protein mixture of the 33 kDa protein
was directly applied to a MALDI-TOF mass spectrometer (JEOL JMS
HX-110), with the matrix of either sinapic acid (SIGMA, MO, U.S.A.)
and 2,5-dihydroxybenzoic acid (SIGMA, MO, U.S.A.) (4 : 1) in 50%
ethanol/0.1% TFA or 10 mg ml–1 a-cyano-4-hydroxyciannamic acid
(SIGMA, MO, U.S.A.) in 50% ethanol/0.1% TFA. Spectra were calibrated using myoglobin (SIGMA, MO, U.S.A.) for measurements of
the molecular masses above 1,500 Da or angiotensin (SIGMA, MO,
U.S.A.) and adrenocorticotropic hormone fragment 7–38 (SIGMA,
MO, U.S.A.) for measurements of those below 1,500 Da.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture
of Japan to I.E. (13640658).
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(Received September 25, 2001; Accepted January 31, 2002)