Low-Molecular-Mass Polypeptide Components of a Photosystem II ...

Plant Cell Physiol. 43(11): 1366–1373 (2002) JSPP © 2002

Low-Molecular-Mass Polypeptide Components of a Photosystem II Preparation from the Thermophilic Cyanobacterium Thermosynechococcus vulcanus Yasuhiro Kashino 1, 4, Hiroyuki Koike 1, Maki Yoshio 1, Hirokazu Egashira 1, Masahiko Ikeuchi 2, Himadri B. Pakrasi 3 and Kazuhiko Satoh 1 1

Himeji Institute of Technology, Faculty of Science, Harima Science Garden City, Hyogo, 678-1297 Japan Department of Life Science (Biology), University of Tokyo, Komaba 3-8-1, Meguro, Tokyo, 153-8902 Japan 3 Department of Biology, Washington University at St. Louis, St. Louis, MO 63130-4899, U.S.A. 2

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Using a recently introduced electrophoresis system [Kashino et al. (2001) Electrophoresis 22: 1004], components of low-molecular-mass polypeptides were analyzed in detail in photosystem II (PSII) complexes isolated from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (formerly, Synechococcus vulcanus). PsbE, the large subunit polypeptide of cytochrome b559, showed an apparent molecular mass much lower than the expected one. The unusually large mobility could be attributed to the large intrinsic net electronic charge. All other Coomassie-stained polypeptides were identified by N-terminal sequencing. In addition to the well-known cyanobacterial PSII polypeptides, such as PsbE, F, H, I, L, M, U, V and X, the presence of PsbY, PsbZ and Psb27 was also confirmed in the isolated PSII complexes. Furthermore, the whole amino acid sequence was determined for the polypeptide which was known as PsbN. The whole amino acid sequence revealed that this polypeptide was identical to PsbTc which has been found in higher plants and green algae. These results strongly suggest that PsbN is not a member of the PSII complex. It is also shown that cyanobacteria have cytochrome b559 in the high potential form as in higher plants. Keywords: Low-molecular-mass polypeptide — Photosystem II — PsbE — PsbN — PsbTc — Thermosynechococcus vulcanus. Abbreviations: PSI, photosystem I; PSII, photosystem II. The nucleotide sequence reported in this paper has been submitted to DDBJ under accession number AB086860.

Introduction Photosystem II (PSII) is one of the most important components for energy metabolism in biosphere. It contributes to the first step in converting light energy to chemical energy in the thylakoid membranes, and catalyses water cleavage, which results in oxygen evolution. This complex reaction is performed on a multi-subunit polypeptide complex, which com4

prises over 20 subunit polypeptides (Zouni et al. 2001, Ikeuchi 1992). Recently, Zouni et al. (2001) reported a crystallographic structure of the PSII complex from a thermophilic cyanobacterium, Thermosynechococcus elongatus. This is a quite important step in the research on the structure and function of PSII complexes. The reported structure contained 36 ahelices (Zouni et al. 2001), out of which, 22 a-helices were assigned to CP47, CP43 and the D1 and D2 proteins. However, uncertainty remains for the assignment of the remaining 14a-helices, all of which should belong to low-molecular-mass intrinsic polypeptides. They claimed that the crystallized PSII complex contained PsbE, PsbF, PsbH, PsbI, PsbJ, PsbK, PsbL, PsbM, PsbN and PsbX as low-molecular-mass intrinsic polypeptides. Very recently, Shen and Kamiya (2000) also reported a crystallographic structure of PSII complexes from a thermophilic cyanobacterium, T. vulcanus, and claimed that the complexes have the same polypeptide composition as that reported by Zouni et al. (2001). If we assume that a unit of PSII core complex contained one copy of each small-size polypeptide and each of them has only one a-helix (this is supported by hydropathy plots of these polypeptides, data not shown), the number of calculated a-helices (10a-helices) is smaller than the number of 14 for the unassigned a-helices. Until now, several low-molecular-mass polypeptides of PSII complexes other than those listed above have been reported (summarized in Funk 2000). Therefore, it is highly probable that the crystallized cyanobacterial PSII complexes have some more low-molecular-mass polypeptides. In fact, we recently clarified the precise polypeptide composition of PSII complexes purified from HT-3 mutant of Synechocystis sp. PCC 6803, which contained newly determined polypeptides (Kashino et al. 2002). It is very important to elucidate the complete polypeptide composition of cyanobacterial PSII core complexes for the determination of crystallographic structure. In this work, we investigated the polypeptides in a lowmolecular-mass region (less than about 20 kDa) of PSII complexes from T. vulcanus using a recently introduced SDSPAGE system, which can resolve polypeptides from less than 3 kDa to over 100 kDa (Kashino et al. 2001).

Corresponding author: e-mail, [email protected]; Fax, +81-791-58-0185. 1366

Small proteins in cyanobacterial PSII complex

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Fig. 1 Polypeptide profiles of thylakoid membranes and isolated PSII complexes. (A) lane 1, thylakoid membranes from T. vulcanus; 2, isolated PSII complexes; 3, PSII complexes after Ca2+-wash; 4, supernatant of Ca2+-wash. Dots on the bands indicate the polypeptides which were subjected to N-terminal analysis. Serial numbers of the dots correspond to those in Table 1. The positions of molecular weight standards are shown on the left; from top to bottom, 36.5, 27.9, 17.8, 8.6 and 4.1 kDa. (B) lane 1, isolated PSII complexes from a HT-3 mutant of Synechocystis 6803; lane 2, Ca2+-washed PSII complexes from T. vulcanus. Asterisks indicate PsbE. Samples equivalent to 7 mg Chl a were applied to each lane, and the polypeptides were stained with Coomassie blue.

Results Polypeptide profile of PSII complexes isolated from T. vulcanus More than 30 polypeptides were highly enriched in the PSII fraction (Fig. 1A, lane 2) from the initial thylakoid membranes (Fig. 1A, lane 1) of T. vulcanus. In the PSII fraction, 16 polypeptide bands were found in the region below about 20 kDa (dotted bands). The N-terminal sequences of such polypeptides were determined as shown in Table 1. From these 16 Coomassie-stained bands, 17 polypeptides were identified by comparison with reported sequences using fasta program (for PsbZ, Psb27 and AtpL, sequence data of T. elongatus was kindly provided from Drs. Tabata and Kaneko (Nakamura et al. 2002)). In addition to the polypeptides reported to be present in the PSII complexes of T. vulcanus, the presence of PsbX, PsbY, PsbZ and Psb27 was confirmed, which indicated that the PSII complex of T. vulcanus contains 14 polypeptides smaller than about 20 kDa (i.e. PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbM, PsbTc, PsbU, PsbV, PsbX, PsbY, PsbZ and Psb27). PsbJ and

PsbN were not detected in this PSII complexes. The presence of AtpL shows some contamination of ATPase. However, no PSI subunit polypeptide was detected although the PSI complex has several low-molecular-mass polypeptides (Jordan et al. 2001). To test the hydrophobicity of these polypeptides, the isolated PSII complexes were treated with 1.0 M CaCl2. Using the Ca2+-wash, peripheral polypeptides were removed (Fig. 1A, lane 4), including the large linker polypeptide (~110 kDa), the linker polypeptides (~50 kDa), the extrinsic 33 kDa polypeptide, phycocyanin and allophycocyanin (~16 kDa). PsbV (band #16) and PsbU (band #13) were also removed by this Ca2+wash. In contrast, all of the polypeptides smaller than the polypeptide #12 were not removed by the Ca2+-wash (Fig. 1, lane 3), which indicates that the 12 polypeptides smaller than PsbU were intrinsic polypeptides. Recently, we have determined the precise polypeptide composition of PSII complexes purified from an HT-3 mutant of Synechocystis 6803 (Kashino et al. 2002). So, we compared the

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Table 1 N-terminal amino acid sequences of low-molecularmass polypeptides of PSII complexes from T. vulcanus Number 1 2 3 4 5 6

7

8 9 10 11 12 13 14 15 16

Sequence KLPEA YAIF MEVNQ LGLIA TALFV TITPS LKGF MDwRV LVVLL PVLLA A METIT YVFIF AxIIA LFFFA IFFRE PPRIT KK MEPNP NRQPV ELN METIT YVFIF AxI METLK ITVYI VVT MEPNP NRQPV ELN METLK ITVYI VVT TSNT PNQ MtILF QLALA ALVI MNPLI ASASV LAASL S ARRTW LGDI AGTTG ER —k LNDFF ALYRR DDxLR —k IDLNN TNIAA MQDAI TAVIN ASDV SVVTK SIVNA DAEAR AELTP EVLTV PLN

Protein PsbK PsbM PsbX PsbY PsbTc* PsbL PsbTc* PsbI PsbL PsbI PsbF PsbZ AtpL PsbH PsbE Psb27 PsbU ApcB ApcA PsbV

The polypeptide numbers are as described in Fig. 1A. The polypeptides 12 and 13 were sequenced after cleavage by lysylendopeptidase. x, unidentified amino acid. *For PsbTc, see the text. “—k” indicates the presence of presequence preceding to the Lys residue.

polypeptide profile of PSII complexes from T. vulcanus with that from Synechocystis 6803 (Fig. 1B). Concerning the lowmolecular-mass region, corresponding polypeptides in Synechocystis 6803 were found for almost all polypeptides in T. vulcanus, and those polypeptides migrated at almost the same rates as those of Synechocystis 6803. One distinct difference found between them was the apparent molecular mass of PsbE; it was remarkably smaller (around 2 kDa) in T. vulcanus than in Synechocystis 6803 (Fig. 1B, shown by asterisks). To clarify the reason for this large difference in the apparent molecular mass of PsbE, the psbE gene in T. vulcanus was sequenced. Gene structure of psbE The psbE gene was found to form a gene cluster with other open reading frames (orfs) (Fig. 2); those orfs were identified as psbE, F, L and J. They were assembled in a DNA region of 700 bp similar to that of other cyanobacteria and plastids (Kaneko et al. 1996, Ohyama et al. 1986, Shinozaki et al. 1986). The deduced amino acid sequences are shown in Fig. 2 in comparison with the homologues of Synechocystis 6803 and a higher plant (Spinacea oleracea). As reported previously (Ikeuchi et al. 1989b), the N-terminal Met residue was cleaved off in PsbE, while it was retained in PsbF and L (Table 1).

PsbJ, however, was not detected in the present study. The predicted molecular mass of PsbE in T. vulcanus was 9.4 kDa, almost the same size as in Synechocystis 6803 (9.3 kDa). However, unlike PsbE in Synechocystis 6803, the apparent molecular mass of PsbE in T. vulcanus on the electrophoresis (Fig. 1) was quite small (7 kDa, data not shown). Only the Met residue of the N-termini was cleaved off in mature forms of both of them (Table 1). It was possible that post-transcriptional cleavage of the C-terminus of PsbE occurs in T. vulcanus similar to the C-terminal processing of the D1 protein (Takahashi et al. 1988). Thus the amino acid sequence of its C-terminus was analyzed. Because PsbE in T. vulcanus has one Lys(73) residue (Fig. 2), the lysylendopeptidase was effective for the limited digestion of this polypeptide (Fig. 3). The determined amino acid sequence following Lys(73) was QQVETFLEQLK, which indicated that the mature PsbE in T. vulcanus had whole amino acid residues. Components of PSII core complexes in cyanobacteria The amount of cytochrome b559, which is composed of PsbE and PsbF, in a crude PSII preparation is important to determine the constituents of PSII because the heme b can be released during the purification process. The oxidation–reduction difference spectra showed semi-symmetrical Gaussian curves centered at around 560 nm (Fig. 4). Interestingly, chemical reduction by hydroquinone of the PSII preparations showed the presence of a high potential form of cytochrome b559, which was equivalent to about 50% and 20% of the total amount of cytochrome b559 in the PSII preparations before (data not shown) and after Ca2+-wash, respectively. The full reduction by dithionite in Ca2+-washed PSII enabled us to calculate the total amount of cytochrome b559 (Fig. 4), which was 1.87 per 41 Chl a molecules (i.e. per PSII reaction center; (Tang and Diner 1994, Kashino et al. 2002)). It was also revealed that the PSII complex contained 3.8 Fe/41 Chl a after the Ca2+-wash by atomic absorption spectroscopy.

Discussion Characteristics of PsbE The large subunit of cytochrome b559 (PsbE) from T. vulcanus showed an unusual migration pattern compared to that of Synechocystis 6803. Because the molecular masses of them are quite similar, it is reasonable to expect the two PsbEs to show similar mobility. As is easily recognized from the data in Fig. 2, the predicted amino acid sequence from Met(1) to Tyr(44) of PsbE in T. vulcanus is highly homologous to that of Synechocystis 6803, and they showed a similar pattern of hydropathy plot and similar values of isoelectric points in this region (data not shown). However, the homology was somewhat decreased from Asp(45) onwards; the resulting isoelectric points were 4.55 in T. vulcanus and 3.92 in Synechocystis 6803, respectively. The intrinsic net electric charges at pH 9.0 (pH in the resolving gel) were calculated to be –2.5 and –5.5 e–-equiv-


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Fig. 2 Structure of psbEFLJ gene cluster in T. vulcanus (S. vulcanus in this figure) and predicted amino acid sequences of PsbE, F, L and J, in comparison with those from Synechocystis 6803 (http://www.kazusa.or.jp/cyano/cyano.html) and spinach (accession number in EMBL: AJ400848). The predicted a-helical regions have been underlined. Asterisks show the identical amino acids.

alent per polypeptide, respectively (the total charge of the whole polypeptides were –2.5 and –6.0, respectively). This large difference of the intrinsic charges can result in the difference of the mobility of PsbEs. This situation was indicated by Weber et al. (1972) and Swank and Munkers (1971); the intrinsic charge might remarkably influence the electric mobility of proteins in SDS gels. Weber et al. (1972) suggested that the use of high concentration of urea in the resolving gel might result in the good linearity of molecular mass versus mobility. However, the PsbE did not conform to this rule because, in this electrophoresis system, the gel contained 6 M urea. Components of PSII core complexes in cyanobacteria Sixteen polypeptide bands of less than about 20 kDa were identified using the SDS-PAGE system for the PSII complexes isolated from T. vulcanus (Fig. 1A). Among these polypeptides, 14 were assigned to be the constituents of the PSII core complex. Nine out of them had already been reported in the PSII core complexes of T. vulcanus; those were PsbE, PsbF, PsbH, PsbI, PsbK, PsbL, PsbM, PsbU and PsbV (Ikeuchi et al. 1989b, Ikeuchi et al. 1989a, Koike et al. 1989, Shen and Inoue 1993). Present data clearly indicates that PsbX, PsbY, PsbZ and Psb27 are also present in T. vulcanus PSII complexes (Table 2), which suggests that these polypeptides are common constituents of cyanobacterial PSII complexes. The crystallographic data reported by Zouni et al. (2001) indicated that the PSII complex contained 14 unassigned transmembrane a-helices, which must belong to small-molecularmass polypeptides. They claimed that 10 polypeptides of small

sizes were bound to the PSII complex, including PsbN (Table 2). From our results, PsbN in their crystallized PSII complexes can be PsbTc (see below). They also described that the crystallized PSII complex contained PsbJ. In this study, it was confirmed that psbJ consisted of a gene cluster with three other genes, psbE, F and L (Fig. 2), suggesting that this gene cluster has the same structure throughout the oxygenic photosynthetic organisms. However, we could not detect PsbJ in our PSII preparation (Table 2). There are several possibilities for the failure to detect PsbJ: (1) PsbJ is not bound stoichiometrically to the PSII complex; (2) PsbJ was removed during the isolation process; (3) the N-terminus of PsbJ was blocked in T. vulcanus; and (4) PsbJ is not easily stained by Coomassie blue. Because we detected only 12 polypeptides which were not removed by salt wash (Fig. 1, Table 1), the a-helices of these low-molecular-mass polypeptides are less than 14 even after taking PsbJ into account. There may be a possibility that some of the determined polypeptides are present as more than two copies in the PSII complex. Cytochrome b559 (PsbE and PsbF) is one candidate since contradictory values have been reported for the number of cytochrome b559 molecules per unit PSII complex; one or two (Whitmarsh and Pakrasi 1996). Zouni et al. (2001) and Shen and Kamiya (2000) indicated that their PSII core complexes have only two heme iron atoms (cytochrome b559 and c550) and one non-heme iron atom per reaction center. However, Zouni et al. (2001) reported that the number of cytochrome b559 molecules is unresolved. In the present study, using Ca2+-washed PSII complexes which were free from cytochrome c550, the oxidation–reduction difference spec-

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Table 2 Comparison of polypeptide composition smaller than about 20 kDa of T. vulcanus (this study), T. elongatus (Zouni et al. 2001) and Synechocystis 6803 (Kashino et al. 2002) Protein (gene) D1 protein (psbA) CP47 (psbB) CP43 (psbC) D2 protein (psbD) Cytochrome b559 (L) (psbE) Cytochrome b559 (S) (psbF) PsbH (psbH) PsbI protein (psbI) PsbJ protein (psbJ) PsbK protein (psbK) PsbL protein (psbL) PsbM protein (psbM) PsbN protein (psbN) Mn-stabilizing protein (psbO) PsbTc protein (psbTc) 12 kDa protein (psbU) Cytochrome c550 (psbV) PsbX protein (psbX) PsbY protein (psbY) PsbZ protein (psbZ, ycf9) Psb27 protein (psb27) Psb28 protein (psb28, ycf79)

T. vulcanus (thermophilic) O O O O O O O O O O O X O O O O O O O O

T. elongatus (thermophilic) O O O O O O O (O) (O) (O) (O) (O) (O) O O O (O)

Synechocysis 6803 (mesophilic) O O O O O O O O O O O O X O O O O O O O O O

“O” indicates proteins detected. Brackets in T. elongatus indicate the polypeptides which were shown without individual data. For PsbN and PsbTc, see the text.

tra showed that the PSII complexes isolated here contained two cytochrome b559 per unit PSII reaction center. Atomic absorption spectroscopy indicated that the Ca2+-washed PSII core complexes had four iron atoms per PSII complex isolated from T. vulcanus (this study) and purified from Synechocystis 6803 (Kashino et al. 2002). Among these iron atoms, at least two iron atoms can be attributed to the non-heme iron (Q400) and heme iron of cytochrome b559 if we assume the stoichiometry of one cytochrome b559 per unit PSII. But, two iron atoms in the Ca2+-washed PSII still remained unidentified. Although it should be kept in mind that the number of cytochrome b559 molecules depends on the extinction coefficient, we cannot exclude the possibility that there are two copies of PsbE and PsbF per PSII reaction center. From the oxidation–reduction difference spectrum of chemical titration, we found that about a half of the cytochrome b559 was in a high potential form in PSII complexes isolated from the thermophilic cyanobacterium, T. vulcanus (data not shown). Furthermore, 20% of the total cytochrome b559 was retained in a high-potential form even after the CaCl2wash (Fig. 4). In cyanobacteria, a high-potential form of cytochrome b559 has not been reported (Stewart and Brudvig 1998). Our results confirmed that cyanobacteria also have cyto-

chrome b559 of a high-potential form, and at least half of the cytochrome b559 is in a high-potential form in situ. Nomenclature of PsbN Among the polypeptides which were reported as PSII

Fig. 3 Limited digestion of PsbE. After electrophoresis of PSII complexes from T. vulcanus, the Coomassie-stained PsbE band was cut out and then incubated in a reaction mixture [100 mM Tris-HCl (pH 9.0) and 0.2% SDS] for 30 min, which was followed by an incubation with lysylendopeptidase at 37°C for 9 h. Then, after an addition of denaturing solution, the gel band was loaded to the second gel and electrophoresed. Lane 1, PSII complexes (7 mg Chl a); lanes 2–7, PsbE incubated with 0, 0.6, 3, 12, 30 and 60 mg ml–1 of lysylendopeptidase, respectively. Each sliced gel-band was derived from PSII complexes equivalent to 16 mg Chl a.


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be called “psbTc” because “psbTc” (Monod et al. 1994) is now widely accepted. Accordingly, there has been no data for the presence of PsbN in PSII complexes. Zouni et al. (2001) claimed that their PSII complex contained PsbN (Table 2), without showing the data. Ikeuchi et al. (1995) made a specific antibody against over-expressed polypeptide of PsbN. However, they failed to detect the polypeptide in the purified PSII complexes from Synechocystis 6803 although the specific antibody reacted with a polypeptide in thylakoid membranes (Ikeuchi et al. 1995). Kohchi et al. (1988) demonstrated the divergent overlapping transcription of ORF43 (psbN) in liverwort chloroplast. They discussed the possible function of this divergent overlapping transcription to be the regulation of gene expression in the psbB operon which is present on the opposite DNA strand. Taking these data into account, we propose that “PsbN” is not a component of PSII.

Materials and Methods

2+

Fig. 4 Absorbance difference spectra of the Ca -washed PSII complexes. HQ-Fe, hydroquinone-reduced minus ferricyanide-oxidized difference spectrum; Asc-Fe, ascorbate-reduced minus ferricyanideoxidized difference spectrum; DT-Fe, dithionite-reduced minus ferricyanide-oxidized difference spectrum. The samples were adjusted to 20 mg Chl a ml–1.

components, some polypeptides were not detected in the PSII complexes from T. vulcanus. PsbN is one of such polypeptides. The reason for this is that the psbN and psbTc genes were originally miss-assigned. In this work, we obtained the whole amino acid sequence of the 4.7 kDa polypeptide (band #5), whose N-terminal sequence was identical to the sequence originally reported as PsbN (4.7 kDa-II, Fig. 5A) (Ikeuchi et al. 1989a). Surprisingly, the whole amino acid sequence was identical to that of “PsbTc” predicted from the gene of T. elongatus (Dr. Iwai, personal communication; Fig. 5B). Furthermore, the whole amino acid sequence of it does not match to the deduced amino acid sequence of the “psbN” gene in T. elongatus (Nakamura et al. 2002, Fig. 5C). Ikeuchi et al. (1989a) found 4.7 kDa polypeptide (4.7 kDa-II) in PSII complexes from T. vulcanus, and named it “PsbN”. They assigned the ORF43 in the chloroplast DNA of liverwort as the gene for PsbN. This was because the homology of the obtained partial sequence of this 4.7 kDa polypeptides was highly homologous to PsbN (ORF43) rather than to PsbTc in liverwort (Fig. 5D, E). Due to the limited information on the amino acid sequence of the 4.7 kDa-II (the N-terminal 19 amino acid sequence) and database available in 1989, the miss-assignment was inevitable. According to the rules of nomenclature, “psbTc” should be called “psbN”. However, to avoid confusion, we propose that the gene should continue to

Isolation of PSII complexes PSII complexes of a thermophilic cyanobacterium, T. vulcanus, were purified according to Satoh et al. (1995) with slight modification. Thylakoid membranes (2 mg Chl ml–1) were solubilized with 1.1– 1.2% n-heptyl-b-D-thioglucoside (Dojin, Kumamoto, Japan) in solution A (50 mM 2-(N-morpholino)ethanesulfonic acid–NaOH (pH 6.7), 10 mM NaCl, 5 mM MgCl2) supplemented with 0.5 M sucrose. After removal of unsolubilized membrane by centrifugation three times (35,000´g for 15 min at 4°C), the PSII complexes were precipitated by centrifugation at 50,000´g for 2 h at 4°C. The resulting pellet was suspended in solution A supplemented with 1 M sucrose. These PSII complexes evolved oxygen at the rate of about 4,000 mmol O2 (mg Chl)–1 h–1. The extrinsic polypeptides of the purified PSII complexes of T. vulcanus were released by incubation with 1 M CaCl2 at a concentration of 1 mg Chl ml–1 for 30 min on ice in the dark (Ono and Inoue 1983). After centrifugation at 360,000´g, the precipitated PSII complexes were washed once and resuspended in solution A supplemented with 0.5 M sucrose. PSII complexes of the HT-3 mutant of Synechocystis 6803 were isolated after Kashino et al. (2002). Electrophoresis and amino acid sequencing of polypeptides Electrophoresis and amino acid sequencing of polypeptides were performed as reported in Kashino et al. (2001). Determined amino acid sequences were compared with reported sequences by fasta program equipped in the GCG software package (Lipman and Pearson 1985). Molecular mass standards (Kaleidoscope Polypeptide Standards) were supplied from Bio-Rad (CA, U.S.A.). Limited digestion of polypeptides After electrophoresis of PSII complexes from T. vulcanus, the Coomassie-stained band (from a sample equivalent to 16 mg Chl a) was cut out and then incubated in a reaction mixture (100 mM TrisHCl (pH 9.0) and 0.2% SDS) for 30 min. After an addition of an appropriate volume of stock solution (1 mg ml–1) of lysylendopeptidase (Wako, Japan), limited digestion was performed at 37°C for 9 h. Then, following an addition of a denaturing solution (5.2% LDS, 172 mM Tris-HCl (pH 8.0), 40 mM dithiothreitol, 0.5 M sucrose, 0.01% pyronine (Kashino et al. 2001)), the sliced gel band was loaded on a second gel and electrophoresed.

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Fig. 5 Homology of the polypeptide number 5 (#5) from T. vulcanus to 4.7 kDa-II polypeptide from T. vulcanus (A), PsbN and PsbTc (B-E). The amino acid sequence which was reported by Ikeuchi et al. (1989a) is shown as “4.7 kDa-II”. Identical amino acids are indicated by “|”, the homologous amino acid residues are indicated by “:”. x, unidentified amino acid; M. polymorpha, Marchantia polymorpha. Amino acid sequencing of the middle of polypeptides After the limited digestion by lysylendopeptidase and SDSPAGE, the polypeptides were sequenced as above. In the case of PsbE, after SDS-PAGE and electroblotting of the polypeptide, an amidoblack stained band on a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, CA, U.S.A.) was cut out, and the polypeptide was released by incubation with 70% (v/v) acetic acid. After drying, the released polypeptides were digested by lysylendopeptidase and then subjected to amino acid sequencing.

fragment which contained psbEFLJ fragment was cloned into pUC118 using DH5aF’ as the host Eschericia coli strain. An internal 1.2 kb DNA fragment which was digested by KpnI–PstI was subcloned into pUC118. A physical map was constructed by determining the fragments that had been digested by restriction endonucleases. DNA sequencing was performed by the chain-termination method using a Sequenase Kit (United States Biochemical, OH, U.S.A.) or Taq Dye Primer Cycle sequencing Kit (Applied Biosystems, CA, U.S.A.). The sequence was determined from both strands.

Optical measurements Cytochrome b559 was assayed by means of reduced-minus-oxidized absorption difference spectra using a UVIKON 922 spectrophotometer (Kontron Instruments, Milan, Italy) at a concentration of 20 mg Chl ml–1. The difference extinction coefficient of 17.5 mM–1 cm–1 for the wavelength at 559 nm versus 570 nm (Cramer et al. 1986) was used. Chl a concentration was determined by the method of Porra et al. (1989).

Acknowledgments

Iron content The content of bound iron was determined using a flameless atomic absorption spectrophotometer (AA-660G) equipped with a GFA-4B graphite furnace atomizer (Shimadzu, Kyoto, Japan) (Kashino et al. 1986). Cloning and sequences of photosynthetic genes A lgt11 genomic library of T. vulcanus was screened with a heterologous probe obtained from pKW1261 which contained psbEFLJ fragment derived from Synechocystis 6803 (Pakrasi et al. 1988). Phage particles were recovered from positive plaques, and the 2.5 kb DNA

We are grateful to Prof. Terry Bricker for the generous gift of the HT-3 strain. We also thank Drs. Tabata and Kaneko for giving the deduced amino acid sequence of psbZ, psb27, psb28 and atpL prior to the release of the database of T. elongatus. This research was supported by grants from the Hyogo prefecture, Japan to Y. K. and to H. K., and by grants from the National Institutes of Health (GM45797), and United States Department of Energy (to H. B. P.).

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(Received July 20, 2002; Accepted September 9, 2002)