©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Nuclear-encoded Subunit of the Photosystem II Reaction Center (*)

(Received for publication, February 16, 1995; and in revised form, May 2, 1995)

Klaus-Dieter Irrgang (§) , Lan-Xin Shi , Christiane Funk (§) , Wolfgang P. Schröder (¶)

From the Department of Biochemistry, Arrhenius Laboratories for Natural Sciences, Stockholm University, S-106 91 Stockholm, Sweden

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A nuclear-encoded polypeptide of 6.1 kDa was identified in isolated photosystem II (PSII) reaction center from Spinacia oleracea. The hydrophobic membrane protein easily escapes staining procedures such as Coomassie R-250 or silver staining, but it is clearly detected by immunodecoration with peptide-directed IgG. This additional subunit was found to be present in PSII reaction centers previously known to contain only the D1/D2/cytb proteins and the psbI gene product. Furthermore, cross-linking experiments using 1-(3-dimethylaminopropyl-)3-ethylcarbodiimide showed that the nearest neighbors were the D1 and D2 proteins and the cytb. The 6.1-kDa protein was purified by immune affinity chromatography. N-terminal sequence analysis of the isolated protein confirmed the identity of the 6.1-kDa protein and enabled finding of strong similarities with a randomly obtained cDNA from Arabidopsis thaliana. Using enzyme-linked immunosorbent assay in combination with thylakoid membrane preparations of different orientation, the N terminus of the protein, predicted to span the membrane once, is suggested to be exposed at the lumen side of the membrane. Consequently the 6.1-kDa protein seems to be the only subunit in the PSII reaction center that is nuclear encoded and has its N terminus on the lumen side of the membrane. These findings open for new interesting suggestions concerning the properties of photosystem II reaction center with respect to the photosynthetic activity, regulation and assembly in higher plants.


INTRODUCTION

Light-induced photosynthetic water oxidation and plastoquinone reduction takes place in the thylakoids of cyanobacteria, algae, and plants. These redox mediated reactions are catalyzed by a multisubunit membrane complex designated as photosystem II(1, 2, 3, 4) . This membrane protein complex has been shown to consist of more than 25 different polypeptides with relative molecular masses ranging from 47 down to 3 kDa. The minimum subcomplex that can evolve oxygen and release protons is referred to as the PSII()core complex. This detergent-solubilized complex contains 10-13 different polypeptides, four manganese ions, at least one calcium ion, and about 50 chlorophyll a molecules.

However, based on sequence homology between the L and M subunits of the reaction center complex of purple bacteria and the D1 and D2 protein, the concept of a D1/D2 reaction center heterodimer was proposed also for higher plants(5, 6, 7) . Indeed, such a PSII reaction center complex consisting of the D1, D2 proteins, cytochrome b has been isolated from higher plants (8, 9, 10) . This so-called D1/D2 reaction center heterodimer has now been shown to bind all of the redox components needed for the primary photochemistry of PSII(11) . Notably all the protein subunits in this complex have been found to be chloroplast encoded.

Previously several low molecular mass polypeptides of 10-3 kDa have been identified in various types of photosystem II preparations (12, 13, 14) using polyacrylamide gels of high resolution. Most of them have one hydrophobic stretch predicted to be a transmembrane -helix. They have been found to be encoded in the chloroplast genome and referred to as psbH-psbN gene products (for a summary see (2) ). One, the psbI gene product, has been found to be present in the D1/D2 heterodimer of higher plants(15, 16) . The function of these small subunits, as well as the organization within the PSII complex are, however, hitherto unknown. Using mutants from Synechocystis PCC 6803 where the psbH, psbK, or psbJ genes have been deleted, it was recently shown that these subunits were not essential for water oxidation and/or electron transport. Therefore, it was suggested that they could fulfill a regulatory or structural function within PSII (17, 18, 19) . The psbL gene product (with an approximately molecular mass of 5 kDa) has been suggested to be involved in stabilizing the Q binding niche(20) . Besides these, at least three further nuclear-encoded low molecular mass polypeptides have been suggested to be present or associated with various types of PSII preparations(12, 13, 14) .

In this paper we report on the identification and isolation of a nuclear-encoded 6.1-kDa polypeptide. It is shown that the 6.1-kDa protein is an additional, previously undetected subunit of the PSII reaction center of higher plants. The molecular mass of the polypeptide was calculated on the basis of the number of amino acids of the homologous protein deduced from the Arabidopsis thaliana gene ((37) ). Although the purified spinach polypeptide has a molecular mass of 4.6 kDa as estimated from SDS-urea polyacrylamide gels, we prefer to designate it 6.1-kDa polypeptide throughout the manuscript.()


MATERIALS AND METHODS

Isolation of PSII Preparations

Thylakoid membranes and PSII membrane fragments were isolated from spinach chloroplasts(21) . PSII membrane fragments contained 220 Chl/reaction center with a Chl a/b (w/w) ratio of 2.0. Oxygen evolving PSII core complexes were either purified after -N-octyl glucoside solubilization using the procedure described by Ghanotakis and Yocum (22) or -dodecylmaltoside as reported by Haag et al.(23) . PSII reaction center complexes have been purified from PSII membrane fragments using three different types of protocols: 1) following the method originally described by Nanba et al.(8) , 2) a modified procedure reported by Seibert et al.(10) , and 3) finally following a technique recently developed by Bóza et al. (24). Light-harvesting complexes of PSII were purified following the method described by Burke et al. (25) . Inside-out and right-side-out thylakoid membranes were isolated using the aqueous polymer two-phase system (Dextran T-500/polyethylene glycol, Carbowax 3350) as described previously(26, 27) .

To remove excess detergent, the PSII membrane fragments were washed with medium: A, 10 mM MES-NaOH, pH 6.5, 5 mM MgCl, 15 mM NaCl, 2 mM sucrose. To release extrinsic polypeptides or partly integrated proteins from the thylakoids the following media were used: 1 M NaCl, 10 mM MES-NaOH, pH 6.5; 1 M CaCl, 10 mM MES-NaOH, pH 6.5; 0.8 M Tris, pH 8.4. The samples were centrifuged and finally resuspended in storage medium (medium A containing 400 mM sucrose). The Chl contents as well as the Chl a/b ratios were determined using the method described by Porra et al.(28) .

Protein Analysis

Polypeptide analyses were either carried out by SDS-urea-PAGE using the buffer system described by Laemmli (29) using a resolving gel of 17.5% acrylamide containing 0.1% (w/v) SDS and 4 M urea or using the system developed by Schägger and von Jagow(30) . Polyacrylamide gels were stained with silver according to Oakley et al.(31) . The relative molecular mass of the polypeptide was determined by plotting the log of relative molecular masses as a function of the relative mobilities. Molecular markers were purchased from Pharmacia (low range marker, 16.9-2.5 kDa) or Amersham (Rainbow marker ). Protein concentrations were measured by Markwell et al.(32) .

N-terminal Sequence Analysis

N-terminal sequencing of the 6.1-kDa polypeptide was either directly performed from the PVDF membranes using essentially the method described by Matsudeira(33) . The sequence was obtained by Edman degradation and pulse liquid phase sequencing using an Applied Biosystems sequenator (ABS 477A).

Immunological Experiments

A polyclonal antiserum was raised in a rabbit against the N-terminal 15 meric oligopeptide (LVDERMSTEGTGLPF) derived from a partial sequence obtained for the mature polypeptide(13, 14) . The purity of the oligopeptide was tested by reversed phase high performance liquid chromatography using an isocratic gradient of acetonitrile and 0.1% trichloroacetic acid and ion spray mass spectrometry (calculated and measured M = 1754). The oligopeptide was coupled to keyhole limpet hemocyanin by chemical cross-linking using m-maleimidobenzoic acid N-hydroxysuccinimide ester. The immunization was carried out following standard procedures. IgG was purified from the antiserum using protein A-Sepharose chromatography according to standard techniques.

Immunoblotting was performed onto PVDF membranes (0.2 µm) according to Towbin et al.(34) using a semidry blotting system (Millipore). Immunodecorations were visualized either using the ECL (enhanced chemoluminescence) technique (Amersham) or the alkaline phosphatase system (Bio-Rad).

Purified IgG was used in dilutions from 1/500 up to 1/10. ELISA multititer plates were first coated with a variety of different PSII samples (usually 0.5-1 µg Chl for PSII membrane fragments, salt-washed PSII membrane fragments, and O-evolving PSII core complexes). After that the plates were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS). The cavities were blocked with 5% (w/v) skimmed milk in PBS for 1 h at 37 °C and washed again as described above. The first antibodies were then added and incubated at 4 °C overnight. The cavities have been washed as described, horseradish peroxidase coupled to goat-anti-rabbit IgG was added in 5% (w/v) skimmed milk in PBS (dilution 1/20,000), and incubated for 1 h at room temperature. The immunodecorations were detected in situ using the ECL method.

Chromatographic Procedures

Approximately 1 mg ml purified IgG has been coupled onto CNBr-activated Sepharose using standard techniques. When using PSII membrane fragments (2 mg/ml Chl) as starting material, they were solubilized in 1% (w/v) -dodecylmaltoside and 1% (v/v) Triton X-100 for 30 min. Unsolubilized material was sedimented by low speed centrifugation and an aliquot of the supernatant (approximately 1-2 mg protein) loaded onto the immunoaffinity column that was equilibrated in a buffer containing 20 mM MES-NaOH, pH 6.5, 4 mM sucrose, 10 mM NaCl, 10 mM CaCl, 0.1% (w/v) -dodecylmaltoside. Subsequently PSII polypeptides have been isocratically eluted by 0.25 M NaCl, 0.5 M NaCl, 0.5 M NaCl, and 0.5% Triton X-100 in the same buffer. The bound 6.1-kDa polypeptide was finally detached from the affinity column by 0.2 M glycine-HCl, pH 2.5, 0.1% (w/v) -dodecylmaltoside. After elution the fractions were immediately titrated to pH 7.5 with an aliquot of 2 M Tris, pH 9.8.

Additionally, O-evolving PSII core complexes were used as starting material. In this case the PSII core complexes were applied directly on to the column without any presolubilization. The 6.1-kDa protein was separated from the other subunits and detached from the column following the same procedure as described above for PSII membrane fragments. The protein was concentrated by vacuum centrifugation, usually followed by precipitation in precooled acetone at -20 °C to remove surplus detergent and salt.

Absorption Spectroscopy

The absorption spectrum of the purified polypeptide was recorded at room temperature using a Shimadzu UV 3000 spectrophotometer from 200 to 800 nm (slit width, 1 nm). Cytb was determined by difference spectroscopy from the reduced (hydroquinone-reduced for the high potential and NaSO-reduced for total cytb) minus K[Fe(CN)]-oxidized form using an extinction coefficient of 17,500 M cm(35) . The absorption spectra of the PSII reaction center complexes (solubilized in 25 mM MES-NaOH, pH 6.5, 10 mM CaCl, 0.025% (w/v) -dodecylmaltoside) were recorded at room temperature with a Chl concentration of 5 µM using a Beckman DU 64 spectrophotometer. The optical path length was 1 cm and the scan speed 500 nm/min.

Cross-linking Experiments

PSII membrane fragments were cross-linked at constant Chl concentrations of 250 µg/ml in assay medium (50 mM MES-NaOH, pH 6.5, 15 mM NaCl, 5 mM MgCl, 400 mM sucrose) on ice for 30 min in the dark using EDC/Chl ratios (w/w) from 1:1 up to 20:1. The samples were centrifuged (12,000 g, 10 min, 4 °C), the supernatants saved for control, and the pellets resuspended in 1 ml of assay medium. The washing procedure was repeated twice under the same conditions, and the pellets were finally resuspended in 250 µl of assay medium. O-evolving PSII core complexes were cross-linked at a constant Chl concentration of 25 µg/ml using the same EDC/Chl ratios as described above. Cross-linking was performed on ice for 30 min in the dark. The polypeptides were precipitated in precooled acetone at -20 °C for 30 min (total volume 1.25 ml). The polypeptides were sedimented for 20 min (12,000 g), the supernatants discarded, and the pellets prepared for electrophoretic analysis.

Quantification of the 6.1-kDa Polypeptide

The 6.1-kDa protein was immunodecorated with known amounts of peptide specific IgG and the immuneresponse was quantified using a densitometer (Molecular Dynamics Densitometer and Image Quant software). The amount of 6.1-kDa protein was calculated at the saturation level of the immuno decoration. The protein was then referred to the number of chlorophyll molecules/PSII reaction center. The following mean values were measured and used for the various PSII preparations; PSII membrane fragments 220 Chl and PSII core complexes 50 Chl. For comparison the same procedure was applied for cytb. In the case of cytb, the calculations based on immunotitration experiments were confirmed by difference spectroscopic determination


RESULTS

Identification and Purification of the 6.1-kDa Polypeptide

A N-terminal site-specific IgG was used to produce an immunoaffinity column. To purify the protein, PSII core complexes were loaded onto the affinity column. The PSII core complexes were found to bind tightly to the column, indicating that the epitope was surface-exposed in these preparations. The low molecular mass polypeptide was purified by first stepwise isocratically detaching other PSII polypeptides from the column by increasing the NaCl concentration from 10 to 500 mM and adding Triton X-100 to a final concentration of 0.5% (v/v). Finally, the low molecular mass protein was eluted in glycine-containing buffer at low pH. The purification method was highly efficient since almost no protein was lost while detaching other PSII polypeptides from the affinity column. The low molecular mass component was exclusively detected in the glycine-eluted fraction (Fig. 1, lanes 1 and 2). Fractions obtained after each step were routinely probed by SDS-urea-PAGE followed by immunoblotting.


Figure 1: Denaturing, silver stained SDS-PAGE of the purified low molecular mass protein (lanes 1 and 2), and PSII membrane fragments (lane 3). Lane 1 shows an immunoblot using the site-specific antiserum raised against the 6.1-kDa protein.



Notably, the low molecular mass polypeptide did not stain with Coomassie R-250 and only very weakly with silver stain after prolonged development (Fig. 1, lane 2). In addition, the isolated protein tends to bind high concentrations of lipids and/or detergent that leads to a distorted band after electrophoresis. A focused band, however, was obtained after acetone precipitation of the polypeptide and resolubilizing it in SDS-containing buffer (Fig. 1, lane 2).

At room temperature the absorption spectrum of the purified low molecular mass protein had a maximum at = 276 ± 1 nm with a shoulder at 280 ± 1 nm (not shown) indicating that no chromophores were associated with the isolated form of the protein. Using immunotitration, the number of 6.1-kDa proteins/PSII reaction center was estimated to be 1-2.

N-terminal amino acid sequencing of the isolated protein was performed after SDS-PAGE followed by electroblotting onto PVDF membranes. Nineteen amino acids could be determined (Table 1) supporting and extending preliminary data(13, 14) , but also confirming that the site-directed antibody indeed identified the 6.1-kDa protein. A comparison of the obtained N-terminal sequence with those from Triticum aestivum(14) , Chlamydomonas reinhardtii(36) , and that deduced from randomly obtained cDNA from A. thaliana(37) revealed that the first 10 amino acids were almost identical. With respect to C. reinhardtii(36) only three deviations were found in: position 7 (S/N), 8 (T/G) and 9 (E/D). The comparison with A. thaliana revealed only one deviation in position 18 (M/S). Interestingly, in position 6 of the purified 6.1-kDa protein a double, equally sized, signal was obtained, suggesting 50% of methionine and glutamine in this position. The significance and reason for this are not clear at the moment.



Localization of the 6.1-kDa Polypeptide

In an attempt to localize the low molecular mass protein of 6.1 kDa within PSII, and to gain information on its nearest neighbors, different thylakoid membrane preparations and PSII complexes were investigated by means of immunoscreening. Under denaturing conditions, i.e. after SDS-PAGE followed by Western blotting, the 6.1-kDa polypeptide was unambiguously identified in all types of PSII complexes, but not in LHCII preparations. Of particular interest is that the 6.1-kDa protein was found in stoicheiometric amounts in PSII reaction center complexes (see Fig. 2A, lanes 1-7 and Table 2). The purity of the used PSII preparations is shown by a silver-stained SDS-polyacrylamide gel in Fig. 2B and additionally for the PSII reaction center complex by its absorption spectrum (Fig. 2C). The presence of the 6.1-kDa protein in the PSII reaction center complex was also further established by analyzing three different types of preparations (see ``Materials and Methods''). All three gave a strong positive immunoreaction with the 6.1-kDa site-directed IgG (only one of these is shown in Fig. 2). The 6.1-kDa protein could neither be removed from the PSII membrane fragments by Tris washing at high pH nor by high salt concentrations indicating that the 6.1-kDa protein is an integral membrane protein component.


Figure 2: A, immunoblots of purified 6.1-kDa protein (1), PSII reaction center (2), O-evolving PSII core complex (3), LHCII (4), PSII membrane fragments (5), Tris-washed PSII membrane fragments (6), and thylakoids, using the antiserum raised against the 6.1-kDa protein. B, silver-stained SDS-PAGE of PSII membrane fragments (7), PSII reaction center (8). The upper arrow indicates aggregated PSII reaction centers at the interface between the spacer gel and the separation gel. C, absorption spectrum of the PSII reaction center complex used in A and B.





The Western blot analysis clearly shows that the 6.1-kDa protein is present in all PSII samples; however, to obtain information on the topology of the protein it is necessary to preserve it from denaturation. Therefore, we used a more appropriated ELISA technique (Table 2). Again the strongest immunodecoration was observed in PSII reaction center and in the PSII core complexes, meaning that the N-terminal tail of the 6.1-kDa protein was highly surface-exposed in these two preparations (see Table 2). Interestingly, the PSII membrane fragments revealed a rather weak antibody reaction in ELISA. After washing the PSII membrane fragments with NaCl to remove the two extrinsic 23- and 16-kDa proteins, the immunoresponse was also very weak. However, in PSII membrane fragments completely deprived of all three extrinsic proteins (33, 23, and 16 kDa) either by Tris or CaCl washing, the epitope was clearly accessible and a strong and distinct immunoresponse was detected. Obviously a removal of the extrinsic 23- and 16-kDa proteins did not expose the N terminus of the 6.1-kDa protein, while an elimination of the extrinsic 33 kDa (psbO) polypeptide did. This indicates that the N terminus of the 6.1-kDa protein would be located somewhere in the vicinity of the 33-kDa protein. However, the O-evolving PSII core complexes contained the psbO gene product(23) , and the N terminus of the 6.1-kDa protein was still recognized by the peptide-directed IgG. This could be due to an induced conformational change in the 33-kDa protein binding region or that the 33-kDa protein is partly lost during the preparation or the ELISA procedure.

Intact normal thylakoids and right-side-out thylakoids reacted only very weakly with the 6.1-kDa directed antibodies, while inside-out thylakoids showed a very strong reaction (roughly 10 times stronger compared to thylakoids). The much stronger IgG immune response with the inside-out thylakoids suggests that the N terminus of the 6.1-kDa protein is on the lumen side of the membrane. This finding leads us to further investigate the gene recently obtained from A. thaliana(37) , corresponding to the isolated 6.1-kDa protein. An analysis of the presequences of the precursor of the 6.1-kDa protein deduced from A. thaliana turned out to have a predicted length of 79 amino acids. This should be compared to the mature protein that is predicted to consist of only 54 amino acids. The presequence reveals some typical features common with other transit sequences found for lumen polypeptides such as the extrinsic 10-, 16-, 23-, and 33-kDa subunits of photosystem II. Some of the typical features are: it starts with the dipeptide composed of methionine- alanine and has a central part enriched in the hydroxylated amino acids serine and threonine as well as in positively charged amino acids arginine and lysine(38, 39) . It ends with the consensus sequence tripeptide alanine-X-alanine, where X represents a variable amino acid residue.

The mature 6.1-kDa protein deduced from A. thaliana gene (37) contains a hydrophobic region in the middle, which is predicted to be a transmembrane -helix. Interestingly, just at the outer surface of the thylakoid membrane, close to the hydrophobic region, four negatively charged amino acids are located (Fig. 4). A sequence homology search against the Swissprot and Prosit database did not reveal any significant homology to other proteins or functional motifs, except for the corresponding 6.1-kDa protein in C. reinhardtii, T. aestivum, and spinach. The calculated molecular mass of the deduced mature protein from A. thaliana gene (37) is 6.043 kDa. The relative molecular mass of the purified polypeptide was estimated to be 4.6 kDa on the applied SDS-PAGE system. The reason for the discrepancy in relative molecular masses between the purified polypeptide and that previously described is not clear at the moment, however, different gel systems have been used(13, 14) .


Figure 4: Schematic drawing of the 6.1-kDa protein with respect to its special features concerning localization, orientation, and nearest neighbors.



Nearest Neighbor Analysis by Chemical Cross-linking with EDC

To further establish that the 6.1-kDa protein is in close contact with polypeptides of the PSII reaction center, chemical cross-linking experiments were performed to investigate the nearest neighbors of the small subunit in PSII. Using a zero cross-linker, EDC, that contains no spacer between the two reactive groups and mainly conjugates proteins via -, -amino groups, and in addition via carboxyl groups, it was possible to identify the proteins next to the 6.1-kDa subunit. PSII membrane fragments were cross-linked with various EDC/Chl (w/w) or EDC/protein (w/w) ratios. The conjugates were analyzed by SDS-urea-PAGE and screened with peptide-specific IgG directed against the 6.1 protein after electroblotting and visualized by ECL. The result of such an experiment using PSII membrane fragments is demonstrated in Fig. 3(lanes 1-6). It was found that with increasing ratios of EDC/Chl the intensity of two bands in the 36-38 kDa and 14-16 kDa region enhanced in parallel. The main part of the 6.1-kDa protein, however, remained not cross-linked, showing that the cross-linking reaction was not quantitative. Using a set of different polyclonal antisera, we tested various PSII polypeptides for being cross-linked by EDC with the 6.1-kDa protein. The D1 and D2 proteins were identified in the cross-linked complex with a relative molecular mass of 36-38 kDa and Cytb was found in the smaller cross-linked complexes of 14-16 kDa (see Fig. 3, lanes 7-9). Using PSII core complexes instead of PSII membrane fragments in the cross-linking experiment, the same cross-linked products were detected with the 6.1-kDa protein IgG (not shown), which further supports the conclusion deduced from the experiments using PSII membrane fragments.


Figure 3: Immunoblots of PSII membrane fragments cross-linked with EDC/Chl ratio (w/w). Control (lane 1), 1:1, lane 2; 2:1, lane 3; 4:1, lane 4; 10:1, lane 5; and 20:1, lanes 6-9, using the antiserum against the 6.1-kDa protein (lanes 1-6), D1 (lane 7), D2 (lane 8), and cytb (lane 9).



It is interesting that chemical cross-linking of PSII membrane fragments with EDC did neither influence the redox properties of the heme group of Cytb nor did it shift its maximum ( = 560 ± 1 nm, data not shown). As the heme group of cytb559 is considered to be located toward the outer surface of the thylakoid membrane(40) , this suggests that the EDC-mediated cross-linking between the 6.1-kDa protein and Cytb takes place preferentially on the lumen side via the N- and C-terminal regions of the two polypeptides, respectively.


DISCUSSION

In this work it is shown that PSII reaction center complexes contain one additional, previously not detected low molecular mass subunit. This finding was affirmed by analyzing several types of PSII reaction center complex preparations and using two different immunoassays (immunoblotting and ELISA). Using both methods unambiguous immunodecorations of the 6.1-kDa polypeptide were obtained in stoicheiometric amounts for the PSII reaction center complex preparations thus far analyzed. The purity of the used PSII reaction center complexes was assured by silver-stained SDS-urea polyacrylamide gels, their absorption spectra (Fig. 2), and by determining the number of chlorophyll a molecules/P680 (4-6 chlorophyll/reaction center, not shown). Furthermore cross-linking experiments in combination with specific immunodetections using anti-D1, anti-D2 and anti-psbE gene product showed a close association of the 6.1-kDa protein with the heterodimeric D1/D2 proteins of the PSII reaction center complex and the subunits of the cytb. The possibility that the 6.1-kDa protein is loosely or accidentally associated with PSII during preparation can be excluded by the fact that the whole PSII complex binds to the affinity column specifically interacting with the N terminus of the 6.1-kDa protein. Only after extensively washing the column (with 0.5 M NaCl and 0.5% Triton X-100) could the other reaction center complex proteins be detached from the 6.1-kDa protein. So far the PSII reaction center complex has been thought to be composed of the D1-, D2-proteins, the - and -subunits of cytb, and the psbI gene product, 4-6 chlorophyll a molecules, two pheophytins and one to two -carotenes(9, 10, 14, 41) . However, due to its unusual amino acid composition the polypeptide stains poorly with Coomassie R-250 and only very weakly with silver. This probably provides an explanation that the 6.1-kDa protein has previously escaped detection. On the basis of the finding presented here, it is suggested that the 6.1-kDa protein is an additional integral component of the PSII reaction center complex.

The 6.1-kDa protein is likely to be an integral membrane protein, as various salt wash treatments, including Tris, were not able to remove the 6.1-kDa protein from PSII membrane fragments. The high hydrophobicity of the protein was further established by its low polarity index (32%) determined from an amino acid analysis of the isolated protein. The corresponding protein deduced from the A. thaliana gene (37) was predicted to be a membrane spanning protein with one -helix. The N terminus of the 6.1-kDa protein was found to be oriented into the lumen of the thylakoids (Fig. 4) since it is not recognized by the N-terminal site-directed IgG in right-side-out thylakoids, but clearly in inside-out thylakoids. No immunodecorations were observed with PSII membrane fragments. The reason for this discrepancy is not yet clarified (see Table 2). One possibility for the detectability of the N terminus in inside-out thylakoids could be that either the 33-kDa protein was partly lost during the preparation of the inverted vesicles or while washing the samples in the cavities of the microtiter plates and therefore the epitope was exposed to the surface. This would be in line with our observation that extrinsic polypeptides can easily be removed from inside-out thylakoids by salt concentrations of 100-200 mM NaCl (data not shown). The orientation of the 6.1-kDa protein is supported by an analysis of its presequence that was found to have high homology with those of other known lumen-directed polypeptides of PSII, like the three extrinsic subunits of 33, 23, and 16 kDa. Interestingly, this means that the orientation of the 6.1-kDa protein is opposite to that of the other proteins found in the PSII reaction center complex. The N termini of both the D1 and the D2 polypeptides are on the stroma side of the membrane(43, 44) . Also the psbI gene product, another monotopic membrane protein, has its N terminus on the outside of the thylakoid membrane(45) . The topology of cytb has recently been determined by means of a peptide-directed antiserum specifically reacting with the C terminus of the -subunit (42) and using hybrid -subunits of cytb in combination with site-specific antisera(40) . Both the N termini are located on the stroma side of the thylakoid membrane(40, 42) .

At the inner thylakoid surface the N-terminal tail of the 6.1-kDa subunit appears to be shielded by the extrinsic 33-kDa protein which in turn is in close association with PSII reaction center complex. This would suggest that the N terminus of the 6.1-kDa protein is in close vicinity to the extrinsic 33-kDa protein. However, in isolated O-evolving PSII core complexes the epitope is recognized by the IgG despite the 33-kDa protein being present. This is probably a reflection of the fact that the N terminus is more susceptible to the environment in isolated PSII core complexes than in PSII membrane fragments.

At the moment no specific function can be ascribed to the 6.1-kDa protein. From its absorption spectrum with a maximum in the UV, no prosthetic group could be inferred to be associated with the isolated protein, but it cannot be totally excluded that it could have been lost during the isolation. However, a data base sequence homology search for functional motifs did not give any suggestions or indications for the function of the protein. In summary, these findings seem to exclude the possibility that the 6.1-kDa protein is directly involved in redox processes of PSII. This polypeptide could provide ligands for binding manganese as the site of the water oxidase is considered to be located near to the D1 and D2 proteins and to CP47.

Recently Tang et al.(46) described the purification of a photochemically active reaction center complex deprived of the psbI gene product and the heterooligomeric cytb. The implication of this finding is that at least these small subunits cannot be involved in pigment binding (46) .

Considering the interesting result that the 6.1-kDa protein is the only nuclear encoded subunit in the PSII reaction center complex, one tends to speculate that it could play a regulatory role in the assembly of PSII. In this case the four negatively charged residues of the protein, located on the outer surface of the thylakoid membrane, may have a crucial function. Other interesting functional roles for the 6.1-kDa protein could be to provide docking domains for other proteins, like the extrinsic subunits regulating the water oxidizing enzyme, or CAB proteins of the inner antennae. Alternatively, the 6.1-kDa protein could be involved in the high turnover of the D1 protein within the PSII reaction center under high light. Further experiments are in progress to elucidate the function of this newly discovered component of the PSII reaction center complex.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a DAAD research exchange grant within the framework of a Swedish/German exchange program. Permanent address: Max-Volmer-Institute, TU Berlin, Strasse des 17, Juni 135, D-106 23 Berlin, Germany.

Supported by the Swedish Forestry and Agricultural Research Council and the Swedish Institute. To whom correspondence should be addressed. Tel.: +46-8-164392; Fax: +46-8-153679.

The abbreviations used are: PSII, photosystem II; Chl, chlorophyll; cytb, cytochrome b 559; EDC, 1-(3-dimethylaminopropyl-)3-ethylcarbodiimide); MES, 2-N-morpholinoethanesulfonic acid; PBS, phosphate-buffered saline; psb, photosystem b or II; PVDF, polyvinylidene difluoride; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

The calculated molecular mass of the mature protein from spinach deduced from its gene is 5.6 kDa. The polypeptide will be designated psbW gene product (Lorkovic, Z. J., Schröder, W. P., Pakrasi, H. B., Irrgang, K.-D., Herrmann, R. G., and Oelmüller, R. (1995) Proc. Natl. Acad. Sci. U. S. A., in press.


ACKNOWLEDGEMENTS

We thank Dr. H. Salter for his help in data base searching, Prof. W. Cramer and Dr. A. Szczepaniak for providing a peptide-directed antiserum elicited against the psbE gene product, and Prof. B. Andersson and Prof. G. Renger for their interest and support. We also thank Dr. P.-I. Ohlsson (University of Umeå) for performing the sequence and amino acid analyses.


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