Title: A Nuclear-encoded Subunit of the Photosystem II Reaction Center
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/cytb559 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 cytb559. 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. 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/cytb559 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 cytb559. 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. 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(1Debus R. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1085) Google Scholar, 2Andersson B. Franzen L.-G. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Publishers, Amsterdam1992: 121-143Google Scholar, 3Hansson . Wydrzinski T. Photosynth. Res. 1990; 23: 131-162Crossref PubMed Scopus (234) Google Scholar, 4Renger G. Photosynth. Res. 1993; 38: 229-247Crossref PubMed Scopus (84) Google Scholar). 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 PSII1 1The abbreviations used are: PSIIphotosystem IIChlchlorophyllcytb559cytochrome b 559EDC1-(3-dimethylaminopropyl-)3-ethylcarbodiimide)MES2-N-morpholinoethanesulfonic acidPBSphosphate-buffered salinepsbphotosystem b or IIPVDFpolyvinylidene difluoridePAGEpolyacrylamide gel electrophoresisELISAenzyme-linked immunosorbent assay. 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. photosystem II chlorophyll cytochrome b 559 1-(3-dimethylaminopropyl-)3-ethylcarbodiimide) 2-N-morpholinoethanesulfonic acid phosphate-buffered saline photosystem b or II polyvinylidene difluoride polyacrylamide gel electrophoresis enzyme-linked immunosorbent assay. 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(5Rochaix J.-D. Dron M. Rahire M. Malnoe P. Plant Mol. Biol. 1984; 3: 363-370Crossref PubMed Scopus (113) Google Scholar, 6Michel H. Deisenhofer J. Staehelin L.A. Arntzen C.J. Encyclopedia of Plant Physiol.Vol. 19. New Series, Springer-Verlag, Berlin1986: 371-381Google Scholar, 7Michel H. Deisenhofer J. Biochemistry. 1988; 27: 1-7Crossref Scopus (649) Google Scholar). Indeed, such a PSII reaction center complex consisting of the D1, D2 proteins, cytochrome b559 has been isolated from higher plants (8Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar, 9Barber J. Chapman D.J. Telfer A. FEBS Lett. 1987; 220: 67-73Crossref Scopus (221) Google Scholar, 10Seibert M. Picorel R. Rubin A. Connolly J.S. Plant Physiol. 1988; 87: 303-306Crossref PubMed Scopus (120) Google Scholar). 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(11Andersson B. Styring S. Lee C.P. Current Topics in Bioenergetics. Vol. 16. Academic press, San Diego1991: 1-81Crossref Google Scholar). 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 (12Ljungberg U. Henrysson T. Rochester C.P.H.-E. Andersson B. Biochim. Biophys. Acta. 1986; 849: 112-120Crossref Scopus (38) Google Scholar, 13Schröder W.P. Henrysson T. and H.-E. FEBS Lett. 1988; 235: 289-292Crossref Scopus (20) Google Scholar, 14Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar) using polyacrylamide gels of high resolution. Most of them have one hydrophobic stretch predicted to be a transmembrane α-helix. They have been found to be encoded in the chloroplast genome and referred to as psbH-psbN gene products (for a summary see (2Andersson B. Franzen L.-G. Ernster L. Molecular Mechanisms in Bioenergetics. Elsevier Publishers, Amsterdam1992: 121-143Google Scholar)). One, the psbI gene product, has been found to be present in the D1/D2 heterodimer of higher plants(15Ikeuchi M. Inoue Y. FEBS Lett. 1988; 242: 99-104Crossref Scopus (149) Google Scholar, 16Webber A.N. Packman L. Chapman D.J. Barber J. Gray J.C. FEBS Lett. 1989; 242: 259-262Crossref Scopus (71) Google Scholar). 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 (17Ikeuchi M. Eggers B. Shen G. Webber A. Yu J. Hirano A. Inoue Y. Vermaas W.F.J. J. Biol. Chem. 1991; 266: 11111-11115Abstract Full Text PDF PubMed Google Scholar, 18Mayes S. Dubbs J.M. Vass I. Hideg E. Nagy L. Barber J. Biochemistry. 1993; 32: 1454-1465Crossref PubMed Scopus (82) Google Scholar, 19Lind L.K. Shukla V.K. Nyhus K.J. Pakrasi H.B. J. Biol. Chem. 1993; 268: 1575-1579Abstract Full Text PDF PubMed Google Scholar). The psbL gene product (with an approximately molecular mass of 5 kDa) has been suggested to be involved in stabilizing the QA binding niche(20Nagatsuka T. Fukuhara S. Akabori K. Toyoshima Y. Biochim. Biophys. Acta. 1991; 1057: 223-231Crossref Scopus (29) Google Scholar). 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(12Ljungberg U. Henrysson T. Rochester C.P.H.-E. Andersson B. Biochim. Biophys. Acta. 1986; 849: 112-120Crossref Scopus (38) Google Scholar, 13Schröder W.P. Henrysson T. and H.-E. FEBS Lett. 1988; 235: 289-292Crossref Scopus (20) Google Scholar, 14Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar). 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 ((37Höfte H. Desprez T. Amselem J. Chiapello H. Coboche M. Moisan A. Jourjon M.-F. Charpenteau L. Berthomieu P. Guerrier D. Giraudat J. Quigley F. Thomas F. Yu D.-Y. Mache R. Raynal M. Cooke R. Grellet F. Delseny M. Parmentier Y. Marcillac G. Gigot C. Fleck J. Philipps G. Axelos M. Bardet C. Tremousaygue D. Lescure B. Plant J. 1993; 4: 1051-1061Crossref PubMed Scopus (218) Google Scholar)). 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.2 2The 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. Thylakoid membranes and PSII membrane fragments were isolated from spinach chloroplasts(21Berthold A.D. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1651) Google Scholar). 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 β-N-octyl glucoside solubilization using the procedure described by Ghanotakis and Yocum (22Ghanotakis D. Yocum C.F. FEBS Lett. 1986; 197: 244-248Crossref Scopus (81) Google Scholar) or β-dodecylmaltoside as reported by Haag et al.(23Haag E. Irrgang K.-D. Boekema E.J. Renger G. Eur. J. Biochem. 1990; 189: 47-53Crossref PubMed Scopus (99) Google Scholar). 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.(8Nanba O. Satoh K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 109-112Crossref PubMed Google Scholar), 2) a modified procedure reported by Seibert et al.(10Seibert M. Picorel R. Rubin A. Connolly J.S. Plant Physiol. 1988; 87: 303-306Crossref PubMed Scopus (120) Google Scholar), 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. (25Burke J.J. Ditto C.L. Arntzen C.J. Arch. Biochem. Biophys. 1978; 187: 252-263Crossref PubMed Scopus (278) Google Scholar). 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(26Andersson H.-E.B. Albertsson P.-Å. Biochim. Biophys. Acta. 1976; 449: 525-535Crossref PubMed Scopus (95) Google Scholar, 27Andersson B.H.-E. Albertsson P.-Å. Biochim. Biophys. Acta. 1976; 423: 122-132Crossref PubMed Scopus (82) Google Scholar). To remove excess detergent, the PSII membrane fragments were washed with medium: A, 10 mM MES-NaOH, pH 6.5, 5 mM MgCl2, 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 CaCl2, 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.(28Porra R.J. Thompson W.A. Kriedemann P.E. Biochim. Biophys. Acta. 1989; 975: 384-394Crossref Scopus (4693) Google Scholar). Polypeptide analyses were either carried out by SDS-urea-PAGE using the buffer system described by Laemmli (29Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207192) Google Scholar) 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(30Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10478) Google Scholar). Polyacrylamide gels were stained with silver according to Oakley et al.(31Oakley B.R. Kirsch D.R. Morris N.R. Anal. Biochem. 1980; 105: 361-363Crossref PubMed Scopus (2448) Google Scholar). 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.(32Markwell M.A. Haas S.M. Bieber L.L. Tolbert N.E. Anal. Biochem. 1978; 87: 206-210Crossref PubMed Scopus (5331) Google Scholar). N-terminal sequencing of the 6.1-kDa polypeptide was either directly performed from the PVDF membranes using essentially the method described by Matsudeira(33Matsudeira P. J. Biol. Chem. 1987; 262: 10035-10038Abstract Full Text PDF PubMed Google Scholar). The sequence was obtained by Edman degradation and pulse liquid phase sequencing using an Applied Biosystems sequenator (ABS 477A). 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(13Schröder W.P. Henrysson T. and H.-E. FEBS Lett. 1988; 235: 289-292Crossref Scopus (20) Google Scholar, 14Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar). 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 Mr = 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 εm) according to Towbin et al.(34Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44918) Google Scholar) 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 1/106. ELISA multititer plates were first coated with a variety of different PSII samples (usually 0.5-1 εg Chl for PSII membrane fragments, salt-washed PSII membrane fragments, and O2-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. Approximately 1 mg ml-1 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) β-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 CaCl2, 0.1% (w/v) β-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) β-dodecylmaltoside. After elution the fractions were immediately titrated to pH 7.5 with an aliquot of 2 M Tris, pH 9.8. Additionally, O2-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. 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). Cytb559 was determined by difference spectroscopy from the reduced (hydroquinone-reduced for the high potential and Na2S2O4-reduced for total cytb559) minus K3[Fe(CN)6]-oxidized form using an extinction coefficient of 17,500 M-1 cm-1(35Cramer W.A. Whitmarsh J. Ann. Rev. Plant Physiol. 1977; 28: 133-172Crossref Google Scholar). The absorption spectra of the PSII reaction center complexes (solubilized in 25 mM MES-NaOH, pH 6.5, 10 mM CaCl2, 0.025% (w/v) β-dodecylmaltoside) were recorded at room temperature with a Chl concentration of 5 εM using a Beckman DU 64 spectrophotometer. The optical path length was 1 cm and the scan speed 500 nm/min. PSII membrane fragments were cross-linked at constant Chl concentrations of 250 εg/ml in assay medium (50 mM MES-NaOH, pH 6.5, 15 mM NaCl, 5 mM MgCl2, 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 εl of assay medium. O2-evolving PSII core complexes were cross-linked at a constant Chl concentration of 25 εg/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. 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 cytb559. In the case of cytb559, the calculations based on immunotitration experiments were confirmed by difference spectroscopic determination 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. 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 λ = 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(13Schröder W.P. Henrysson T. and H.-E. FEBS Lett. 1988; 235: 289-292Crossref Scopus (20) Google Scholar, 14Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar), 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(14Ikeuchi M. Koike H. Inoue Y. FEBS Lett. 1989; 242: 263-269Crossref PubMed Scopus (86) Google Scholar), Chlamydomonas reinhardtii(36de Vitry C. Diner B. Popot J.-L. J. Biol. Chem. 1991; 266: 16614-16621Abstract Full Text PDF PubMed Google Scholar), and that deduced from randomly obtained cDNA from A. thaliana(37Höfte H. Desprez T. Amselem J. Chiapello H. Coboche M. Moisan A. Jourjon M.-F. Charpenteau L. Berthomieu P. Guerrier D. Giraudat J. Quigley F. Thomas F. Yu D.-Y. Mache R. Raynal M. Cooke R. Grellet F. Delseny M. Parmentier Y. Marcillac G. Gigot C. Fleck J. Philipps G. Axelos M. Bardet C. Tremousaygue D. Lescure B. Plant J. 1993; 4: 1051-1061Crossref PubMed Scopus (218) Google Scholar) revealed that the first 10 amino acids were almost identical. With respect to C. reinhardtii(36de Vitry C. Diner B. Popot J.-L. J. Biol. Chem. 1991; 266: 16614-16621Abstract Full Text PDF PubMed Google Scholar) 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.Tabled 1 Open table in a new tab 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.Tabled 1 Open table in a new tab 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 CaCl2 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 O2-evolving PSII core complexes contained the psbO gene product(23Haag E. Irrgang K.-D. Boekema E.J. Renger G. Eur. J. Biochem. 1990; 189: 47-53Crossref PubMed Scopus (99) Google Scholar), 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(37Höfte H. Desprez T. Amselem J. Chiapello H. Coboche M. Moisan A. Jourjon M.-F. Charpenteau L. Berthomieu P. Guerrier D. Giraudat J. Quigley F. Thomas F. Yu D.-Y. Mache R. Raynal M. Cooke R. Grellet F. Delseny M. Parmentier Y. Marcillac G. Gigot C. Fleck J. Philipps G. Axelos M. Bardet C. Tremousaygue D. Lescure B. Plant J. 1993; 4: 1051-1061Crossref PubMed Scopus (218) Google Scholar), 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(38von Hejne G. Steppuhn J. Herrmann R.G. Eur. J. Biochem. 1989; 180: 535-545Crossref PubMed Scopus (909) Google Scholar, 39de Boer D. Weisbeek P. Sundqvist C. Ryberg M. Pigment-Protein Complexes in Plastids: Synthesis and Assembly. Academic Press Inc., San Diego1993: 311-334Google Scholar). 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 (37Höfte H. Desprez T. Amselem J. Chiapello H. Coboche M. Moisan A. Jourjon M.-F. Charpenteau L. Berthomieu P. Guerrier D. Giraudat J. Quigley F. Thomas F. Yu D.-Y. Mache R. Raynal M. Cooke R. Grellet F. Delseny M. Parmentier Y. Marcillac G. Gigot C. Fleck J. Philipps G. Axelos M. Bardet C. Tremousaygue D. Lescure B. Pla