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First published online November 17, 2006; 10.1105/tpc.106.042671 The Plant Cell 18:3121-3131 (2006) © 2006 American Society of Plant Biologists Psb27, a Cyanobacterial Lipoprotein, Is Involved in the Repair Cycle of Photosystem II
a Plant Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany 1 To whom correspondence should be addressed. E-mail matthias.roegner{at}rub.de; fax 49-234-3214322.
Photosystem II (PSII) performs one of the key reactions on our planet: the light-driven oxidation of water. This fundamental but very complex process requires PSII to act in a highly coordinated fashion. Despite detailed structural information on the fully assembled PSII complex, the dynamic aspects of formation, processing, turnover, and degradation of PSII with at least 19 subunits and various cofactors are still not fully understood. Transient complexes are especially difficult to characterize due to low abundance, potential heterogeneity, and instability. Here, we show that Psb27 is involved in the assembly of the water-splitting site of PSII and in the turnover of the complex. Psb27 is a bacterial lipoprotein with a specific lipid modification as shown by matrix-assisted laser-desorption ionization time of flight mass spectrometry. The combination of HPLC purification of four different PSII subcomplexes and 15N pulse label experiments revealed that lipoprotein Psb27 is part of a preassembled PSII subcomplex that represents a distinct intermediate in the repair cycle of PSII.
The photosynthetic electron transfer chain of cyanobacteria, eukaryotic algae, and vascular plants is located in a specialized membrane system, the thylakoids, and mediated by the integral membrane protein complexes photosystem II (PSII), cytochrome b6f complex, and photosystem I (PSI). Electron transfer is initiated in the PSII complex by light-induced charge separation at the central chlorophyll redox center P680, and electrons are transferred to the quinone B binding site via a short internal redox chain (Diner and Rappaport, 2002  ). P680+ is reduced by electrons provided by the water-splitting system at the luminal side of PSII, which contains four manganese ions and one calcium ion (Rutherford and Boussac, 2004).
Structural studies have provided a detailed static view of PSII (Zouni et al., 2001
Although structural information is provided in great detail, only little is known about the dynamic aspects of the PSII life cycle, including transient complexes and factors involved in the biogenesis, maintenance, repair, and degradation of the complex (Nickelsen et al., 2006). The first step of biogenesis includes the integration of the transmembrane helices and central redox centers into the lipid phase followed by the formation of an initial PSII precomplex built by D1, D2, cytochrome b559, and PsbI (Komenda et al., 2004
Photosynthetic water splitting is inevitable coupled with the formation of reactive oxygen species followed by photooxidative damage of protein subunits even under low light conditions (Anderson et al., 1997
Another intriguing and still unresolved question is the spatial organization of the different parts of the PSII life cycle. The idea of specialized regions for the biogenesis of photosystems was strongly supported by the detection of preassembled PSI and PSII complexes in the cytoplasmic membrane of cyanobacteria (Zak et al., 2001
Here, we report about four different transient complexes that represent particular steps in the life cycle of PSII. Among them, the PSII/Psb27 complex is of special interest. It was shown by the combination of intact mass tag analysis, introduced by Gomez et al. (2003)
Combination of Ni-Chelating and Ion Exchange Chromatography Enables the Isolation of Four Different PSII Subcomplexes For the efficient isolation of reaction centers from Thermosynechococcus elongatus, 10 His residues were fused to the C terminus of the PSII subunit CP43. His-tagged PSII complexes were purified via Ni chelate affinity chromatography (Figure 1 ) followed by continuous bed ion exchange chromatography (IEC), which resulted in the isolation of four different PSII subfractions (Figure 2A ). Applying HPLC size exclusion chromatography and native PAGE (Figure 2B), we could identify two characteristic monomeric complexes, termed PSIIM(low) and PSIIM(high) (fractions 1 and 2), and two distinct dimeric complexes, termed PSIID(high) and PSIID(low) (fractions 3 and 4). Activity measurements on the purified subcomplexes revealed as highest activity 4790 µmol O2 (mg Chl)–1 h–1 for PSIID(high) (fraction 3), >50% lower activity of 2070 µmol O2 [mg Chl]–1 h–1 for PSIID(low) (fraction 4), a moderate activity of 2920 µmol O2 [mg Chl]–1 h–1 for PSIIM(high) (fraction 2), and a marginal activity of 210 µmol O2 [mg Chl]–1 h–1 for PSIIM(low) (fraction 1).
Transient Absorption Spectroscopy Reveals Light-Induced Charge Separation in the four PSII Subcomplexes P680+QA–- P680QA (QA for primary electron accepting plastoquinone of PSII) absorption difference spectra of the inactive PSIIM(low) subcomplex and the active PSII subcomplexes did not show significant differences, indicating the integrity of the reaction centers (Figure 3 ). However, an increased number of chlorophylls per reduced QA could be determined for PSIIM(low), which reflects a larger antenna size in comparison with the active PSII subfractions (Table 1 ) and may be either due to an impaired assembly in vivo or a loss of QA.
SDS-PAGE Analysis Shows the Presence of Psb27 in PSIIM(low) Figure 2C shows the SDS-PAGE gels of the four different PSII subpopulations. The polypeptide patterns of active PSIIM(high) and highly active PSIID(high) and PSIID(low) are in good agreement with previous results obtained for a PSII preparation from T. elongatus (Kuhl et al., 2000 ). Distinct bands of the major PSII subunits CP47, CP43 (with a mass shift due to the His-tag), D1, D2, and the extrinsic proteins PsbO, PsbV, and PsbU were consistently observed for all three subcomplexes after gel staining with Coomassie blue. In addition, uniform band patterns were observed in the low molecular mass region for PSIIM(high), PSIID(high), and PSIID(low). By contrast, SDS-PAGE analysis of inactive PSIIM(low) revealed the absence of the three extrinsic subunits PsbO, PsbV, and PsbU. Instead, a novel component of  11 kD was instead observed in the low molecular mass range, which could be identified as Psb27 (Kashino et al., 2002) via mass spectrometric sequence analysis. Interestingly, no manganese could be detected in the PSIIM(low) subcomplex (Table 1), although the C terminus of its D1 subunit seems to be already processed (Figure 4
).
Psb27 Is Shown to Be a Bacterial Lipoprotein Hydropathy plots and sequence analysis with TMHMM (Krogh et al., 2001 ) predict that Psb27 is a soluble protein that lacks membrane-spanning helices. To confirm this prediction, PSIIM(low) complexes were exposed to a variety of different treatments, as summarized in Table 2
. Among them, washing with 1 M CaCl2 (Ono and Inoue, 1983) or 1 M Tris, pH 8.8 (Yamamoto and Ke, 1981), represents well-established procedures to separate the extrinsic, lumen-exposed proteins PsbO, PsbV, and PsbU from PSII. Since all selected washing procedures failed to release Psb27 from PSIIM(low) (Table 2), we propose a strong hydrophobic interaction between Psb27 and the PSII core center. In contrast with a CaCl2-washed PSII complex, binding of the extrinsic protein PsbO, PsbU, or PsbV to the PSIIM(low) complex was not observed in reconstitution assays (Figure 5
). This suggests that the lumen-exposed PSII domain, which is required for binding of extrinsic PSII proteins, is effectively blocked by Psb27. A lumen-exposed localization of Psb27 is also consistent with its bacterial signal sequence as predicted by the SignalP algorithm (Bendtsen et al., 2004). Such a motif is commonly known to direct subunits across the membrane into the luminal space. A detailed sequence analysis of Psb27 from various cyanobacteria (Figure 6
) also showed a highly conserved Cys residue in the N-terminal leader sequence. As predicted by the online servers DOLOP (Madan Babu and Sankaran, 2002) and LIPOP (Juncker et al., 2003), this Cys residue is combined with a sequence motif called lipobox, which is unique for bacterial lipoproteins (Figure 6). Proteomics analysis of the thylakoid lumen of Arabidopsis thaliana revealed the presence of a Psb27 homologue (Peltier et al., 2002); however, in contrast with cyanobacteria, eukaryotes are lacking both the lipobox sequence motif and the maturation system consisting of a diacylglycerol transferase, a lipoprotein signal peptidase, and an apolipoprotein N-acyltransferase, which are required for the specific lipid modification.
To confirm the presence of lipid-modified Psb27 in the PSIIM(low) complex as purified in this work, the isolated intact subcomplex was subjected to enzymatic cleavage with unspecific lipase. Intact mass tag analysis (Whitelegge et al., 1997 , 1998; Gomez et al., 2002) was used to monitor the cleavage of the lipid modification. Spectra of Psb27 present in PSIIM(low) were obtained in situ after various incubation times with Lipolase by quadrupole-time-of-flight mass spectrometry (qTOF-MS) following matrix-assisted laser-desorption ionization (MALDI).
Figure 7
shows sections of MALDI-qTOF-MS spectra from the PSIIM(low) subcomplex in the m/z range of 12 to 14 kD before and after treatment with Lipolase. Intact mass tags of photosynthetic proteins were acquired with high mass accuracy (e.g.,
15N Pulse Label Experiments Indicate Involvement of the PSII/Psb27 Complex in the Repair Process of PSII Time-dependent distribution of the PSII subcomplexes was analyzed by pulse label experiments using 15N-enriched media (98% purity) combined with MALDI-TOF/TOF-MS analyses (Figure 8 ). During the mid-log growth phase, the nitrogen source was shifted from 14N to 15N, and PSIIM(low) and PSIID(high) subcomplexes were isolated at various times after the 15N pulse. Isolated PSIIM(low) and PSIID(high) were separated by SDS-PAGE and 15N incorporation into the D1 subunit of the respective complex was analyzed by MALDI-TOF analysis of the corresponding tryptic peptide mixtures. For PSIIM(low), an 25% 15N incorporation could be observed 3 h after the 15N pulse, while for PSIID(high), no incorporation could be detected (Figure 8, PSIIM(low)/3 h and PSIID(high)/3 h). Accordingly, the amount of incorporated 15N atoms was distinctly higher (75%) in PSIIM(low) than in the active PSII dimer (45%) 10 h after the pulse (Figure 8, PSIIM(low)/10 h and PSIID(high)/10 h).
To further analyze the role of the PSIIM(low) subcomplex in the de novo biogenesis or repair of PSII, the incorporation of 15N was also monitored for different PSII subunits 24 h after the 15N pulse (Figure 9 ). It is obvious that the D1 subunit exhibits the highest amount of newly synthesized protein ( 96%), whereas all other analyzed subunits clearly show a higher proportion of old (15N-free) protein. In conclusion, the PSIIM(low) complex seems mainly determined by the repair cycle of D1, as in case of a de novo biogenesis of the whole complex, a more or less uniform 15N incorporation into all involved subunits would be expected. On the other hand, our data do not exclude the de novo biogenesis for a minor subfraction of PSIIM(low). Our results also show that, interestingly, the 15N incorporation rate differs between the other analyzed subunits. Approximately 75% of newly synthesized protein for CP43 and 65% for the D2 subunit indicate a higher turnover of these subunits compared to CP47 (45%) and PsbE (35%). With its minimal synthesis rate (t1/2 = 37 h), the latter may be regarded as a kind of internal standard, as it corresponds roughly to the doubling time of the cells (35h) under the experimental conditions.
In conclusion, these results provide strong evidence that Psb27 plays a role in the biogenesis of the water-splitting site that is most critical for the function of PSII and especially for the repair of damaged PSII complexes. The PSIIM(low)-Psb27 complex represents an intermediate state in which the integral membrane part is fully assembled and capable of primary charge separation and electron transfer. These results are in agreement with data from Roose and Pakrasi (2004) showing that a  ctpA- strain of Synechocystis is not able to assemble functional PSII and accumulates instead a preassembled PSIIM(low)-Psb27 complex, suggesting a role for Psb27 in the C-terminal processing of D1. In combination with the growing evidence that a partial assembly of the PSII core complex, with a processed D1 subunit (however, without water splitting), occurs in the cyanobacterial plasma membrane (Zak et al., 2001; Klinkert et al., 2004; Keren et al., 2005), the PSIIM(low)-Psb27 complex could also represent a transit complex for the transfer from the cytoplasmic to the thylakoid membrane. Physiologically, such an arrested intermediate could be of vital importance, as a premature start of water splitting could easily lead to the production of dangerous reactive oxygen species or maybe an easy target for destruction. However, up to now, Psb27 was not shown to be associated with the plasma membrane of cyanobacteria, and, interestingly, Gloeobacter violaceus, a primitive cyanobacterium that lacks a separate thylakoid membrane system, exhibits no Psb27 homologue. On the other hand, there is evidence that the PSIIM(low)-Psb27 complex of our study should be located in the thylakoid membrane: It was isolated via a His-Tag attached to subunit CP43, which was reported to be absent in the cytoplasmic membrane (Zak et al., 2001). While we cannot exclude a general role of the PSIIM(low)-Psb27 complex in the biogenesis of PSII, our data strongly suggest that the PSIIM(low)-Psb27 complex is an intermediate in the PSII repair cycle (as reviewed in Aro et al., 2005), which continuously replaces the D1 subunit (and maybe others; Figure 10
). Such a model is consistent with data showing impairment of PSII recovery after photoinhibition in a Psb27 knockout line of Arabidopsis (Chen et al., 2006). The association of Psb27 with dynamic processes of PSII and the fact that many more lipoproteins with still unknown function are predicted from genomic data might reveal a more general role of lipoproteins in photosynthesis and elsewhere. This would be in line with examples for lipidated proteins (Mattoo and Edelman, 1987; Gomez et al., 2002) and with the general importance of lipid–protein interactions (Fyfe et al., 2005) in photosynthesis.
Especially for membrane proteins, this report shows the potential of HPLC purification in combination with state-of-the-art MS for the characterization of transient complexes. Such a combined approach may contribute to better understanding of dynamic processes of membrane protein complexes with transient intermediates of limited stability and low abundance.
Preparation of His-Tagged PSII and Analytical Size Exclusion Chromatography His-tagged PSII complexes (10x His fused to the C terminus of the CP43 subunit) were isolated from cells that had been grown under normal light conditions ( 30 µE) and that had been harvested in the exponential growth phase. Cells were otherwise processed as previously reported by Kuhl et al. (2000). For the 15N pulse label experiments, NaNO3 was substituted by 5 mM 15NH4Cl in the media. Thylakoid membranes were solubilized in 20 mM MES, pH 6.5, 10 mM CaCl2, 10 mM MgCl2, 1.2% n-dodecyl-ß,D-maltoside (ß-DM; Biomol), and 0.5% Na-cholate (Dojindo) at a chlorophyll concentration of 1 mg/mL. After centrifugation at 45,000g for 90 min at 4°C, the supernatant was loaded onto a chelating sepharose fast flow column (Pharmacia) that was equilibrated with buffer (20 mM MES, pH 6.5, 10 mM CaCl2, 10 mM MgCl2, 300 mM NaCl, 500 mM mannitol, 0.03% ß-DM, and 1 mM histidine). The column was then washed with 4 volumes of equilibration buffer at a flow rate of 2 mL min–1. PSII complexes were eluted by a linear gradient of 1 to 100 mM histidine. To separate different PSII subfractions by IEC, the resulting protein solution was dialyzed against buffer (20 mM MES, pH 6.5, 20 mM CaCl2, 20 mM MgCl2, 0.5 M mannitol, and 0.03% ß-DM) overnight and loaded onto a UNO Q6 column (Bio-Rad) as previously reported by Kuhl et al. (2000). The oligomerization status of PSII was monitored by analytical size exclusion chromatography using a TSK-gel 4000 SWXL column (TosoHaas) in a Waters HPLC system (for details, see Kuhl et al., 2000).
PSII Activity Measurements, SDS-PAGE, and Native PAGE
PSII Reconstitution and Dissection Experiments
Mass Spectrometry
For the MALDI peptide mass fingerprint analysis of the D1 subunit, protein bands were excised from the polyacrylamide gel and destained with two to three changes of 50% (v/v) acetonitrile buffered with 25 mM NH4HCO3. After the gel slices had been completely dried in a vacuum concentrator, they were rehydrated in trypsin solution (12.5 ng/µL trypsin and 25 mM NH4HCO3). Enzymatic hydrolyzation was performed overnight at 37°C. Peptide fragments were eluted from the gel matrix by application of 1 volume of elution solution (50% [v/v] acetonitrile and 0.5% [v/v] TFA) and sonication in a water bath for 20 min. The supernatant was spotted on an AnchorChip (Bruker Daltonics) according to the manufacturer's instructions using
Transient Absorption Spectroscopy
Determination of the Mn Content
Accession Numbers
We thank the German Research Council (Sonderforschungsbereich 480, project C1 to M.R. and Sonderforschungsbereich 498, project A6 to E.S.) for financial support and the Protein Center of the Ruhr-University Bochum for infrastructural support. We also thank K.-E. Jaeger and T. Eggert for advising us on the lipase assay, F. Lendzian for the electron paramagnetic resonance measurements, A. Trebst, S. Oeljeklaus, and J. Nickelsen for stimulating discussions, and M. Çetin, C. König, and R. Oworah-Nkruma for excellent technical assistance.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Matthias Rögner (matthias.roegner{at}rub.de). www.plantcell.org/cgi/doi/10.1105/tpc.106.042671 Received March 21, 2006; Revision received September 17, 2006. accepted October 27, 2006.
Anbudurai, P.R., Mor, T.S., Ohad, I., Shestakov, S.V., and Pakrasi, H.B. (1994). The ctpA gene encodes the C-terminal processing protease for the D1 protein of the photosystem II reaction center complex. Proc. Natl. Acad. Sci. USA 91, 8082–8086. Anderson, J.M., Park, Y.I., and Chow, W.S. (1997). Photoinactivation and photoprotection of photosystem II in nature. Physiol. Plant 100, 214–223. Aro, E.M., Suorsa, M., Rokka, A., Allahverdiyeva, Y., Paakkarinen, V., Saleem, A., Battchikova, N., and Rintamaki, E. (2005). Dynamics of photosystem II: A proteomic approach to thylakoid protein complexes. J. Exp. Bot. 56, 347–356. Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340, 783–795.[CrossRef][ISI][Medline] Chen, H., Zhang, D., Guo, J., Wu, H., Jin, M., Lu, Q., Lu, C., and Zhang, L. (2006). A Psb27 homologue in Arabidopsis thaliana is required for efficient repair of photodamaged photosystem II. Plant Mol. Biol. 61, 567–575.[CrossRef][ISI][Medline] Diner, B.A., and Rappaport, F. (2002). Structure, dynamics, and energetics of the primary photochemistry of photosystem II of oxygenic photosynthesis. Annu. Rev. Plant Biol. 53, 551–580.[CrossRef][Medline] Diner, B.A., Schlodder, E., Nixon, P.J., Coleman, W.J., Rappaport, F., Lavergne, J., Vermaas, W.F., and Chisholm, D.A. (2001). Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: Sites of primary charge separation and cation and triplet stabilization. Biochemistry 40, 9265–9281.[CrossRef][Medline] Ferreira, K.N., Iverson, T.M., Maghlaoui, K., Barber, J., and Iwata, S. (2004). Architecture of the photosynthetic oxygen-evolving center. Science 303, 1831–1838. Fyfe, P.K., Hughes, A.V., Heathcote, P., and Jones, M.R. (2005). Proteins, chlorophylls and lipids: X-ray analysis of a three-way relationship. Trends Plant Sci. 10, 275–282.[CrossRef][ISI][Medline] Gomez, S.M., Bil, K.Y., Aguilera, R., Nishio, J.N., Faull, K.F., and Whitelegge, J.P. (2003). Transit peptide cleavage sites of integral thylakoid membrane proteins. Mol. Cell. Proteomics 2, 1068–1085. Gomez, S.M., Nishio, J.N., Faull, K.F., and Whitelegge, J.P. (2002). The chloroplast grana proteome defined by intact mass measurements from liquid chromatography mass spectrometry. Mol. Cell. Proteomics 1, 46–59. Gustavsson, N., Greber, B., Kreitler, T., Himmelbauer, H., Lehrach, H., and Gobom, J. (2005). A proteomic method for the analysis of changes in protein concentrations in response to systemic perturbations using metabolic incorporation of stable isotopes and mass spectrometry. Proteomics 5, 3563–3570.[CrossRef][ISI][Medline] Hillmann, B., Brettel, K., van Mieghem, F., Kamlowski, A., Rutherford, A.W., and Schlodder, E. (1995). Charge recombination reactions in photosystem II. 2. Transient absorbance difference spectra and their temperature dependence. Biochemistry 34, 4814–4827.[CrossRef][Medline] Juncker, A.S., Willenbrock, H., Von Heijne, G., Brunak, S., Nielsen, H., and Krogh, A. (2003). Prediction of lipoprotein signal peptides in Gram-negative bacteria. Protein Sci. 12, 1652–1662. Kamata, T., Hiramoto, H., Morita, N., Shen, J.R., Mann, N.H., and Yamamoto, Y. (2005). Quality control of photosystem II: An FtsH protease plays an essential role in the turnover of the reaction center D1 protein in Synechocystis PCC 6803 under heat stress as well as light stress conditions. Photochem. Photobiol. Sci. 4, 983–990.[CrossRef][ISI][Medline] Kamiya, N., and Shen, J.R. (2003). Crystal structure of oxygen-evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A resolution. Proc. Natl. Acad. Sci. USA 100, 98–103. Kashino, Y., Lauber, W.M., Carroll, J.A., Wang, Q., Whitmarsh, J., Satoh, K., and Pakrasi, H.B. (2002). Proteomic analysis of a highly active photosystem II preparation from the cyanobacterium Synechocystis sp. PCC 6803 reveals the presence of novel polypeptides. Biochemistry 41, 8004–8012.[CrossRef][Medline] Keren, N., Liberton, M., and Pakrasi, H.B. (2005). Photochemical competence of assembled photosystem II core complex in cyanobacterial plasma membrane. J. Biol. Chem. 280, 6548–6553. Klinkert, B., Ossenbuhl, F., Sikorski, M., Berry, S., Eichacker, L., and Nickelsen, J. (2004). PratA, a periplasmic tetratricopeptide repeat protein involved in biogenesis of photosystem II in Synechocystis sp. PCC 6803. J. Biol. Chem. 279, 44639–44644. Komenda, J., Barker, M., Kuvikova, S., de Vries, R., Mullineaux, C.W., Tichy, M., and Nixon, P.J. (2006). The FtsH protease slr0228 is important for quality control of photosystem II in the thylakoid membrane of Synechocystis sp. PCC 6803. J. Biol. Chem. 281, 1145–1151. Komenda, J., Reisinger, V., Muller, B.C., Dobakova, M., Granvogl, B., and Eichacker, L.A. (2004). Accumulation of the D2 protein is a key regulatory step for assembly of the photosystem II reaction center complex in Synechocystis PCC 6803. J. Biol. Chem. 279, 48620–48629. Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, E.L. (2001). Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 305, 567–580.[CrossRef][ISI][Medline] Kuhl, H., Kruip, J., Seidler, A., Krieger-Liszkay, A., Bunker, M., Bald, D., Scheidig, A.J., and Rogner, M. (2000). Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. J. Biol. Chem. 275, 20652–20659. Loll, B., Kern, J., Saenger, W., Zouni, A., and Biesiadka, J. (2005). Towards complete cofactor arrangement in the 3.0 Å resolution structure of photosystem II. Nature 438, 1040–1044.[CrossRef][Medline] Madan Babu, M., and Sankaran, K. (2002). DOLOP—Database of bacterial lipoproteins. Bioinformatics 18, 641–643. Mattoo, A.K., and Edelman, M. (1987). Intramembrane translocation and posttranslational palmitoylation of the chloroplast 32-kDa herbicide-binding protein. Proc. Natl. Acad. Sci. USA 84, 1497–1501. Michel, K.P., Thole, H.H., and Pistorius, E.K. (1996). IdiA, a 34 kDa protein in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942, is required for growth under iron and manganese limitations. Microbiology 142, 2635–2645.[Abstract] Nixon, P.J., Barker, M., Boehm, M., de Vries, R., and Komenda, J. (2005). FtsH-mediated repair of the photosystem II complex in response to light stress. J. Exp. Bot. 56, 357–363. Ono, T., and Inoue, Y. (1983). Mn-preserving extraction of 33-, 24- and 16-kDa proteins from O2-evolving PS II particles by divalent salt-washing. FEBS Lett. 164, 255–260.[CrossRef] Peltier, J.B., Emanuelsson, O., Kalume, D.E., Ytterberg, J., Friso, G., Rudella, A., Liberles, D.A., Soderberg, L., Roepstorff, P., von Heijne, G., and van Wijk, K.J. (2002). Central functions of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14, 211–236. Peter, G.F., and Thornber, J.P. (1991). Biochemical composition and organization of higher plant photosystem II light-harvesting pigment-proteins. J. Biol. Chem. 266, 16745–16754. Plucken, H., Muller, B., Grohmann, D., Westhoff, P., and Eichacker, L.A. (2002). The HCF136 protein is essential for assembly of the photosystem II reaction center in Arabidopsis thaliana. FEBS Lett. 532, 85–90.[CrossRef][ISI][Medline] Roose, J.L., and Pakrasi, H.B. (2004). Evidence that D1 processing is required for manganese binding and extrinsic protein assembly into photosystem II. J. Biol. Chem. 279, 45417–45422. Rutherford, A.W., and Boussac, A. (2004). Biochemistry. Water photolysis in biology. Science 303, 1782–1784. Schagger, H., and von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.[CrossRef][ISI][Medline] Seidler, A. (1996). The extrinsic polypeptides of photosystem II. Biochim. Biophys. Acta 1277, 35–60.[Medline] Shi, L.X., and Schroder, W.P. (2004). The low molecular mass subunits of the photosynthetic supracomplex, photosystem II. Biochim. Biophys. Acta 1608, 75–96.[Medline] Summerfield, T.C., Shand, J.A., Bentley, F.K., and Eaton-Rye, J.J. (2005b). PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions. Biochemistry 44, 805–815.[CrossRef][Medline] Summerfield, T.C., Winter, R.T., and Eaton-Rye, J.J. (2005a). Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803. Photosynth. Res. 84, 263–268.[CrossRef][ISI][Medline] Thornton, L.E., Ohkawa, H., Roose, J.L., Kashino, Y., Keren, N., and Pakrasi, H.B. (2004). Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem ii activity in the cyanobacterium Synechocystis 6803. Plant Cell 16, 2164–2175. Whitelegge, J.P., Gomez, S.M., Stevens, R.L., Mastrogiacomo, A., Gundersen, C., and Faull, K.F. (1997). Electrospray-ionization mass spectrometry of lipidated intrinsic membrane proteins. FASEB J. 11, A1088. Whitelegge, J.P., Gundersen, C.B., and Faull, K.F. (1998). Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci. 7, 1423–1430.[Abstract] Yamamoto, Y., and Ke, B. (1981). Membrane-surface electric properties of triton-fractionated spinach subchloroplast fragments. Biochim. Biophys. Acta 636, 175–184.[Medline] Zak, E., Norling, B., Maitra, R., Huang, F., Andersson, B., and Pakrasi, H.B. (2001). The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes. Proc. Natl. Acad. Sci. USA 98, 13443–13448. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001). Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution. Nature 409, 739–743.[CrossRef][Medline] This article has been cited by other articles:
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-cyano-4-hydroxycynamic acid as the MALDI matrix. MALDI-TOF-MS analysis of tryptic peptides was performed using the UltraFlex2.0 mass spectrometer according to the manufacturer's instructions. The UltraFlex2.0 was equipped with a Scout MTP MALDI target. The spectra were acquired in a mass range from m/z 400 to m/z 3500 in the positive mode with a target voltage of 25 kV and a pulsed ion extraction of 21.85 kV. The laser frequency was set to 50 Hz, and the spectra shown were a sum of 200 to 400 laser shots. The reflector voltage was set to 26.4 kV and detector voltage to 1.7 kV. For external calibration of the instrument, a peptide standard with m/z of 757.399, 1296.684, 1619.822, and 2093.086 D was used. The relative amount of newly synthesized protein after the 15N pulse was calculated according to Gustavsson et al. (2005)
