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Originally published In Press as doi:10.1074/jbc.M605463200 on November 14, 2006

J. Biol. Chem., Vol. 282, Issue 1, 267-276, January 5, 2007
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Localization of the Small CAB-like Proteins in Photosystem II*

Danny Yao{ddagger}§, Thomas Kieselbach{ddagger}, Josef Komenda||**, Kamoltip Promnares||**, Miguel A. Hernández Prieto{ddagger}§, Martin Tichy||**, Wim Vermaas, and Christiane Funk{ddagger}§1

From the {ddagger}Department of Biochemistry and the §Umeå Plant Science Centre, Umeå University, SE-901 87 Umeå, Sweden, the School of Life Sciences and the Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-4501, the ||Institute of Physical Biology, University of South Bohemia, 373 33 Nové Hrady, Czech Republic, and the **Laboratory of Photosynthesis, Institute of Microbiology, Academy of Sciences, 379 81 Trebon, Czech Republic

Received for publication, June 7, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In organisms performing oxygenic photosynthesis, sunlight is absorbed by chlorophylls and other pigments, and absorbed excitation energy is transferred to the reaction centers, where the photochemical process of converting excitation energy to chemical (redox) energy takes place. These pigments are bound to proteins to keep them in their proper location and orientation so that the energy transfer is efficient and rapid and so that toxic triplet states can be quenched effectively. In plants, the vast majority of pigments, including chlorophylls a and b and various carotenoids, are bound to a family of integral membrane proteins called the light-harvesting complex (LHC).2 Most abundant is LHCII, the main light-harvesting complex of Photosystem (PS) II, which has been crystallized and is known to consist of three transmembrane helices (B, C, and A), each of which is composed of 20–34 amino acids (1, 2). The sequences of helices A and B are very similar and comprise the CAB (chlorophyll a/b-binding) motif, which is composed of ~25 amino acid residues and includes the domain involved in chlorophyll binding (3). Each individual LHCII apoprotein molecule binds an array of about eight chlorophylls a, six chlorophylls b, three to four carotenoids, and two lipids (4). Several other closely related chlorophyll a/b-binding polypeptides function as light-harvesting antenna for PSII and PSI in plants. Together, these proteins are known as CAB proteins (3). The CAB proteins in plants display a high degree of sequence similarity and are believed to share a common evolutionary origin (5, 6). Their corresponding nuclear encoded genes belong to an extended cab family that includes also the genes coding for early light-inducible proteins, which are stress-induced (see Ref. 7) and probably bind chlorophyll a and lutein (8). The CAB family also includes the PsbS protein (reviewed in Ref. 9), which has an important function in non-photochemical quenching (10). PsbS is predicted to have four thylakoid membrane-spanning regions, and it binds chlorophylls a and b as well as carotenoids (11). Moreover, related genes coding for polypeptides with one or two transmembrane {alpha}-helices have been detected in the genomes of Arabidopsis thaliana (12, 13), rice and poplar (14), Chlamydomonas reinhardtii (15), and the red alga Cyanidioschyzon merolae (16).

In contrast to plants, cyanobacteria lack the multihelix CAB proteins. The major peripheral LHC in some cyanobacteria is the phycobilisome, which is in the cytoplasm, is bound to the thylakoid membrane, and contributes to the deep blue-green color of cyanobacteria. However, small CAB-like proteins of <8 kDa have recently been identified in the genomes of marine and freshwater cyanobacteria (reviewed in Ref. 17). These proteins are predicted to have a single membrane-spanning {alpha}-helix, which shows significant sequence similarity to the first and third membrane-spanning regions of the green plant CAB proteins, giving them the name small CAB-like proteins (SCPs) (18). They were also designated high light-inducible proteins because their RNA level was found to increase after transfer of cells to high light and many other stress conditions (19, 20). In the small genome of the cyanobacterium Prochlorococcus marinus MED4, as many as 23 scp or hli genes were identified (17), and these genes have recently been detected in the genomes of Prochlorococcus cyanophages (21, 22), where they are believed to maintain the photosynthetic activity of the host during an infection (21). Although the function of the SCPs is not fully understood, these findings indicate their importance.

In the cyanobacterium Synechocystis sp. PCC 6803, five SCPs were identified (18); four of them (ScpB–E) encode proteins of ~6 kDa, whereas the fifth (ScpA) is the C-terminal extension of the ferrochelatase. The genes coding for ScpB–E are induced under various different stress conditions, including very high light intensity (>500 µmol m–2 s–1), low temperature, and nitrogen and sulfur starvation (19, 20). A mutant with these four genes inactivated is sensitive to high intensity illumination and shows alteration in pigmentation and in the ability to perform non-photochemical dissipation of absorbed light energy (23). The enhanced expression of the scp genes in response to high intensity illumination is consistent with the putative function of SCPs in protection against light stress (19). It was suggested that SCPs play a role in energy dissipation that is analogous to the process of non-photochemical quenching of higher plants (23), but the absence of scp genes does not affect fluorescence characteristics (24). On the other hand, IsiA and/or a carotenoid closely associated with phycobilin energy transfer is now recognized to be involved with energy transfer regulation (25–27). It also has been hypothesized that SCPs prevent the formation of reactive oxygen species by serving as transient carriers of chlorophyll (24, 28).

The presence of the CAB motif in SCPs suggests that SCPs bind chlorophyll molecules in a similar way as the LHCII of plants. Furthermore, the SCPs seem to participate in tetrapyrrole biosynthesis and regulate pigment availability. The chlorophyll synthesis rates in the PSI-less/chlL–/scpB–, PSI-less/chlL–/scpE–, and PSI-less/chlL–/scpC–/scpD– strains decrease when these three mutants are transferred from darkness to light (24, 28). Interestingly, ScpC and ScpD seem to be functionally complementary (24). These two genes are most similar (87.1% identity) (19), indicating a rather recent gene duplication (17) or a reasonably strict primary structure requirement.

ScpD was immunologically detected in thylakoid membranes of Synechocystis sp. PCC 6803 (29). To understand the function of this and other SCPs, it is important to know which complexes in the membrane they interact with. Here, we used His-tagged ScpD proteins to identify the main complexes with which ScpD is associated. After two-dimensional PAGE (blue native (BN) PAGE followed by SDS-PAGE), ScpD was found to be associated with monomeric PSII, its closest neighbor being CP47. CP43 and Psb28 were also found to interact with ScpD. Although ScpC could be identified in the PSII fraction and ScpB was found to co-fractionate with ScpD to some degree, ScpE was found in thylakoids, but did not seem to be associated with PSII.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Growth Conditions—Synechocystis sp. PCC 6803 strains (wild-type, the PSI-less strain ({Delta}psaAB) (30), the PSII-less strain ({Delta}psbDIC/{Delta}psbDII) (31), the PSI-less/PSII-less strain ({Delta}psaAB/{Delta}psbDIC/{Delta}psbDII) (32), the CP47-His-tagged HT-3 strain (33), and the ScpD-His strain (see below)) were cultivated at 30 °C in BG-11 medium (34). The PSI-less and PSII-less mutants were provided with 15 mM glucose. All strains except the PSI-less strain were grown at normal (50 µmol photons m–2 s–1) or high (500 µmol photons m–2 s–1) light intensity as indicated. Because of its light sensitivity, the PSI-less strain was cultured at 10 µmol photons m–2 s–1. To induce the SCPs, the wild-type and PSII-less strains were grown at high light intensity for 7 h. In the PSI-less/PSII-less and PSI-less strains, SCPs are induced also at light intensities of 50 and 10 µmol photons m–2 s–1, respectively (18).

Mutant Construction—To generate the ScpD-His strain, a plasmid construct was made to tag the ScpD protein in Synechocystis with an His6 epitope on its N terminus and to express the corresponding gene construct under the control of the psbAII promoter. To construct this plasmid, the scpD gene was amplified by PCR using a mixture of Taq and Pfu DNA polymerases and gene-specific primers (forward, 5'-TTATACATATGCATCATCATCATCATCATGGAACTAGCCGCGGATTTCGCCT-3'; and reverse, 5'-TCGGATCCTTAGAGAGGAGAGCAACCAACCC-3') with artificially generated restriction sites for NdeI and BamHI and containing six histidine codons (CAT) in the forward primer. After restriction, the PCR fragment was cloned into the NdeI and BamHI sites of the pPSBA plasmid; the resulting plasmid contains the scpD-His gene construct right behind the psbAII start codon (35) and retains the upstream and downstream regions of the Synechocystis psbAII gene. The ligation mixture was amplified by PCR using pPSBA primers amplifying the entire psbAII/scpD-His region, and DNA of the desired size was selected. Amplification by PCR was chosen because transformation of Escherichia coli with the ligation mixture yielded no colonies, presumably reflecting toxicity of the plasmid to E. coli. The PCR product containing the scpD-His gene was transformed into the Synechocystis psbAII-KS strain, in which the psbAII gene was replaced with a kanamycin resistance/sacB cartridge (35). The sacB gene codes for a levansucrase, leading to sucrose sensitivity of this strain (36). After transformation, Synechocystis cells were grown on BG-11 plates for 4 days. Transformants were then transferred to plates with 5% sucrose, and sucrose-resistant colonies were checked for kanamycin sensitivity. The resulting strain expressing both wild-type and His-tagged forms of the ScpD protein was subsequently transformed with chromosomal DNA from a {Delta}scpD strain carrying a spectinomycin resistance cassette insertion (37), and spectinomycin-resistant transformants were selected (28). Insertion of the scpD-His gene at the desired location was confirmed by DNA sequencing, and deletion of the wild-type scpD gene was confirmed by PCR.

Biochemical Preparations—Total membranes from the different Synechocystis strains were isolated as described (18). Radioactive labeling of cells using a mixture of L-[35S]methionine and L-[35S]cysteine (>1000 Ci/mmol, final activity of 400 µCi/ml; Tran35S-label, ICN Biomedicals) and isolation of membranes were performed as described (38). Isolated membranes were solubilized with n-dodecyl beta-maltoside (n-dodecyl beta-maltoside/chlorophyll ratios were 20 and 100 (w/w) in the PSI-containing and PSI-less strains, respectively), and extracted complexes were separated by BN gel electrophoresis (39).

Isolation of His-tagged Complexes—Cells from Synechocystis sp. PCC 6803 strains carrying a His tag were pelleted after 4 h of exposure to high light intensity (500 µmol photons m–2 s–1), resuspended in Buffer A (50 mM MES-NaOH (pH 6.0), 10 mM MgCl2, 5 mM CaCl2, and 25% glycerol), and broken. Thylakoids were prepared as described (33). The cell homogenate (at 1 mg/ml chlorophyll) was brought to 1.28% beta-dodecyl maltoside and incubated for 25 min at 4 °C. The sample was then loaded onto an Ni2+ metal affinity column. The column was washed with 9 column volumes (45 ml) of Buffer A containing 0.04% beta-dodecyl maltoside and 10 mM imidazole. Subsequently, the column was washed with 10 ml of Buffer A with 0.04% beta-dodecyl maltoside and 30 mM imidazole. Bound ScpD-His was eluted with 0.04% beta-dodecyl maltoside and 100 mM imidazole in Buffer A. The eluate was precipitated by the addition of an equal volume of 25% polyethylene glycol 8000 in 50 mM MES-NaOH (pH 6.0) and then resuspended in Buffer A containing 0.04% beta-dodecyl maltoside.

PAGE—To the resuspended Ni2+ column eluate was added 0.1 volume of loading solution containing 750 mM aminocaproic acid and 5% Coomassie Brilliant Blue G-250. Protein complexes in the eluate were separated by BN-PAGE at 4 °C as described (39) using a 5–14% polyacrylamide gradient gel. For the second dimension, the BN gel lane of interest was incubated for 20 min in a solution containing 25 mM Tris-HCl (pH 7.5) and 1% SDS and then placed on top of an SDS-12–20% polyacrylamide gel containing 7 M urea (40). After electrophoresis, gels were either stained with silver nitrate (41) or transferred onto polyvinyl difluoride membrane for further analysis by Western blotting.

Immunoblotting—For immunoblotting, the proteins were transferred onto polyvinyl difluoride membrane (42). Anti-ScpC antibody raised in rabbits against residues 1–17 of the ScpC protein (MTTRGFRLDQDNRLNNF) was a gift from Dr. A. Sokolenko (University of Munich). A peptide-directed antibody against a region near the N terminus of ScpE (ELQPNQTPVQEDPKFG) was made commercially by Innovagen AB (Lund, Sweden).

Pigment Analysis—Chlorophyll content was determined in 80% acetone and was calculated as described (43).

Protein Analysis by Matrix-assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry—Protein identification by peptide mass fingerprinting and post-source decay tandem mass spectrometry (MS/MS) analysis was carried out using a Voyager-DE STR mass spectrometer (Applied Biosystems, Stockholm). In-gel digestion to produce peptides for analysis by mass spectrometry was carried out essentially as described (44) using sequencing-grade modified trypsin (Promega/SDS Biosciences, Falkenberg, Sweden) or sequencing-grade chymotrypsin (Roche Diagnostics, Bromma, Sweden). Silver-stained protein bands were destained prior to in-gel digestion using the method previously described (45). To analyze the in gel-digested proteins by MALDI-TOF mass spectrometry, dried droplet preparations were applied as described (46). The matrices used were readymade solutions of {alpha}-cyano-4-hydroxycinnamic acid (G2037A) and 2,5-dihydroxybenzoic acid (G2039A) from Agilent Technologies (Stockholm). Samples were concentrated and desalted as needed using homemade Stop-and-Go extraction columns as described (47). Data base searches were carried out on an in-house Mascot server that was licensed to Umeå University by Matrix Science (www.matrixscience.com) using the current version of the NCBInr Database and the Synechocystis Protein Database of the European Bioinformatics Institute. The data bases were searched using peptide mass fingerprint spectra and post-source decay MS/MS spectra. If appropriate, proteins were identified by sequence queries that included both types of data. The search parameters restricted the error for peptide masses to 50 ppm and for MS/MS fragments to 0.5 Da. The instrument type specified for MS/MS ion searches was MALDI-TOF/TOF. By default, the search parameters permitted one missed cleavage site and variable oxidation states of methionine. If appropriate, two or more missed cleavage sites were allowed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Proteins Co-purifying with ScpD-His—Based on two-phase separation experiments, ScpD is a prevalent SCP member in the thylakoid membrane (29), but its association with protein complexes in this membrane system remains unknown. To learn about the function of the SCPs, we decided to tag ScpD with His, to determine its interaction partners, and to analyze the SCP composition of thylakoid complexes. As described under "Experimental Procedures," we created a Synechocystis mutant in which scpD had been deleted and replaced with a His-tagged scpD copy, the expression of which was under the control of the psbAII promoter. After harvesting and rupturing the cells, the total membranes were solubilized using beta-dodecyl maltoside, and ScpD-His-containing complexes were isolated via nickel column chromatography (33). Subsequently, the proteins were separated by SDS-PAGE and analyzed by MALDI-TOF mass spectrometry.

To test the validity of this protocol, we also isolated PSII complexes via CP47-His using the HT-3 mutant (33) and analyzed them by SDS-PAGE (data not shown). They were composed of essentially the same subunits as shown in originally (33, 57).

The SDS gel in Fig. 1 shows the washed off fractions and eventual eluate resulting from the affinity purification of the ScpD-His complex (lanes C–E) and similar fractions of a chromatography control using wild-type ScpD (lanes A and B). Although the wild-type fraction collected upon washing the column (lane A) showed no recognizable pattern, the corresponding fraction from the ScpD-His strain showed a pattern of components resembling that of PSII (lane C). Indeed, upon elution with 100 mM imidazole, such components co-eluted with ScpD-His (lanes D and E; representing results from two independent preparations). The presence of two distinct bands with apparent masses of 47.3 and 6 kDa was clearly visible in these lanes, and fainter bands migrating with apparent masses between 4 and 6, 30 and 45, and 70 and 80 kDa could be observed. The only visible differences between the PSII complexes washed off the column at 30 mM imidazole (lane C) and at 100 mM imidazole (lane D) were the presence of ScpD-His and an enrichment of CP47 in the latter fraction. It therefore seems that most of the PSII complexes were washed off the column and that only a minor fraction was bound to ScpD. The corresponding eluent fraction from the wild-type control in lane B did not display protein bands, demonstrating the specificity of retention of the proteins in lanes D and E by ScpD-His.

To identify the proteins that co-purified with ScpD-His, the individual bands in Fig. 1 (lanes D and E) were digested with trypsin and analyzed by MALDI-TOF mass spectrometry. If the peptide mass fingerprint spectra of the individual bands were not sufficient for unequivocal protein identification, post-source decay MS/MS spectra of individual peptides were acquired for protein identification by sequence queries (48, 49). Table 1 summarizes the results of this analysis. As expected, the mass spectra showed the presence of ScpD (ssr2595) in the major band at an apparent mass of 6 kDa; ScpD was identified with high confidence by its peptide mass fingerprint spectrum in combination with an MS/MS analysis of the peptide GFRLDQDNR. The major band at an apparent mass of 47.3 kDa was found to contain CP47 (slr0906) and the hypothetical protein slr0909. Mass spectrometry analysis also identified CP43 (sll0851) with an apparent mass of 39.8 kDa, and D2 (sll0849/slr0927) and D1 (sll1867; two bands) with apparent masses of 36.7 kDa, 34.3 kDa, and 33.5 kDa, respectively. The band of the D2 protein also contained slr1128, annotated as a hypothetical integral membrane protein. In the high mass range, the FtsH proteases sll1463 and slr0228 were present at an apparent mass of ~77 kDa, and the FtsH protease slr1604 was found at an apparent mass of 71 kDa. Furthermore, sll1021 and slr0798 (both annotated as hypothetical proteins) were found at apparent masses of 88.1 and 95.1 kDa, respectively. In the low mass range, ScpB (ssl1633) and the small subunit of cytochrome b559 (smr0006) were identified at an apparent mass of 4.8 kDa.


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  		TABLE 1
Mass spectrometry identification of proteins apparently forming a complex with ScpD-His (data shown in Fig. 1)

 


Figure 1
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  		FIGURE 1.
Proteins co-purifying with ScpD-His. This Coomassie Blue-stained SDS-polyacrylamide gel displays total membrane fractions purified via nickel chromatography. Fractions from wild-type cells (control) are shown in lanes A and B, and fractions from the ScpD-His mutant strain are shown in lanes C–E. Lanes A and C, fractions obtained during the second washing step (0.04% beta-dodecyl maltoside and 30 mM imidazole in Buffer A); lanes B, D, and E, fractions obtained in the elution step (0.04% beta-dodecyl maltoside and 100 mM imidazole in Buffer A). Lanes D and E show the results of two separate experiments, and the protein bands shown in lane E were analyzed by MALDI-TOF mass spectrometry, resulting in identification as indicated to the right (also see Table 1). Cytb559, cytochrome b559.

 
ScpD-His Associates with PSII Subunits—The composition of the affinity-purified ScpD-His complex indicated that ScpD associated with PSII components. However, fractions that are isolated by a one-step affinity purification may contain contamination by nonspecifically bound proteins. For this reason, we subjected the purified ScpD-His complex to two-dimensional BN/SDS gel electrophoresis. This technique is an accepted approach to separate protein complexes and their subunits, and it has been successfully used to study protein complexes from the thylakoid membranes of higher plants (50, 51). Fig. 2 shows an example for the analysis of the ScpD-His complex at ~200 kDa by two-dimensional BN/SDS gel electrophoresis. The pattern of BN gel separation in the first dimension showed two main bands (Fig. 2). Upon SDS-PAGE in the second dimension and silver staining of the gel followed by MALDI-TOF mass spectrometry of bands, the corresponding proteins could be identified. The results are summarized in Table 2. In the high mass region, we found the PSII core subunits CP47, CP43, D1, and D2 at apparent masses of 52, 39, 33, and 32 kDa, respectively. In the low mass region, we detected PsbH, PsbZ, and ScpC/ScpD as a broader band spanning the 6–8 kDa range. It is interesting to note that only the band with faster migration in the first dimension provided clear evidence for small subunits (Fig. 2). The slower migrating band may represent PSII dimers at ~500 kDa (50). The relatively strong affinity of the putative PSII dimer on BN gel for the dye Coomassie Blue (as seen by the intense coloration of this band relative to the more rapidly migrating PSII fraction that represents PSII monomers) was unexpected. Equally unexpected was the depletion of ScpD and other low mass polypeptides in this fraction, as this fraction was isolated by retention on a nickel-nitrilotriacetic acid column, indicating the association with ScpD-His at the time of isolation. We hypothesize that ScpD (and possibly ScpC) is associated with monomeric PSII in a way that affects Coomassie Blue affinity (which would place the SCPs around the PSII monomer) and that the PSII monomers and dimers/multimers are in dynamic exchange. Another interesting feature of Fig. 2 is the heavy staining of low mass proteins in the more rapidly migrating band. Although quantitation of proteins based on silver staining is tenuous, the high staining intensity of these proteins suggests the association of multiple polypeptide copies with PSII.


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  		TABLE 2
Mass spectrometry identification of proteins that co-purified with ScpD-His upon separation by two-dimensional BN/SDS-PAGE after nickel chromatography and solubilization with 0.04% beta-dodecyl maltoside

 


Figure 2
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  		FIGURE 2.
ScpD-His is associated specifically with PSII. ScpD-His and copurified proteins were separated by two-dimensional BN/SDS-PAGE after nickel chromatography and solubilization with 0.04% beta-dodecyl maltoside. Proteins were identified by mass spectrometry (see Table 2).

 
The limited amount of the ScpD-His complex material on the BN gel shown in Fig. 2 made the analysis of the low mass proteins difficult. Although the identification of PsbH and PsbZ was unambiguous, MS/MS analysis identified the peptide GFRLDQDNR, which matches the sequence of ScpD and of its close homolog ScpC. For this reason, our mass spectrometry data do not allow us to distinguish between these two SCPs. The purification of the protein complex using His-tagged ScpD implies that ScpD is present. However, on the basis of the mass spectrometry data, we can neither confirm nor exclude the presence of ScpC.

Nearest Neighbors of ScpD—The purification of ScpD-His complexes by nickel affinity chromatography and BN gel electrophoresis showed a clear association of ScpD with PSII, but did not provide evidence regarding the localization of ScpD within the PSII complex. To obtain information regarding the neighbors of ScpD-His in the PSII complex, we used a higher beta-dodecyl maltoside concentration (0.8% rather than 0.04%) for solubilization of the ScpD-His complex before separation by two-dimensional BN/SDS gel electrophoresis. The BN/SDS gel in Fig. 3 shows that the stronger solubilization of the purified ScpD-His complex resulted in the formation of smaller subcomplexes. To monitor the separation of different ScpD-His complexes, the SDS gel was probed by immunoblotting using antibodies directed against the His tag. To make sure no ScpD-His was overlooked, the antibody concentration used was high, and therefore, the signal was not linear with the amount of His tag. The composition of the ScpD-containing complexes was analyzed by MALDI-TOF mass spectrometry. Table 3 shows the results from the analysis of two different gels and corresponds to protein bands as indicated in Fig. 3.


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  		TABLE 3
Mass spectrometry identification of proteins that co-purified with ScpD-His upon separation by two-dimensional BN/SDS-PAGE after nickel chromatography and solubilization with 0.8% beta-dodecyl maltoside

The results from the analysis of two different gels shown in Fig. 3 are presented. ORF, open reading frame; r.m.s., root mean square; SQ, sequence query including peptide mass fingerprint (PMF) and MS/MS ion search (MIS) data.

 
ScpD-His was found to be prominently associated with CP47, which was consistently detected to co-purify with ScpD-His. The weak band below that of CP47 in Fig. 3 was assigned to CP43 (Table 3). The diffuse band in the 7–8-kDa region contained ScpD-His (see immunoblot in the lower panel of Fig. 3), and as this band was more diffuse than that of the immunoblot, it also might contain ScpC, as our mass spectra do not allow us to exclude the presence of this protein. A smaller complex toward the right in Fig. 3 clearly contained ScpD in the diffuse band in the 7–8-kDa range. In addition, we found Psb28 in a band at an apparent mass of 12.4 kDa. The distinct spot close to Psb28 is probably an artifact and does not seem to display this protein.

ScpC Co-migrates with PSII—As indicated, the detected mass fragment of ScpD is exactly identical to that of ScpC. Indeed, the primary structures of ScpD and ScpC are 87% identical, and the compelling similarity between these two SCPs indicates that they may have not only a similar function, but also similar binding partners. Therefore, it was important to determine the location of ScpC in the thylakoid membrane.


Figure 3
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  		FIGURE 3.
Closest neighbors of ScpD. ScpD-His and co-purified proteins were separated by BN/SDS-PAGE after nickel chromatography and solubilization with 0.8% beta-dodecyl maltoside. The lower panel shows immunostaining of ScpD-His after two-dimensional PAGE using an antibody directed against the His tag. Proteins were identified by mass spectrometry (see Table 3). Note that Psb28 does not correspond to the sharp dot on the gel, but rather is an underlying band.

 
Toward this goal, Synechocystis wild-type cells were pulse-labeled with [35S]Met/Cys for 30 min while growing at 500 µmol photons m–2 s–1, and subsequently, thylakoid membranes were isolated and analyzed by two-dimensional BN gel electrophoresis in combination with autoradiography. The autoradiogram of the wild-type strain in Fig. 4A (first panel) displayed a strong band in the ScpC/ScpD region at 6 kDa that was present in two complexes. The first complex, RCC1, was identified previously as monomeric PSII consisting of the CP47, CP43, D2, and D1 proteins (38). The second complex, termed RC47, was smaller and was depleted in CP43. A comparison with SCP deletion mutants showed that the band with a molecular mass of 6 kDa was reduced in the {Delta}scpC/{Delta}scpD strain (second panel), but present in the {Delta}scpB and {Delta}scpE strains (third and fourth panels, respectively). These observations indicate that the high light-induced 6-kDa band of the RCC1 and RC47 complexes contained ScpD and/or ScpC, but most likely not ScpB or ScpE.

To distinguish between ScpD and ScpC in the 6-kDa band of the RCC1 and RC47 complexes, an antibody was raised against the N terminus of ScpC (MTTRGFRLDQDNRLNNF), which is identical to that of ScpD except for the third residue (S in ScpD). Indeed, immunostaining of high light-induced wild-type cells identified the bands in the 6-kDa region as ScpC and ScpD (Fig. 4B, left panel). The bands were absent in thylakoids of the high light-induced {Delta}scpC/{Delta}scpD strain (right panel). Therefore, the minor band with a molecular mass of 6 kDa seen in the autoradiogram of the {Delta}scpC/{Delta}scpD strain (Fig. 4A, second panel) belongs to other co-migrating proteins. In the {Delta}scpD deletion mutant, the antibody unambiguously identified ScpC co-migrating with RCC1 and RC47 (Fig. 4B, middle panel). Interestingly, after applying the same procedure to a PSII-less mutant, ScpC and ScpD were found to co-migrate with small complexes or as free proteins (data not shown), suggesting that these SCPs do not readily associate with large complexes such as PSI. Separation of thylakoid preparations from high light-induced {Delta}scpC and {Delta}scpD strains by one-dimensional SDS electrophoresis made it possible to identify the lower migrating band as ScpD (Fig. 4C, third lane) and the upper band as ScpC (fourth lane). In the wild-type strain, the ScpD protein appeared to be dominant (Fig. 4C), but the ratio between ScpC and ScpD amounts was highly variable among various strains and conditions, suggesting that ScpC and ScpD are indeed functionally equivalent. Interestingly, in the {Delta}scpB strain, the level of ScpD was decreased, and ScpC appeared to be absent altogether (fifth lane). ScpC/ScpD immunodetection by two-dimensional BN/SDS-PAGE of extracts from the {Delta}scpB strain showed that ScpD remained associated with PSII complexes (RCC1 and RC47) as in the wild-type control (data not shown).


Figure 4
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  		FIGURE 4.
ScpC co-migrates with PSII. A, autoradiograms of thylakoid membrane proteins from the high light-treated wild-type (WT) strain (whole gel) and mutant strains {Delta}scpC/{Delta}scpD, {Delta}scpB, and {Delta}scpE (only the PSII region is shown) after pulse radiolabeling with [35S]Met/Cys for 30 min. Proteins were separated by two-dimensional BN/SDS-PAGE prior to autoradiography. B, immunodetection of ScpC/ScpD in the wild-type strain (whole gel) and mutant strains {Delta}scpD and {Delta}scpC/{Delta}scpD (only the PSII region is shown) using anti-ScpC/ScpD antibody. C, immunoblot using anti-ScpC/ScpD antibody after one-dimensional SDS-PAGE of thylakoid membrane proteins from high light-induced cells. The loaded samples contained 4 µg of chlorophyll/lane and correspond to the wild-type strain, {Delta}scpC/{Delta}scpD, {Delta}scpC, {Delta}scpD, and {Delta}scpB as indicated.

 
ScpE Is Not Associated with PSII—Now that ScpD and ScpC have been positively correlated with PSII complexes, we set out to determine the location of the other two small SCPs as well (ScpB and ScpE; ScpA is a C-terminal extension of ferrochelatase). We were unable to generate antibodies against ScpB. However, we could elicit antibodies against the peptide ELQPNQTPVQEDPKFG, which is a sequence that is part of the N-terminal region of ScpE. These antibodies were used for detection of ScpE on SDS-polyacrylamide gels of membrane preparations from the wild-type and PSII-less strains (31) that were grown at 500 µmol photons m–2 s–1 (high light) for 7 h to induce the expression of ScpE from the PSI-less/PSII-less strain (32), in which SCPs are induced also at light intensities of 50 µmol photons m–2 s–1 (18), and from the PSI-less strain that, because of its light sensitivity, was grown at 10 µmol photons m–2 s–1. As negative controls, membranes from the wild-type strain grown at normal light intensity (50 µmol photons m–2 s–1) and from the {Delta}scpE strain grown at 500 µmol photons m–2 s–1 were included (Fig. 5, A and D). Although no ScpE was detected in membranes from the {Delta}scpE strain or the wild-type strain grown at 50 µmol m–2 s–1, the antibody immunostained ScpE in the other strains and in the wild-type strain grown at high light intensity.


Figure 5
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  		FIGURE 5.
ScpE is located in the thylakoid membrane. The subcellular location of ScpE was detected by immunoblotting. After breaking the cells, the total membranes were separated by one-dimensional SDS-PAGE and analyzed using anti-ScpE antibody. A, wild-type (WT), PSI-less (PSI–), PSII-less (PSII–), and PSI-less/PSII-less mutant cells were grown at 50 (Control), 500 (HL), and 10 (PSI-less mutant) µmol m–2 s–1. B, thylakoid (TM), cytoplasmic (PM), and outer (OM) membranes were purified by two-phase partitioning (51); separated by SDS-PAGE; and analyzed by immunoblotting using anti-ScpE antibody. C, total membranes of high light-treated wild-type and nickel chromatography-purified PSII complexes (CP47-His) (33) and ScpD-His complexes were analyzed by immunoblotting using anti-ScpE antibody. D, shown is the accumulation of ScpE in the high light-induced wild-type, {Delta}scpB, {Delta}scpC/{Delta}scpD, and {Delta}scpE strains. Equal amounts of cells were loaded in each lane. The proteins were separated by denaturing SDS-PAGE, transferred onto polyvinyl difluoride membrane, and probed with anti-ScpE antibody.

 
To further localize ScpE, the total membrane fraction of the wild-type strain grown at high light intensity was separated into fractions enriched in thylakoid membranes, plasma membranes, and outer membranes using two-phase partitioning (52). In these fractions, ScpE was immunodetected exclusively in the thylakoid membrane fraction (Fig. 5B). To investigate whether ScpE is associated with PSII, oxygen-evolving PSII was isolated from the HT-3 mutant (33), which contains a hexahistidine tag at the C terminus of the CP47 protein. Also the ScpD-His fraction eluted after nickel column purification (Fig. 1) was analyzed. Although an immunoreaction was obtained from the positive control (total membranes isolated from high light-stressed wild-type strain), ScpE could not be detected in either PSII or the ScpD-His fraction (Fig. 5C). Deletion of ScpB or ScpC and ScpD did not alter the presence of ScpE, even though the ScpE abundance was decreased in the {Delta}scpC/{Delta}scpD strain (Fig. 5D).


Figure 6
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  		FIGURE 6.
ScpE is not associated with PSII. Shown is the localization of ScpE after two-dimensional BN/SDS-PAGE analysis of thylakoid membrane proteins from the PSI-less and high light-induced wild-type (WT) strains. Thylakoid membrane proteins were separated in the first dimension by BN-PAGE and in the second dimension by denaturing SDS-PAGE using a 12–20% linear gradient polyacrylamide gel, blotted onto polyvinyl difluoride membrane, and immunostained using anti-ScpE antibodies.

 
To determine the potential association of ScpE with membrane complexes, a two-dimensional BN/SDS-polyacrylamide gel was challenged with anti-ScpE antibodies. As indicated in Fig. 6, ScpE was not stably associated with one of the photosystems, but instead was present in small complexes and in free form in the wild-type strain as well as in the PSI-less mutant. This is in agreement with the fact that no ScpE protein was detected in isolated PSII (Fig. 5).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In various plant genomes, one-helix proteins with high similarity to the first and third helices of the plant chlorophyll a/b-binding antenna proteins have been detected (12–16). However, their function still remains enigmatic. In this work, we have shown that ScpC–E of Synechocystis sp. PCC 6803 are found in thylakoid membranes. ScpC and ScpD are associated with PSII, whereas ScpE is not associated with larger membrane complexes.

Plant one-helix proteins (13) are apparently involved in pigment-related processes other than light harvesting, and a light-harvesting function can also be excluded for the SCPs of Synechocystis sp. PCC 6803 (24, 28). The whole cab gene family has been suggested to have originally evolved to serve a function in photoprotection, and the role in light harvesting may be a derived function (53).

Instead, the SCPs affect steps in the chlorophyll biosynthesis pathway (28) and chlorophyll stability in the cell, even in darkness (24). Interestingly, the one-helix CAB-like proteins in different organisms exhibit regulatory responses opposite to those of their relatives, the light-harvesting proteins: at high light intensity, when the expression of the LHC proteins is repressed, the one-helix proteins/SCPs are up-regulated. Also, the SCPs are up-regulated under many stress conditions (19). This indicates a function in protection in a broad sense; they might provide either direct protection (for example, as a pigment carrier) or indirect protection by regulating pigment metabolism. From a sequence perspective, it is likely that they bind pigments (chlorophylls and carotenoids), as chlorophyll-binding residues in the CAB family are conserved in SCPs, and deletion of SCPs leads to a decrease in chlorophyll and carotenoid content of cells (24).


Figure 7
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  		FIGURE 7.
Model of ScpD binding to PSII. Stress conditions cause PSII monomerization. ScpD and probably also ScpC bind to the monomers and are located close to CP47. Substoichiometric Psb28 might stabilize the binding between CP47 and ScpC/ScpD.

 
According to the results of this study, ScpD is clearly associated with PSII. PSII core proteins co-purified with ScpD-His and formed a complex that was retained during BN-PAGE at least when PSII was monomeric. Association of SCPs with PSII has not been observed thus far in crystallography studies of PSII (54–56) or in proteomic PSII association studies (57), as SCPs are apparent only under conditions of high light exposure. In PSII, ScpD seems to be associated most closely with CP47; CP47 was the most prominent band after purification of ScpD-His. However, after solubilization in the presence of increased detergent concentration, also Psb28 was detected in association with ScpD. Psb28 (sll1398) has been found to be a substoichiometric subunit of PSII (57, 58) and is thought to have a regulatory function. As it is considered to reside on the stromal side of PSII, it might stabilize the complex between ScpD and CP47 (Fig. 7).

Another interesting association of the light-stressed ScpD-His/PSII complex is that with several FtsH proteases. Deletion analysis of single FtsH protease in Synechocystis has shown that slr1604 is crucial for the survival of cells, a phenotype that also has been observed in {Delta}slr0228 (59). However, deletion of sll1463 does not show a phenotype. Our mass spectrometry analysis showed that this gene was expressed and that its product was apparently associated with PSII. Therefore, it is most likely involved in repair of light-stressed PSII. The co-purification of PSII and FtsH with ScpD-His suggests that ScpD is associated with the PSII repair process. This provides an explanation for the fact that ScpD was found to be associated with monomeric PSII (Fig. 2), which is often correlated with PSII repair processes. Indeed, the relatively weak Coomassie Blue staining of the monomeric PSII band suggests that ScpD forms a protective shield around PSII, most prominently interacting with the chlorophyll-binding protein CP47, which is near the periphery of the complex.

As ScpD has chlorophyll-binding potential but does not contribute to light harvesting (24), an attractive hypothesis is that ScpD polypeptides serve as a chlorophyll storage device while PSII is repaired and components are replaced. Indeed, the rate of turnover of chlorophyll is much lower than that of the PSII protein components with which it is associated (60). Multiple ScpD and/or ScpC proteins may be associated with PSII, thus providing an explanation for the high level of SCPs relative to PSII (Fig. 2).

ScpC is part of PSII as well. Upon BN/SDS-PAGE of complexes from a PSII-less mutant, ScpC and ScpD migrated as low molecular mass complexes or as free proteins (data not shown). After denaturing PAGE, ScpC migrated at a slightly higher molecular mass compared with ScpD. ScpC and ScpD substitute for each other's function (24) and have been hypothesized to form a complex (19). Although ScpD is generally more abundant than ScpC in light-stressed wild-type cells, the ScpC/ScpD ratio may vary. ScpB was found by mass spectrometry analysis in the fraction co-isolating with ScpD-His (Fig. 1). Although this suggests that ScpB is associated with ScpD, the pulse-labeling pattern of PSII in the absence of scpB was similar to that of the control (Fig. 4). ScpE is present in thylakoids, but is not found to be associated with ScpD. Interestingly, ScpB and ScpE have the strongest influence on chlorophyll biosynthesis (28). ScpA is the C-terminal part of ferrochelatase. In this study, we did not include ScpA, suggested to have an important function in regulating the tetrapyrrole pathway at the branch point between chlorophyll biosynthesis versus heme/phycobilin biosynthesis. It has been shown that decreased ferrochelatase activity improves photoautotrophic growth of a PSII mutant because of increased supply of chlorophyll (61) and that the ScpA domain is important for the ferrochelatase function.3

Based on the considerations provided here, the most plausible explanation regarding the function of ScpC and ScpD in light-stressed PSII is pigment storage during protein turnover. As such pigments may not be able to transfer energy to functional photosystems and as there is no evidence for high chlorophyll fluorescence from pigments while photosystems are being repaired, excitations of pigments associated with SCPs should be quenched efficiently. However, these pigments should not be in excitation transfer contact with PSII pigments so that they do not decrease light-harvesting efficiency under conditions in which light is not in excess. There may be parallels between PSII interaction with SCPs versus the interaction between IsiA and PSI. IsiA has been found to form a ring around PSI, which is functional in light harvesting, but also empty rings that are effective energy dissipaters have been detected (62). Similarly, ScpD and ScpC may form a ring around damaged PSII centers, aid in repair, and dissipate absorbed energy as needed.


   FOOTNOTES
 
* The work was supported by the Swedish Foundation for International Cooperation in Research and Higher Education, the Swedish Research Council, Department of Energy Grant DE-FG02-04ER15543 (to W. R.), and Czech Academy of Sciences Institutional Research Concept AV0Z50200510 and Project MSM6007665808 (to J. K. and M. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 46-90-786-7633; Fax: 46-90-786-7661; E-mail: Christiane.Funk{at}chem.umu.se.

2 The abbreviations used are: LHC, light-harvesting complex; PS, Photosystem; SCPs, small CAB-like proteins; BN, blue native; MES, 4-morpholineethanesulfonic acid; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry. Back

3 R. Sobotka, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Birgitta Norling (Stockholm University) for samples enriched in thylakoid, plasma, and outer membranes purified via two-phase partition; Dr. Dmitry Sveshnikov (Umeå University) for the localization of ScpE on cyanobacterial membranes; and Dr. A. Sokolenko for anti-ScpC antibody. Parts of this work were carried out at the Umeå Life Science Platform and the Umeå Protein Analysis Facility.



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