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Originally published In Press as doi:10.1074/jbc.M412967200 on April 1, 2005

J. Biol. Chem., Vol. 280, Issue 22, 21667-21672, June 3, 2005
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Phycobilisome Linker Proteins Are Phosphorylated in Synechocystis sp. PCC 6803*

Irina Piven{ddagger}, Ghada Ajlani§, and Anna Sokolenko{ddagger}

From the {ddagger}Department für Biologie I, Bereich Botanik, Ludwig-Maximilians-Universität, Menzingerstrasse 67, 80638 München, Germany and the §Departmenet de Biologie Joliot-Curie, Section de Biophysique des Fonctions Membranaires, Commissariat à l'Energie Atomique Saclay, F-91191 Gif-sur Yvette, France

Received for publication, November 16, 2004 , and in revised form, February 18, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The controversial issue of protein phosphorylation from the photosynthetic apparatus of Synechocystis sp. PCC 6803 has been reinvestigated using new detection tools that include various immunological and in vivo labeling approaches. The set of phosphoproteins detected with these methods includes ferredoxin-NADPH reductase and the linker proteins of the phycobilisome antenna. Using mutants that lack a specific set of linker proteins and are affected in phycobilisome assembly, we show that the phosphoproteins from the phycobilisomes correspond to the membrane, rod, and rod-core linkers. These proteins are in a phosphorylated state within the assembled phycobilisomes. Their dephosphorylation requires partial disassembly of the phycobilisomes and further contributes to their complete disassembly in vitro. In vivo we observed linker dephosphorylation upon long-term exposure to higher light intensities and under nitrogen limitation, two conditions that lead to remodeling and turnover of phycobilisomes. We conclude that this phosphorylation process is instrumental in the regulation of assembly/disassembly of phycobilisomes and should participate in signaling for their proteolytic cleavage and degradation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Phycobilisomes (PBSs)1 are large multimeric protein structures that function as an extrinsic light-harvesting antenna in cyanobacteria and red algae. They are located at the outer surface of thylakoid membranes where they transfer their excitation energy to the photosynthetic reaction centers that are embedded within the lipid bilayer. Our knowledge, generated over >30 years from biochemical and biophysical analysis, genetics, electron microscopy, and x-ray crystallography, led to improvement of the PBS structural model (1). In Synechocystis sp. PCC 6803, PBSs are comprised of the bilin-containing proteins allophycocyanin (APC) and phycocyanin (PC) and colorless linker proteins that assemble PC and APC substructures and tune their properties to optimize energy transfer (2). Linker proteins can be divided into four groups: (i) rod-core linkers (LRC) that attach the peripheral rods to the PBS core; (ii) rod linkers () that associate PC substructures into rod segments; (iii) the small core linkers () that are associated with trimeric APC at the peripheries of the core cylinders; and (iv) the core-membrane linker () that acts in the organization of the PBS core and is the major terminal energy emitter to photosystem II (1–3).

PBSs represent a major biosynthetic commitment of a cyanobacterial cell; apart from their light-harvesting function in photosynthesis, PBSs are recruited as a principal nutrient source under starvation conditions. This dual function implies specific regulation for assembling, disassembling, and remodeling the PBS structure according to changes in metabolic and energy requirements. Cyanobacteria have evolved several molecular mechanisms for acclimation of the PBS antenna that operates at the transcriptional (4, 5), translational, and post-translational levels (6, 7). Taken together, these regulatory mechanisms contribute to remodeling PBS composition, size, and number per thylakoid membrane when cells are exposed to environmental changes.

It has been suggested that chaperones may be essential in the first steps of PBS assembly when the degradation of biliproteins competes with protein biosynthesis (8). The subsequent modulation of PBS structures upon acclimation to various stresses leads in some cases to partial or full disassembly, degradation, and reutilization of phycobiliproteins. For instance, high rates of PBS degradation were observed for various cyanobacterial strains upon nutrient deprivation and high light stress (6, 9–11). The nbl gene family encodes a major group of proteins that is involved in stress signaling and the control of PBS degradation. Two signaling components, a response regulator and a histidine kinase, are encoded by the nblR and nblS genes, respectively (12, 13), whereas two other genes code for NblA and NblB, which are required for coordination of PBS degradation under nutrient deprivation (14–17). The cleavage of linker proteins has been suggested to be a prerequisite for the complete degradation of phycobiliproteins (7, 18–20). Because PBSs represent tightly organized structures, it is not yet clear which protein determinants can trigger the initiation of the disassembly process and how such structural components as linker proteins, which are embedded in rod discs (21–23), can become accessible to regulatory proteins and proteolytic enzymes.

The facility by which phosphate groups can be added to a variety of amino acids within proteins and thereby induce changes in enzymatic activity, stability, or binding properties makes protein phosphorylation an attractive way of regulating cellular responses to the environment (24). In plant and algal chloroplasts, the light-harvesting antenna is a flexible structure that can accommodate changes in environmental conditions. Its post-translational modification by phosphorylation at some threonine residues triggers changes in protein conformation and redistribution of the antenna between the two photosystems in a process known as state transition (25, 26). Changes in protein phosphorylation also control the rate of proteolysis of the antenna proteins under excess illumination (27). Phosphorylation changes had been suggested as contributing to some mechanisms for PBS modifications a decade ago, but the experimental support to this view remained controversial (28). Most attempts to detect subunit phosphorylation within the PBS antenna in cyanobacteria were unsuccessful, with one exception in which the phosphorylation of {beta}-PC in Synechococcus sp. PCC 6301 was reported (29).

In the present study we show that some linker proteins are permanently phosphorylated within the assembled phycobilisomes in Synechocystis sp. PCC 6803. The phosphorylation of these proteins can be detected serologically with phosphospecific antisera as well as by the use of a fluorescence dye that recognizes phosphoresidues. We show that mutant strains that lack specific sets of linker proteins also lose these phosphoprotein signals. We demonstrate both in vivo and in vitro that the dephosphorylation of linkers accompanies partial PBS disassembly and eventually leads to their total disassembly, a step that may be a prerequisite for protein degradation. Based on these data we discuss the physiological significance of linker phosphorylation in the response to changes in irradiance levels and to nitrogen deprivation, two environmental changes that cause remodeling and degradation of PBSs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—Synechocystis sp. PCC 6803 and its derivatives were cultivated in BG11 medium (30) under a white light of 50 µE m-2 s-1 at 30 °C. For phosphorylation studies, wild-type was grown at a low light (LL; 50 µE m-2 s-1) up to OD750 = 0.5 and then transferred to high light (HL; 700 µE m-2 s-1) or to nitrogen deprivation for 24 h. For nitrogen deprivation, cells were harvested by centrifugation at 3,500 x g for 5 min and resuspended in nitrogen-depleted media. The cells were kept under LL at 30 °C for a further 24 h. Media lacking nitrogen were prepared by replacing ferric ammonium citrate, NaNO3, and Co(NO3)2 with FeCl3, NaCl, and CoCl2.

Extraction of Thylakoid Membrane and Phycobilisome Proteins— Thylakoid membrane proteins were isolated as described (31). For separation of soluble and membrane-bound proteins from cell cultures normalized to equal OD750 = 0.7, cells were disrupted by sonification with a Sonifier B-12 from Branson Sonic Power Co. (Danbury, CT) in a four-cycle regime at 20 s each (70 watts) with a pause of 20 s in between. PBSs were isolated as described previously (18) with some modifications (7). The PBS preparation was based on cell disruption with glass beads, solubilization by 2% (v/v) Triton X-100, and subsequent separation of PBSs by 0.25–0.79 M sucrose density gradient ultracentrifugation using 0.9 M phosphate buffer, pH 7.0. The fraction of intact PBSs that formed the lower band in the gradient was diluted in 0.9 M phosphate buffer and centrifuged at 80,000 x g for 4 h at 4 °C. The PBS pellet was dissolved in H2O and frozen at -70 °C.

Protein Gel Electrophoresis, Protein Staining, and Immunological Analysis—Thylakoid (3–5 µg chlorophyll per lane) and PBS proteins were separated by 12.5% SDS-PAGE according to (32). For PBS loading, the absorbance of the samples was measured at 620 nm, and an amount of biliproteins equivalent to OD620 = 1.5 was loaded per well. Cell cultures acclimated to HL and nitrogen deprivation were adjusted to OD750 = 0.7, and cells were fractionated into membrane and soluble fractions as described above. For protein visualization, gels were stained with Coomassie Brilliant Blue, silver nitrate, or the luminescent dye SYPRO® Ruby (Molecular Probes, Leiden, Netherlands). Phosphoproteins were detected either by immunological analysis with phosphothreonine/phosphoserine antisera (Zymed Laboratories Inc.) or staining with the phosphoprotein gel dye Pro-QTM Diamond (Molecular Probes, Leiden, Netherlands). For blocking unspecific reactions of phosphothreonine antisera, the inhibitors for phosphoserine residues (Zymed Laboratories Inc.) were incubated with polyvinylidene difluoride membranes prior to the immunological reaction. Antisera against PBS proteins were kindly provided by A. Grossman. The SYPRO® Ruby and Pro-QTM Diamond stains were detected by the fluorescent image analyzer FLA-300 (Fujifilm). Gels stained with SYPRO® Ruby and Pro-QTM Diamond were scanned by orange filter O580 with excitation/emission wavelengths at 473/580 nm. For detection of PBS bilin-containing proteins by self-fluorescence, non-stained gels were scanned by red filter with excitation and emission at 635/675 nm.



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  		FIG. 1.
Detection of phospho-substrates in isolated thylakoids (A) and the purified PBS antenna (B) of the Synechocystis wild-type strain. Thylakoid and phycobilisome proteins were isolated from wild-type Synechocystis cells grown under a standard light regime and separated by electrophoresis on 12.5% SDS-polyacrylamide gels. Protein profiles were visualized by silver staining (A and B) or by the fluorescence dye SYPRO Ruby (B). Protein phosphorylation was detected for thylakoid membrane and PBS proteins with phosphothreonine (Phospho-thr) antisera (A and B), the fluorescent dye Pro-Q Diamond (B), or by in vivo labeling (A). The fluorescence of the bilin-containing proteins (PBP) APC and PC was detected by scanning the non-stained gels with a red laser as described under "Materials and Methods" (B, lane labeled Autofluo). Protein molecular mass markers are indicated at the left and right of panels A and B, respectively. PBS proteins are marked at the left of the panel B (includes the membrane linker , the rod linkers and , and the rod-core linker LRC). The phosphorylated protein bands in panel A that are co-migrating with PBS proteins are marked by arrows.

 
Phosphorylation of Cyanobacterial Proteins in Vivo—The phosphorylation of Synechocystis cellular proteins in vivo was performed according to (33). Cells were grown in BG11 medium under white LL (50 µE m-2 s-1) to an OD750 of 0.6. Cells were harvested by centrifugation and resuspended in BG11 medium without phosphate to an OD750 of 3. For each sample, 1 ml of cell culture was transferred to a small Petri dish to provide equal illumination. The phosphorylation was performed by the addition of 30 µCi of carrier-free [32Pi] with subsequent incubation under LL for 1.5 h. After incubation, cyanobacterial proteins were precipitated with 5% (w/v) trichloroacetic acid, washed with 100% acetone, and dissolved in SDS sample buffer for the Laemmli gel system.

Dephosphorylation by Alkaline Phosphatase and Proteolysis in Vitro—For dephosphorylation of thylakoid and PBS proteins, samples were incubated with bovine alkaline phosphatase (Sigma) according to (34). Phycobilisome proteins were incubated with alkaline phosphatase in a buffer containing 0.1 M glycine, pH 10.4, 1 mM MgCl2, and 1 mM ZnCl2 for 30 min at room temperature. The reaction was stopped by the addition of 20 mM EDTA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of Thylakoid Proteins in the Wild-type Synechocystis Strain—Phosphorylation of photosynthetic proteins in Synechocystis was analyzed by two assays, immunological detection with antisera raised against phosphorylated amino acid residues or in vivo phosphorylation with carrier-free [32Pi]. For immunological analyses, thylakoid-enriched protein fractions were extracted from cyanobacterial cells grown under standard conditions. After SDS-PAGE, protein profiles were visualized by silver-staining (Fig. 1A, left lane) and transferred onto polyvinylidene fluoride membrane for immunological detection of phosphorylated proteins (Fig. 1A, middle lane). The application of phosphothreonine and phosphoserine antisera resulted in similar phosphoprotein profiles (data not shown). To check whether similar phosphoprotein patterns resulted from serological cross-reactivity of phosphothreonine and phosphoserine residues or from the presence of both phosphoresidues, inhibitors blocking phosphothreonine or phosphoserine antisera were applied during the immunological assay. Because these treatments did not change significantly the profile of phosphorylated proteins (results not shown), we assumed that phosphorylation occurred at both the threonine residue and the serine residue. In subsequent studies we restricted analysis to the use of phosphothreonine antisera. Approximately 15 phosphoproteins were detected in thylakoid preparations with antisera elicited against phosphothreonine residues (Fig. 1A). The strongest phosphorylation signals were obtained for proteins with molecular masses of 16, 20, 23, 29, 33, 35, 40, 50, and 90 kDa. Obviously, these proteins represent a set of phosphoproteins that are stable enough to be detected by an immunological approach. To detect phosphoproteins that undergo rapid phosphate exchanges we employed an in vivo labeling approach with [32Pi]. This experiment identified another set of phosphoproteins in thylakoid membranes (Fig. 1A), predominantly in the ranges of 18–20 and 30–36 kDa and a highly phosphorylated band of 66 kDa. Comparison of silver-stained thylakoid membrane polypeptides with those of the immunological analysis suggested that the major phosphorylated proteins migrated in an electrophoretic position of phycobilisome subunits. Because the phosphorylation of the PBS antenna has been a controversial issue for more than a decade, we applied the same immunological assay to a purified PBS fraction. PBS antenna proteins were extracted from Synechocystis cells on sucrose gradients and separated by SDS-PAGE. Proteins were then visualized by staining with silver nitrate or the fluorescence dye SYPRO Ruby (Fig. 1B). The preparation of PBSs contains predominantly two groups of proteins, heavily stained phycobilin-containing proteins in the molecular mass range of 16–22 kDa and less abundant proteins corresponding to the FNR protein and non-bilin-containing linker peptides, the rod linkers and , and the rod-core linkers LRC as well as the mature form and the truncated form of the membrane linker, and , respectively (18). SYPRO Ruby, which is a quantitative and highly sensitive dye for protein detection, stained saturating amounts of phycobiliproteins (APC and PC) negatively. Phosphorylated proteins were visualized with phosphothreonine antisera or the fluorescence dye Pro-Q Diamond, which reacts with phosphorylated residues (Fig. 1B). Both assays revealed five heavily phosphorylated bands that corresponded to the linker proteins (, , , and LRC) and FNR, which were also markedly labeled in thylakoid membrane preparations assayed with phosphospecific serum. Note on Fig. 1B the limited shift in position of the FNR between the lanes labeled Pro-Q and Autofluo versus the lanes labeled Silver and SYPRO Ruby that is due to changes in gel sizes during various staining and blotting procedures. Although two highly phosphorylated bands in the molecular mass range between 16 and 22 kDa were also detected in thylakoid membrane preparations (Fig. 1A, lane labeled Phospho-thr and 32Pi), no signals in the same region could be visualized in PBS preparation at least with the phosphothreonine antisera (Fig. 1B). Comparable results were obtained with the fluorescent Pro-Q Diamond dye, although it displayed additional signals in the range of PC/APC. To understand the nature of the signals originating in PC/APC proteins, the non-stained gel loaded with PBS proteins was scanned with a red laser set at an excitation/emission wavelength suitable for detection of bilin-containing proteins. (Fig. 1B, lane labeled Autofluo). We observed a very weak signal co-migrating with FNR and intense signals from the PC and APC bands. This observation showed that the PC/APC signals obtained from Pro-Q staining corresponded to the selffluorescence of bilin proteins and not to the phosphorylation of PC/APC.



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  		FIG. 2.
Analysis of phosphoproteins in the wild-type and mutant strains deficient in PBS proteins. A, PBS antenna extracted from the wild-type and linker-deficient CK strain. PBS proteins separated by electrophoresis on 8–18% SDS-polyacrylamide gels were stained with Coomassie Brilliant Blue. B, Western analysis of PBS proteins in thylakoid membranes of the wild-type, PAL, and CK strains. Thylakoid (Thyl) proteins from wild-type (WT), PAL, and CK strains were separated by electrophoresis in a 12.5% SDS-polyacrylamide gel, and proteins were probed with antisera raised against various coremembrane, rod, rod-core linkers, and APC/PC (indicated at the left). C, phosphoproteins in thylakoid membranes of the wild-type and PBS-deficient strains. Phosphorylated proteins were identified by immunodetection with phosphothreonine antisera. PBS extracted from wild-type cells were used as a control to identify linker proteins among other phosphorylated thylakoid proteins.

 
Phosphorylation of Thylakoid Proteins in PBS-deficient Mutants—To assess whether the phosphorylation signals actually originated from the PBS linkers rather than from minor co-migrating contaminating minor polypeptides, the immunological assay was applied to mutant strains that either had a decrease in PBS sizes because of a lack of rods (CK mutant) or that fully lacked PBS antenna (PAL mutant). The PAL mutant bears a deletion in genes encoding the and APC proteins in a PC-deficient background (35). The CK mutant is deleted in the entire cpc-operon.2 PBSs were still present in CK but contained only APC and were smaller in size (Fig. 2A). Both strains were analyzed for the presence of all linker proteins by immunodetection with antisera raised against various linker and bilin-containing polypeptides (Fig. 2B). According to immunological analysis, none of the PBS proteins was present in the PAL strain. In the CK strain the absence of PC rods and the rod linkers and prevented the accumulation of newly synthesized LRC, which connects the APC-containing core and the PC rods (Fig. 2B). These rod-core linkers were still detectable in the CK strain but to a much lower extent than in the wild-type. The CK strain still contained APC in the PBS core and the linker in a similar amount as in the wild-type. The phosphorylation pattern in thylakoid membranes of the wild-type, PAL, and CK mutant strains was also probed with phosphothreonine antisera (Fig. 2C). PBSs extracted from the wild-type were loaded as a control for the migration of linker proteins. The differences in the intensity of the protein phosphorylation profile of wild-type thylakoid membranes as compared with the one presented in Fig. 1A resulted from a weaker exposure of enhanced chemiluminescence-labeled films to visualize a better contrast with the protein patterns in the PBS-deficient mutants. High resolution of the 29–36-kDa region revealed three phosphorylated bands in the range of rod linkers, with the central one showing no correspondence to PBS subunits. No phosphorylated bands were detected at any linker position in the PAL mutant (Fig. 2C). The core-membrane linker remained detectable as a phosphoprotein in the CK mutant at the same rate as in the wild-type, whereas no phosphorylated band remained at the positions of the other linkers. Because the absence of linker polypeptides in the mutants resulted in the loss of the corresponding phosphothreonine signals, we concluded that the rod linkers were phosphorylated in the PBSs of the wild-type.



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  		FIG. 3.
Dephosphorylation of PBS linker proteins by alkaline phosphatase and their separation in sucrose gradients. A, isolated PBSs were incubated in the absence (-) or presence (+) of alkaline phosphatase and separated by electrophoresis on a 12% polyacrylamide gel. The degree of phosphorylation was immunologically detected with anti-phosphothreonine serum. B, PBS fractions treated (+AP) and not treated with alkaline phosphatase (-AP) were separated on 0.25–0.79 M sucrose gradients. Fractions of 0.2 ml were collected from the bottom of the gradient and separated by electrophoresis on a 12% polyacrylamide gel. Proteins were detected by silver staining (the upper sections of the experiments with and without alkaline phosphatase) and by immunological analysis with phosphothreonine (Phospho-thr) antiserum (the upper sections). From silver staining, only the 16–22-kDa region with APC/PC proteins (phycobiliproteins, labeled PBP) is presented. For immunological analysis, the part of the gel of 24–38 kDa with rod and rod-core linkers is shown.

 
Dephosphorylation Occurs Only in Partially Disassembled Phycobilisomes—As an additional proof of linker phosphorylation in vivo, PBSs were dephosphorylated in vitro by incubation with an alkaline phosphatase that is specific for O-phosphorylation of threonine or serine residues. We observed only partial dephosphorylation of the linker proteins (Fig. 3A). Because linker proteins are embedded in PC hexamers, we reasoned that the dephosphorylation sites might not be easily accessible to the phosphatase in the assembled PBSs. When isolated PBSs are resuspended in water or in phosphate buffer of very low concentration, they are partially disorganized (36). To check whether the dephosphorylation of linker proteins is controlled by the assembly state of the PBSs, we ran the pretreated or non-treated PBS preparations with alkaline phosphatase in sucrose gradients. As shown by the silver-staining pattern of the 16–22-kDa region containing the PC/APC proteins, this experiment separated the intact PBSs found at the bottom of the gradient (fractions 1 in Fig. 3B) from partially or fully disassembled PBS subunits found at the top of the gradient (fractions 2–20 in Fig. 3B). Phosphorylated linker proteins in the region of 24–37 kDa were detected immunologically with an anti-phosphothreonine serum and are presented in the lower sections of Fig. 3B. Without alkaline phosphatase treatment, linker proteins were heavily phosphorylated in fraction 1 but less efficiently in fractions 9–16, where most PBS proteins were detected by silver nitrate staining. After dephosphorylation by alkaline phosphatase, PBS preparation resolved phosphorylated linkers only in intact PBSs (Fig. 3B, fraction 1), but no other phosphorylation was observed all over the gradient fractions (Fig. 3B, section labeled +AP). At the same time the phycobiliproteins were all shifted to the upper part of the gradient, indicating that they underwent further disassembly. This experiment demonstrates that linker proteins are subjected to dephosphorylation only in partially disassembled PBS structures and that their dephosphorylation enhances disassembly of the PBS antenna.



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  		FIG. 4.
Phosphorylation levels of thylakoid proteins upon acclimation to high light and nitrogen deprivation. A and B, Synechocystis wild-type cells were grown under LL and then acclimated to HL and nitrogen deprivation (-N) for 24 h. Cell cultures were normalized to the same optical density, and proteins were separated into membrane (thylakoid-enriched) and soluble (cytoplasm) fractions. Proteins were separated by electrophoresis on a 12% polyacrylamide gel, and the amount of the linker proteins was controlled by antisera elicited against linker proteins (A). The {beta}-subunit of ATP-synthase was used as a loading control. The phosphorylation rate was detected by immunological analysis with phosphothreonine antisera (B). Arrowheads indicate the strongly dephosphorylated and LRC linkers.

 
Dephosphorylation of PBS Linkers Is Enhanced under High Light or in Nitrogen-limiting Conditions—In higher plant chloroplasts, phosphorylation/dephosphorylation of light-harvesting antenna is involved in state transitions and antenna degradation, two phenomena that are controlled by changes in light intensity and light quality. We noted that the phosphorylation/dephosphorylation rate of linker peptides was not changed during the short time acclimation corresponding to state transitions (data not shown). However, the cyanobacterial PBS antenna is highly rearranged and degraded in part upon increasing light intensities as well as in nitrogen-limiting conditions (6). We thus looked for the possible effects of high light and nitrogen deprivation on the in vivo phosphorylation of linker proteins. To this end, the wild-type of Synechocystis was acclimated to these conditions for 24 h. To better distinguish all phospho-linker polypeptides from the other phosphoproteins, the whole cell protein extracts were separated into membrane (thylakoid-enriched) and soluble (cytoplasm) fractions. These fractions were then probed with antibodies raised against linker proteins (Fig. 4A) and phosphothreonine amino acid residues (Fig. 4B) to assess the rates of phosphorylation of each linker relative to its actual content in each growth condition. Our immunological analysis revealed PBS proteins in both the membrane and the soluble fractions with the exception of the linker, which was not detected in the cytoplasm (Fig. 4A). We attribute the absence of to its rapid degradation upon the release PBS from the thylakoid membranes. The acclimation of cyanobacterial cells to HL and nitrogen deprivation leads to shortening of PBS rods and degradation of PBS structures (6, 9–11). Accordingly, we observed that membrane-associated PBSs lost and completely, which results in shortening the PC rods upon acclimation to nitrogen deprivation. The preservation of the LRC linkers and a significant fraction of the linker in the membrane fraction argue for the remaining association of PBSs of smaller size to the thylakoid membrane under these conditions. Under HL we still detected all linkers in the membrane fraction, but the amount of the distal and linkers decreased significantly.

We then probed the phospho-linkers in these cell extracts (Fig. 4B). As we have shown above (Fig. 2C), phosphorylated migrates close to another membrane-bound phosphoprotein that prevents its proper identification in the membrane fraction. However, it can be easily identified in the soluble fraction. For the LRC and linkers, HL and nitrogen deprivation caused a marked decrease in the amount of their phosphorylated forms relative to their quantity in either the soluble or membrane fraction; the drops in the phosphorylated forms were, respectively, two and four times larger than the drops in their total content. However, for and , the dephosphorylation rates were not higher than their degradation rates, which argues for a rapid degradation of the dephosphorylated forms. We thus conclude that the environmental changes that cause an increased disassembly and degradation of PBS are accompanied by a dephosphorylation of several PBS linkers, which include the membrane and rod-core linkers.



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  		FIG. 5.
A model for the putative role of protein linker phosphorylation in the biogenesis of PBS structures. Synthesized linker proteins are phosphorylated by some intracellular kinase(s) either when partially assembled with PC and APC or directly after protein synthesis. The whole PBS structures are fully assembled afterward in a step-by-step process (monomers, trimers, hexamers, and whole PBSs; not shown). Various stress conditions like high light and nutrient deprivation initiate PBS degradation. Linker dephosphorylation may occur either in partially disassembled PBS or within truncated forms of PC-LR. Later on the dephosphorylation of linkers can serve as a signal for protein degradation.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Various in vivo and in vitro labeling studies have indicated phosphorylation of thylakoid membrane proteins in photosynthetic prokaryotic organisms (summarized in Ref. 37), like Calothrix sp. PCC 7601 (38), Synechococcus sp. PCC 6301 (39, 40), Synechococcus sp. PCC 7942 (41), and Synechocystis sp. PCC 6803 (42). The labeling of proteins in Calothrix sp. PCC 7601 revealed three phosphoproteins in thylakoid membranes phosphorylated at serine and threonine residues (38). In Synechococcus sp. PCC 6301, labeling experiments performed in vivo with [32Pi] orthophosphate demonstrated phosphorylation of an 18.5-kDa protein that was also found in a purified PBS fraction and has been proposed to represent {beta}-PC (43, 44). Since these early observations of protein phosphorylation in thylakoid membranes were made, only one additional protein, the PsbH component of photosystem II, was claimed to be phosphorylated in cyanobacteria (45), although conflicting observations were reported ten years later (46).

In the present study the phosphoproteins of Synechocystis thylakoid membranes were analyzed by three strategies. The first two are based on an immunological detection with antisera raised against phosphorylated amino acid residues and on the application of a fluorescent dye recognizing phosphate groups attached to tyrosine, serine, and threonine residues. The third one consists of an in vivo incubation of Synechocystis cells with radiolabeled orthophosphate. Approximately 15 phosphoproteins could be detected using these approaches in the thylakoid membranes of Synechocystis. The profiles of phosphoproteins detected by these procedures were qualitatively and quantitatively different. Phosphospecific antisera and the fluorescent dye detect proteins carrying phosphorylated amino acid groups that may be very long-lived, whereas radiolabeling with [32Pi] identifies those proteins which are post-translationally modified during the incubation time, in particular those residues that are reversibly phosphorylated.

The most easily detectable and highly phosphorylated proteins turned out to be FNR and linker proteins (, , , and LRC). The phosphoprotein with a molecular mass of ~30 kDa detected previously with a phosphothreonine antisera (47) could correspond to one of the rod linker proteins. We did not observe any phosphorylation of the major bilin-containing antenna proteins APC and PC, which contrasts with a previously published report (29). FNR, which was shown to be associated to PBSs (48) was substantially phosphorylated in our antenna preparations. The phosphorylation of FNR from higher plant chloroplasts at serine and threonine residues has been observed, but the function of this process remains poorly understood (49). The profile of phosphorylated proteins in wild-type and cyanobacterial mutant strains deficient in PBSs (PAL strain) or rod linker proteins (CK strain) differed in these bands corresponding to the missing linkers in the mutants. This observation proved that the signals with phosphothreonine antisera corresponded to linker proteins.

The detection of substantial signals of linker proteins with anti-phosphothreonine sera contrasted with their poor phosphorylation by orthophosphate [32Pi]. This indicated that linker phosphorylation at serine or/and threonine residues is quite a stable post-translational modification. Surprisingly, the rod linkers were poorly sensitive to a dephosphorylation treatment, and their dephosphorylation in PBS preparations by alkaline phosphatase did not exceed 50%. This could be explained by the localization of linker proteins connecting PC segments in the internal cavities of the disks (22, 23). Therefore, externally added enzymes have little access to the linkers within the fully assembled PBS structures. The separation of partially disassembled PBSs that were pre-treated with alkaline phosphatase demonstrated that indeed only partially or completely dissociated PBSs can be fully dephosphorylated, whereas linkers in a fully assembled PBSs are not accessible. Moreover, we observed that dephosphorylation of linkers in partially assembled PBSs further disassembled their constitutive subunits. This experiment suggested that dephosphorylation of linkers could participate in the mechanism governing the remodeling and turnover of the PBSs in Synechocystis. Acclimation to higher light intensities as well as nitrogen deprivation induces a down-sizing of the PBS antenna that is due in part to changes in PBS gene expression (4, 5) and in part to proteolytic processes that target some PBS subunits (6, 7). In particular, protease SppA1 causes the cleavage of linker proteins under acclimation to higher light regimes with the subsequent release of distal PC rod segments (7). Indeed, we observed that the rate of linker protein phosphorylation decreased with environmental cues that cause restructuring, disassembly, and degradation of PBSs. The acclimation of cells for at least 24 h to high light or nitrogen starvation conditions induced a marked decrease in the rate of phosphorylation of the LRC and linkers. The net dephosphorylation of these two linkers, as compared with that of and , could be due to their slower degradation rate after full dephosphorylation. This observation argues for a role of linker dephosphorylation in vivo in PBS remodeling.

Linker dephosphorylation may act as a signal for protein degradation once PBS disassembly has started, whereas protein phosphorylation could occur before or during the assembly of PBS hexamers (Fig. 5). The initial steps of biliprotein biosynthesis include competition between protein biosynthesis and degradation. The biliprotein subunits with no attached bilins or those lacking partners can be rapidly subjected to degradation (summarized in Ref. 8). The mechanisms controlling biliprotein degradation are unknown, although one of the suggested models is the binding of chaperone proteins that would activate a degradation pathway (8). In that case, phosphorylation may play an important role in the stabilization of linker polypeptides in the PBS assembly pathway, whereas their unphosphorylated state could be signaling for their degradation. Changes in protein phosphorylation modify folding that, in turn, changes their affinity for proteolytic enzymes and assembly partners. The role of linker (de)phosphorylation in cyanobacteria would then be similar to that of the phosphorylations of the light-harvesting complex II (LHCII) and the D1 protein in higher plant chloroplasts that are considered to be targeted to degradation upon dephosphorylation (27, 50). A long phase of acclimation to higher light intensities of at least 24 h is required for the detection of PBS protein dephosphorylation. Such a lag period in the acclimation was also observed for light-harvesting complex of higher plants (27). This observation argues for an activation of some substrates or/and enzymes involved in the protein dephosphorylation and/or cleavage.

To date it has not been clear how PBS structures can be loosened during nutrient deprivation or light stress, conditions that cause a massive degradation of the cyanobacterial antenna (10, 51, 52). The major protein family known to be involved in the degradation of PBS is encoded by the nbl gene family. One of the components, NblA, is directly involved in PBS degradation under nutrient deprivation (14, 16, 17). It is suggested that the binding of NblA to PC can mark proteins for their further recognition by proteases or can soften the PBS structure by increasing the distances between single PC segments. It is then expected that proteases and phosphatases will get higher access to their substrates. These and other questions could be approached by studies on NblA and cyanobacterial phosphatases.


   FOOTNOTES
 
* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB TR1 and SO 448/2. 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

To whom correspondence should be addressed. Tel.: 49-89-17861242; Fax: 49-891782274; E-mail: Anna.Sokolenko{at}lrz.uni-muenchen.de.

1 The abbreviations used are: PBS, phycobilisome; APC, allophycocyanin; HL, high light; LL, low light; LCM, core-membrane linker; LR, rod linker; LRC, rod-core linker; µE, microeinstein; PC, phycocyanin. Back

2 B. Ughy and G. Ajlani, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank A. R. Grossman for providing the antisera raised against phycobiliproteins.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Crystal Structure of NblA from Anabaena sp. PCC 7120, a Small Protein Playing a Key Role in Phycobilisome Degradation
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