JXB Advance Access originally published online on March 7, 2005
Journal of Experimental Botany 2005 56(414):1239-1244; doi:10.1093/jxb/eri119
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RESEARCH PAPER |
Isolation of phosphorylated and dephosphorylated forms of the CP43 internal antenna of photosystem II in Hordeum vulgare L.
1Dipartimento di Scienze Ambientali e della Vita, Università del Piemonte Orientale ‘Amedeo Avogadro’, Via Bellini 25/G, I-15100 Alessandria, Italy
2Dipartimento di Biologia, Università di Padova, Via Bassi 58/B, I-35131 Padova, Italy
* To whom correspondence should be addressed. Fax: +39 0131 360390; E-mail: roberto.barbato{at}unipmn.it
Received 11 October 2004; Accepted 17 January 2005
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Abstract |
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As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll–protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.
Key words: Hordeum vulgare L., photosynthesis, photosystem II, thylakoid phosphoproteins
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Photosystem II (PSII) is a multiprotein–pigment complex which drives the light-induced oxidation of water, with concomitant reduction of a plastoquinone pool. At least 25 different polypeptides, 200 pigment molecules, and a number of inorganic metal ions (Mn, Fe, Cl, Ca) are assembled to form a functional PSII unit, which exists in the thylakoid membrane as a dimer (Hankamer et al., 1997
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A number of PSII subunits are reversibly phosphorylated by some thylakoid-bound kinases for which at least four different regulatory patterns have been described (Pursiheimo et al., 2003). Phosphoproteins are then dephosphorylated by chloroplast phosphatase(s) (Bennett, 1991). Thylakoid phosphoproteins include the main LHCII complex (Bennett, 1991) and CP29 (Bergantino et al., 1995) among the peripheral antenna proteins, and the D1, D2, CP43, and PsbH subunits among core proteins (Bennett, 1991). An additional 12 kDa protein has recently been identified which, depending on its phosphorylation/dephosphorylation state, may or may be not bound to the thylakoid membrane (Carlberg et al., 2003). The role of reversible phosphorylation is not completely undestood. In the case of Chlamydomonas reinhardtii, phosphorylation of LHCII by the Stt7 kinase (Depege et al., 2003) mediates state I–state II transition; as a consequence of phosphorylation, up to 80% of this antenna complex may be transferred to stroma lamellae, where it acts as an antenna for PSI. Even though homologues of the Stt7 kinase do exist in higher plants, other LHCII kinase(s), called the TAKs kinases, have been described (Snyders and Kohorn, 1999), but their role in state transitions has not been defined yet. In the case of core proteins, the functional meaning of phosphorylation is less clear. As an example, phosphorylation of D1 has been reported either to prevent (Koivuniemi et al., 1995) or to promote (Callahan et al., 1990) its light-induced turnover, whereas phosphorylation of other core proteins might have a role in the PSII repair cycle and in monomer/dimer interconversion (Baena-Gonzalez et al., 1999). Moreover, PSII proteins are not evenly phosphorylated in PSII cores, giving rise to different populations with different sets of phosphoproteins. As an example, cores fully phosphorylated in PsbH are not (or just weakly) phosphorylated at the level of D1 (Giardi et al., 1995). The meaning of this further heterogeneity is not known (see Giardi et al., 1995, for a discussion). A possible approach in understanding the role of phosphorylation of chlorophyll binding-proteins, consists, for a given protein, in the isolatation of both the phosphorylated and dephosphorylated forms, leading to the possibility of comparing their spectroscopic properties. This approach has been succesfully used with CP29 (Croce et al., 1996), and in this case phosphorylation was found to induce conformational change affecting the organization of pigments, possibly modifying its light-harvesting properties. As part of a project raised to characterize the role of phosphorylation of core PSII subunits, some data on CP43, that is the detection and the resolution of the phosphorylated and dephopshorylated forms of CP43, and their isolation in a native state in a homogeneous and highly purified form, is reported here.
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Materials and methods |
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Photosystem II preparation and fractionation
Barley (Hordeum vulgare L.) was grown in a growth chamber under standard conditions (Bergo et al., 2002
). Two-to-three week-old seedlings were harvested during the light phase of the day, and the oxygen-evolving PSII core was prepared as described by Ghanotakis et al. (1987). All buffers contained 5 mM NaF. Fractionation of PSII was carried out as described by Dekker et al. (1989). Fractions of interest were pooled, extensively dialysed against 0.03% dodecyl ß-D-maltoside in 0.5 mM MES pH 6.5, then concentrated using Centricon concentrators. A second PSII preparation was obtained by sucrose gradient centrifugation of the dodecyl ß-D-maltoside solubilized oxygen-evolving PSII core, as described by Barbato et al. (1989). This core preparation contained CP47, CP43, D1, D2, and cytochrome b-559 subunits. Isoelectrofocusing was carried out on a Sephadex bed using a 3.5–6.0 pH gradient, obtained by mixing 3.5–5.0 and 4.0–6.0 Anpholine (2% final concentration), with 0.03% dodecyl ß-D-maltoside as described by Bassi et al. (1988). Focusing was carried out at 4 °C for 14 h at a constant power of 3 W.
SDS-PAGE and immunoblotting
SDS-PAGE was carried out using either the Laemmli (1970) or the TRIS-SO4 method (Bassi et al., 1988). For limited proteolysis, SDS gels were stained with acid-free blue Coomassie (0.5% w/v Coomassie R-250 in 50% methanol), bands of interest were cut out from the gel and then loaded on a second SDS gel. Bands were overlaid with 10 µl of Arg-C protease (1 unit µl–1) dissolved in 62.5 mM TRIS-HCl pH 6.8, 0.1% SDS, 10% glycerol, and a trace of bromophenol blue. The current was switched on until the tracking dye reached the bottom of the stacker, afterwards electrophoresis was stopped for 3 h to allow proteolysis. Current was turned on and electrophoresis accomplished as usual. Proteins were blotted to a polyvinylidene difluoride membrane according to Dunn (1986) and the filters probed with antibodies either to authentic CP43 (Barbato et al., 1992) or antibodies to phosphothreonine-containing proteins (Zymed), as described by Bergo et al. (2002). Immunoreactions were detetcted with a chemiluminescence kit.
Other methods
The number of chlorophyll molecules bound to each CP43 popypeptide was calculated as described by Barbato et al. (1991), where the amount of the protein was determined by the Coomassie blue method of Ball (1986) with bovine serum albumin as the standard, and the corresponding amount of chlorophyll by the method of Wellburn (1994) after extraction in 80% acetone. As the first 14 residues of CP43 are cleaved off during post-translational maturation of the protein (Michel et al., 1988), a molecular weight of 50301 Da (corresponding to residues 15–473) was assumed for calculation. The contribution to molecular mass of any other post-translational modification was not considered. Measurements were performed in triplicate using two independent preparations.
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Results |
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Resolution of the phosphorylated and dephosphorylated form of CP43
In Fig. 1 the polypeptide composition of PSII membranes (lane 1) and oxygen-evolving PSII complexes (lane 2) is shown. The gel in Fig. 1 is a 10–20% acrylamide gradient containing 6 M urea run using the TRIS-glycine/TRIS-SO4 buffer system. In both preparations two distinct bands were observed at the level of CP43, thereafter referred to as 43p1 and 43p2 in order of increasing electrophoretic mobility. For preparative purposes, PSII cores were run in a 14 cm long 12.5% acrylamide/6 M urea TRIS-SO4 gel until the prestained 36 kDa marker (migrating just below the CP43 doublet) reached the bottom of the gel. In this condition the 43p1 and 43p2 bands were separated enough to be independently cut out from the gel with minimal cross-contamination. Proteins were then subjected to further analysis by re-electrophoresis, immunoblotting, and limited proteolysis with an Arg-C specific protease. As shown in Fig. 2, the antibody to CP43 recognized both 43p1 (Fig. 2A, lane 1) and 43p2 (Fig. 2A, lane 2) bands, whereas the antibody to phosphothreonine-containing proteins recognized the 43p1 band (Fig. 2B, lane 1), but not the 43p2 band (Fig. 2B, lane 2). Upon treatment with Arg-C protease, identical immunoreactive patterns were generated from proteolysis of the 43p1 and 43p2 bands (Fig. 2A, lanes 3, 4), with a main fragment of about 35 kDa, and some additional lower molecular weight polypeptides with masses in the range of 10–15 kDa. The antibody to phosphothreonine-containing proteins detected, among digests of the slow migrating form, a band at 35 kDa (the same as that detected by the antibody to CP43), a second digest at 30 kDa (not detected by the antibody to CP43), and two more fragments, at 11 kDa and 14 kDa, respectively. Of course, no proteolytic digests were detected from proteolysis of the 43p2 band.
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This finding strongly supports the possibility that the two bands were actually different forms of the same protein: the slower migrating one (the 43p1 band) was the phosphorylated form of CP43, whereas the faster migrating one (the 43p2 band) was the dephosphorylated form of CP43.
Resolution of different native forms of CP43
As the presence of the phosphorylated and dephosphorylated form of CP43 has been demonstrated, the possibility to isolate them in a native form was taken into account. As starting material, CP43-enriched fractions obtained by ionic exchange chromatography were used. Briefly, PSII cores were solubilized with 0.5% (w/v) dodecyl ß-D-maltoside, passed throught S-Sepharose and then adsorbed onto a Q-Sepharose column. As originally reported by Dekker et al. (1989), S-Sepharose retained mainly the PsbS/22 kDa subunit (Fig. 3, lane 1), besides a number of other unidentified proteins. The eluate was then loaded onto a Q-Sepharose column and eluted with a step gradient of MgSO4 (15 ml each of 0, 2.5, 10, 15, and 30 mM). Polypeptide composition of fractions was analysed by SDS–PAGE (Fig. 3). Fraction 1 (Fig. 3, lane 2, 0 mM MgSO4) contained CP43 polypeptide whereas fraction 2 (Fig. 3, lane 3, 2.5 mM MgSO4), contained, besides CP43, some CP47 and PsbO subunits. Fractions 3–5 (Fig. 3, lanes 4–6, 10, 25, and 30 mM MgSO4, respectively) contained, besides core proteins CP43 and CP47, some chl a/b-binding proteins (CP29 and, possibly, CP26). Fractions 1–3 (45 ml) were pooled, extensively dialysed, concentrated by centrifugation with Centricon concentrators (to 5 ml), and subjected to non-denaturing isoelectrofocusing using a pH gradient of 3.5–6.0. After focusing, seven green bands were resolved (not shown), whose polypeptide composition, determined by SDS–PAGE, is shown in Fig. 4 (lanes 4–10). Green bands with isoelectric points of 4.72 (Fig. 4, lane 4), 4.71 (Fig. 4, lane 5), and 4.60 (Fig. 4, lane 6) were characterized by the presence of a main polypeptide migrating at the level of CP43 (compare with Fig. 4, lane 1). It should be noted that the polypeptide found in the band with pI 4.60 had an electrophoretic mobility slightly lower than polypeptides found in bands with pI 4.70 and 4.71. Other green bands (Fig. 4, lanes 7–10) contained polypeptides with a molecular weight and pI typical of chlorophyll a/b-binding proteins, and their identity was not investigated further. However, the three bands with pIs of 4.72, 4.71, and 4.60, were analysed further by imunoblotting with antibodies to CP43 and phosphothreonine-containing proteins (Fig. 5). All three bands were recognized by the antibody to CP43 (Fig. 5A) whereas the antibody to phosphothreonine-containing proteins only recognized the band with the more acidic pI.
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Properties of phosphorylated and dephosphorylated forms of CP43
To gain additional information on the effect of phosphorylation, the number of chlorophyll molecules bound to each isoform of the CP43 pigment–protein complex, was determined. It was found that isoform with pI 4.72 binds 14.0±3.1 chlorophyll molecules, the one with pI 4.71, 12.6±2.8 chlorophyll molecules, and that with pI 4.60, 13.6±2.9 chlorophyll molecules, indicating that phosphorylation does not affect the number of bound pigments. These values are in line with those previously obtained with a different preparation of CP43 complexes (Barbato et al., 1991
) and similar to that reported from structural studies on cyanobacterial cores (Ferreira et al., 2004). Absorption spectra of the three bands were also recorded. As shown in Fig. 6, they were very similar, if not identical, with a maximum in the red at 670 nm and an asymmetric red band typical of this antenna complex (Zucchelli et al., 1994).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this paper it is shown that, in the thylakoid membranes of higher plants, the CP43 internal antenna of PSII exists in some different forms which may be separated by high resolution SDS-PAGE or, in a native form, by non-denaturing isoelectrofocusing. In denaturing conditions, the presence of a doublet at the level of CP43 has already been reported (Ghanotakis et al., 1987
; Barbato et al., 1989) and attributed to partial proteolytic degradation (Ghanotakis et al., 1987); alternatively, phosphorylation has been suggested as a possible source of heterogeneity (Bergo et al., 2002). Here, evidence for the latter possibility is provided. On the base of reactivity towards antibodies to phosphothreonine-containing proteins and authentic CP43, as well as limited proteolysis with Arg-C specific protease, the two bands are identified as the phosphorylated and dephosphorylated forms of CP43. Therefore, as in the case of D1 (Callahan et al., 1990) and CP29 (Croce et al., 1996), phosphorylation at a single threonine residue may give rise to an electrophoretic mobility shift, producing an increase in apparent molecular weight. This finding has the implication that the level of the phosphorylated and dephosphorylated CP43 can be established simply by high resolution SDS-PAGE, making their quantification in different experimental/environmental conditions much simpler and more reliable than before. As shown in Fig. 4, different forms of CP43 may also be resolved in a native state by non-denaturing isoelectrofocusing. In this case, three bands are resolved. The most acidic one is the phosphorylated form of CP43, as judged by its reactivity with anti-CP43 and anti-phosphothreonine-containing protein antibodies, and its electrophoretic mobility shift. Therefore, this band is identical to the 43p1 polypeptide detected by SDS–PAGE. The other two bands are identified as dephosphorylated forms of CP43, because of their positive immunoreactivity towards anti-CP43 antiserum and the absence of immunoreactivity to polyclonals against phosphothreonine-containing proteins, but that cannot be resolved by SDS-PAGE. Their differences are not clear at the moment. As CP43 is N-acetylated besides O-phosphorylated at its N-terminal threonine (Michel et al., 1988), a possibility is that one of the two dephosphorylated forms could also be deacetylated. The biochemical analyses, i.e. the number of chlorophylls per polypeptide and room temperature absorption spectra, do not show any substantial differences among the various CP43 complexes. A detailed spectroscopic study on isolated CP43 has been carried out by Groot et al. (1999). In this study, three different groups of chlorophyll molecules had been detected using a number of spectroscopic methods. The CP43 preparation used by Groot et al. (1999) was obtained with a method similar to that described here, with the main difference that the isoelectrofocusing step was not performed. Taking into account that once phosphorylated, this form of CP43 is also stable in the dark (Purshieimo et al., 2003), it is likely that such a study was carried out with a mixture of the two forms. Therefore, our method will be most useful when the optical properties of homogeneous populations of CP43 are to be compared, making it possible to understand whether or not, even in this chlorophyll-protein, phosphorylation affects pigment organization, as previously shown with CP29 (Croce et al. 1996). |
Acknowledgements |
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This work was supported by the Italian Minister of Research, FIRB (Fondo Investimento Ricerca di Base) n. RBAU01E3CX_007 to RB. Financial support from the University of Piemonte Orientale ‘Amedeo Avogadro’ and Associazione Territorio e Formazione (ATF) of Provincia di Alessandria is also acknowledged.
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