Occurrence of a Photosystem II polypeptide in non-photosynthetic membranes of cyanobacteria

Cytoplasmic and thylakoid membranes have been purified from the cyanobacteria Anacystis nidulans R2 and Phormidium laminosum by sucrose density gradient centrifugation. Probing of Western blots of proteins from these purified membrane fractions with antibodies directed against the 33 kDa polypeptide of Photosystem II from pea indicates that this protein is present in both the thylakoid and cytoplasmic membranes, rather than just the thylakoid membranes. This has been confirmed by immunogold labelling of cells. Oxygen evolution assays have been used to show that the 33 kDa polypeptide is not assembled into a functional Photosystem II complex in the cytoplasmic membranes. This may be due to the absence of other Photosystem II components.     

Massive breakdown of the Photosystem II polypeptides in a mutant of the cyanobacterium Synechocystis sp. PCC 6803

Degradation of the D1 protein of the Photosystem II (PS II) complex was studied in the Fad6/desA::Km^r mutant of a cyanobacterium Synechocystis sp. PCC 6803. The D1 protein of the mutant was degraded during solubilization of thylakoid membranes with SDS at 0°C in darkness, giving rise to the 23 kDa amino-terminal and 10 kDa carboxy-terminal fragments. Moreover, the D2 and CP43 proteins were also degraded under such conditions of solubilization. Degradation of the D2 protein generated 24, 17 and 15.5 kDa fragments, and degradation of the CP43 protein gave rise to 28, 27.5, 26 and 16 kDa fragments. The presence of Ca²⁺ and urea protected the D1, D2 and CP43 proteins against degradation. Degradation of the D1 protein was also inhibited by the presence of a serine protease inhibitor suggesting that the putative protease involved belonged to the serine class of proteases. The protease had the optimum activity at pH 7.5; it was active at low temperature (0°C) but a brief heating (65°C) during solubilization destroyed the activity. Interestingly, the protease was active in isolated thylakoid membranes in complete darkness, suggesting that proteolysis may be a non-ATP-dependent process. Proteolytic activity present in thylakoid membranes seemed to reside outside of the PS II complex, as demonstrated by the 2-dimensional gel electrophoresis. These results represent the first (in vitro) demonstration of strong activity of a putative ATP-independent serine-type protease that causes degradation of the D1 protein in cyanobacterial thylakoid membranes without any induction by visible or UV light, by active oxygen species or by any chemical treatments.     

Photosystem II characteristics of a constructedSynechocystis 6803 mutant lacking synthesis of the D1 polypeptide

Photosystem II (PSII) composition was studied in a mutant of the cyanobacteriumSynechosystis 6803 in which synthesis of the reaction center polypeptide D1 has been inactivated. The mutant thylakoids had lost also the other reaction center polypeptide D2 and the chlorophylla-binding protein CP47. Cytochromeb559 and the chlorophylla-binding protein CP43 accumulated to almost wild-type amounts in mutant thylakoids. Also the 33 kDa polypeptide involved in water oxidation was present and membrane-bound in mutant thylakoids. The intrinsic 22 kDa polypeptide, so far known only from plants, was detected both in wild-type and mutant thylakoids.     

A chloroplast membrane lacking Photosystem I. Changes in unstacked membrane regions

The structure and polypeptide composition of the photosynthetic membrane of a mutant of maize has been investigated. The thylakoid membranes of the mutant plants are deficient in Photosystem I activity, although Photosystem II is at near normal levels. SDS polyacrylamide gel electrophoresis of thylakoid membranes from the mutant shows them to be deficient in two polypeptide bands which have been associated with Photosystem I. Freeze-fracture studies of the membrane show that the absence of these polypeptides is associated with a measurable reduction in particle diameter on the unstacked protoplasmic fracture face. This fracture face is derived from the splitting of membranes in unstacked regions of the thylakoid membrane system. It is suggested that in membranes stacked by salts in vitro, Photosystem I activity may be confined to this region.     

Different degrees of phosphorylation and lateral mobility of two polypeptides belonging to the light-harvesting complex of Photosystem II

Several studies have shown that a subpopulation of the light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC-II) migrates from the appressed to the stroma-exposed thylakoids upon its phosphorylation. In this study we have analyzed the 27 and 25 kDa apopolypeptides of LHC-II, resolved by two-dimensional electrophoresis, with respect to their relative abundance and phosphorylation in thylakoids and subfractions derived from appressed or stroma-exposed thylakoid regions. The results show that the two polypeptides are heterogeneous with respect to both phosphate incorporation and degree of lateral migration. In intact thylakoids, the specific phosphorylation of the 25 kDa polypeptide exceeded that of the 27 kDa polypeptide by a factor of 3. Following phosphorylation, the 25 kDa polypeptide of the stroma lamellae showed as much as 4–5-times higher specific phosphorylation compared to the 27 kDa polypeptide. Moreover, there was a time-dependent increase in the amount of the 25 kDa polypeptide relative to the 27 kDa polypeptide in the stroma-exposed thylakoids. These results demonstrate a different polypeptide composition of the LHC-II tightly bound to Photosystem II and the free pool of LHC-II able to migrate laterally upon phosphorylation. The mobile pool of LHC-II is estimated to have two 27 kDa polypeptides for every 25 kDa polypeptide, while the ratio in the immobile pool is 4:1.     

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaCl, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem II membranes that retain the 33 kDa protein.Abbreviations Chl chlorophyll - HQ hydroquinone - MES 2(N-morpholino)ethanesulfonic acid - PS II Photosystem II - Tris 2-amino-2-hydroxymethylpropane-1,3-diol     

A chloroplast membrane lacking Photosystem II. Thylakoid stacking in the absence of the Photosystem II particle

The polypeptide composition and membrane structure of a variegated mutant of tobacco have been investigated. The pale green mutant leaf regions contain chloroplasts in which the amount of membrane stacking has been reduced (although not totally eliminated). The mutant membranes are almost totally deficient in Photosystem II when compared to wild-type chloroplast membranes, but still show near-normal levels of Photosystem I activity. The pattern of membrane polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows several differences between mutant and wild-type membranes, although the major chlorophyll-protein complexes described in many other plant species are present in both mutant and wild-type samples. Freeze-fracture analysis of the internal structure of these photosynthetic membranes shows that the Photosystem II-deficient membranes lack the characteristic large particle associated with the E fracture face of the thylakoid. These membranes also lack a tetramer-like particle visible on the inner (ES) surface of the membrane. The other characteristics of the photosynthetic membrane, including the small particles observed on the P fracture faces in both stacked and unstacked regions, and the characteristic changes in the background matrix of the E fracture face which accompany thylakoid stacking, are unaltered in the mutant. From these and other observations we conclude that the large (EF and ES) particle represents an amalgam of many components comprising the Photosystem II reaction complex, that the absence of one or more of its components may prevent the structure from assembling, and that in its absence, Photosystem II activity cannot be observed.     

Does the trimeric form of the Photosystem 1 reaction center of cyanobacteria in vivo exist?

When isolating Photosystem 1 from the thylakoid membranes of cyanobacteria, a multiplicity of photosystem complexes can be encountered, which has not yet been observed in any other phototrophic organisms: After solubilisation of thylakoid membranes with detergents, trimeric, dimeric and monomeric forms of Photosystem 1 can be separated. The question must now be answered, which of the stable Photosystem 1 forms is the functional form in vivo-monomeric or trimeric? The two possibilities are discussed, though we mainly present arguments for the existence of the trimeric form of Photosystem 1 in cyanobacterial thylakoid membranes.     

Selective extraction of CP 26 and CP 29 proteins without affecting the binding of the extrinsic proteins (33, 23 and 17 kDa) and the DCMU sensitivity of a Photosystem II core complex

A highly purified oxygen evolving Photosystem II core complex was isolated from PS II membranes solubilized with the non-ionic detergent n-octyl--D-thioglucoside. The three extrinsic proteins (33, 23 and 17 kDa) were functionally bound to the PS II core complex. Selective extraction of the 22, 10 kDa, CP 26 and CP 29 proteins demonstrated that these species are not involved in the binding of the extrinsic proteins (33, 23 and 17 kDa) or the DCMU sensitivity of the Photosystem II complex.Abbreviations Chl chlorophyll - DCBQ 2,6-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - LHC light-harvesting complex - MES 2-(N-morpholino)ethanesulfonic acid - OGP n-octyl--d-glucoside - OTG n-octyl--d-thioglucoside - PAGE polyacrylamide gel electrophoresis - PS II Photosystem II - SDS sodium dodecyl sulfate     

Organization of chlorophyll-protein complexes of Photosystem I in Chlamydomonas reinhardii

The polypeptide pattern, chlorophyll-protein complexes, fluorescence emission spectra and light intensity required for saturation of electron flow via Photosystem (PS) II and PS I in a pale-green photoautotrophic mutant, y-lp, were compared to those of the parent strain, Chlamydomonas reinhardii y-1 cells. The mutant exhibits a 686 nm fluorescence yield at 25°C and 77 K 2–6-fold higher than that of the parent strain cells, and is deficient in thylakoid polypeptides 14, 17.2, 18 and 22 according to the nomenclature of Chua (Chua, N.-H. (1980) Methods Enzymol. 60C, 434–446). All chlorophyll-protein complexes ascribed to PS II and the CP I complex were present in both type of cells. However, a chlorophyll-protein complex CP Ia containing — in the parent strain — the 66–68 kDa polypeptides of CP I and the four above-mentioned polypeptides, was absent in the mutant. It was previously reported that a chlorophyll-protein complex, CP O, obtained from C. reinhardii contains five polypeptides, namely, 14, 15, 17.2, 18 and 22 (Wollman, F.A. and Bennoun, P. (1982) Biochim. Biophys. Acta 680, 352–360). A CP O-like complex was present also in the mutant y-lp cells but it contains only one polypeptide, 15. Energy transfer from PS II to PS I was not impaired in the mutant, although a 4-fold higher light intensity was required for the saturation of PS I electron flow in the y-lp cells as compared with the parent strain. No difference was found in the light saturation curves for PS II activity between the mutant and parent strain cells. Based on these and additional data (Gershoni, J.M., Shochat, S., Malkin, S. and Ohad, I. (1982) Plant Physiol. 70, 637–644), it is concluded that the chlorophyll-protein complexes of PS I in Chlamydomonas comprise a reaction center-core antenna complex containing the 66–68 kDa polypeptides (CP I), a connecting antenna consisting of four polypeptides (14, 17.2, 18 and 22), and a light-harvesting antenna containing one polypeptide, 15. These appear to be organized as a complex, CP Ia. The interconnecting antenna is deficient in the y-lp mutant and thus the CP Ia complex is unstable and energy is not transferred from CP O to CP I. The effective cross-section of PS I antenna is thus reduced and a high fluorescence is emitted at 686 nm.     

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum. Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria (Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl₂ or MgCl₂ inhibited O₂ evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O₂ evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O₂ evolution in parallel.     

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47–56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O₂ evolving activity without addition of CaCl₂ than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.Abbreviations PS photosystem - chl chlorophyll - LHCP light harvesting chlorophyll a/b protein complex - WT wild type - OEE1, OEE2 and OEE3 oxygen evolution enhancing complex of 29–36 kDa, 21–24 kDa and 16–18 kDa, respectively     

The chlorophyll-protein complexes of Acetabularia. A novel chlorophyll ab complex which forms oligomers

(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll $\text{a}\text{b}$ light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl $\text{a}\text{b}$ ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.     

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.     

The Water Oxidation Complex of Chlamydomonas: Accumulation and Maturation of the Largest Subunit in Photosystem II Mutants

Photosystem II particles of Chlamydomonas reinhardtii contain three extrinsic polypeptides of 29, 20, and 16 kilodaltons, whose functions are incompletely defined. We prepared a monospecific polyclonal antibody against the 29 kilodalton protein and determined that it also specifically recognizes a protein of approximately 33 kilodaltons in thylakoid membrane fractions of several vascular plants, eukaryotic algae, and a cyanobacterium. The cross-reacting 33 kilodalton protein of pea was removed from inverted thylakoid vesicles by CaCl₂ washes demonstrating the structural relationship between the Chlamydomonas polypeptide and the largest subunit of the water oxidation complex of vascular plants. Functional identity of the Chlamydomonas polypeptide was confirmed by antibody inhibition of O₂ evolution in inverted pea vesicles. In contrast to wild-type cells, only low levels of the 29 kilodalton polypeptide are recovered with purified thylakoid membranes of the mutants examined. However, we show that the mature form of the 29 kilodalton polypeptide accumulates to wild-type levels in whole cell extracts of photosystem II deficient mutants and a water oxidation mutant of Chlamydomonas. Impaired membrane assembly has no effect on the maturation or stability of this component of the multi-subunit water oxidation complex.     

Biochemical Characterization of Photosystem II Antenna Polypeptides in Grana and Stroma Membranes of Spinach

The photosystem (PS) II antenna system comprises several biochemically and spectroscopically distinct complexes, including light-harvesting complex II (LHCII), chlorophyll-protein complex (CP) 29, CP26, and CP24. LHCII, the most abundant of these, is both structurally and functionally diverse. The photosynthetic apparatus is laterally segregated within the thylakoid membrane into PSI-rich and PSII-rich domains, and the distribution of antenna complexes between these domains has implications for antenna function. We report a detailed analysis of the differences in the polypeptide composition of LHCII, CP29, and CP26 complexes associated with grana and stroma thylakoid fractions from spinach (Spinacia oleracea L.), making use of a very high-resolution denaturing gel system, coupled with immunoblots using monospecific antibodies to identify specific antenna components. We first show that the polypeptide composition of the PSII antenna system is more complex than previously thought. We resolved at least five type I LHCII apoproteins and two to three type II LHCII apoproteins. We also resolved at least two apoproteins each for CP29 and CP26. In state 1-adapted grana and stroma thylakoid membranes, the spectrum of LHCII apoproteins is surprisingly similar. However, in addition to overall quantitative differences, we saw subtle but reproducible qualitative differences in the spectrum of LHCII apoproteins in grana and stroma membrane domains, including two forms of the major type II apoprotein. The implications of these findings for models of PSII antenna function in spinach are discussed.     

Energy transfer for low temperature fluorescence in PS II mutant thylakoids

The Chl-protein complexes of three maize (Zea mays L.) mutants and one barley (Hordeum vulgare L.) mutant were analyzed using low temperature Chl fluorescence emissions spectroscopy and LDS-polyacrylamide gel electrophoresis. The maize mutants hcf-3, hcf-19, and hcf-114 all exhibited a high Chl fluorescence (hcf) phenotype indicating a disruption of the energy transfer within the photosynthetic apparatus. The mutations in each of these maize mutants affects Photosystem II. The barley mutant analyzed was the well characterized Chl b-less mutant chlorina-f2, which did not exhibit the hcf phenotype. Chlorina-f2 was used because no complete Chl b-less mutant of maize is available. Analysis of hcf-3, hcf-19, and hcf-114 revealed that in the absence of CP43, LHC II can still transfer excitation energy to CP47. These results suggest that in mutant membranes LHC II can interact with CP47 as well as CP43. This functional interaction of LHC II with CP47 may only occur in the absence of CP43, however, it is possible that LHC II is positioned in the thylakoid membranes in a manner which allows association with both CP43 and CP47.Abbreviations hcf high chlorophyll fluorescence - LDS lithium dodecyl sulfate - LHC II light-harvesting complex of Photosystem II - LHC I light-harvesting complex of Photosystem I - CPIa chlorophyll-protein complex consisting of LHC I and the PS I core complex - CPI chlorophyll-protein complex consisting of the PS I core complex - CP47 47 kDa chlorophyll-protein of the Photosystem II core - CP43 43 kDa chlorophyll-protein of the Photosystem II core - CP29 29 kDa chlorophyll-protein of Photosystem II - CP26 26 kDa chlorophyll-protein of Photosystem II - CP24 24 kDa chlorophyll-protein of Photosystem II - fp free pigments     

A new chlorophyll-protein complex related to Photosystem I in Chlamydomonas reinhardii

A new chlorophyll-protein complex, CP O, was isolated from Chlamydomonas reinhardii using lithium dodecyl sulfate polyacrylamide gel electrophoresis run at 4°C. A similar complex is recovered using Triton/digitonin solubilization of thylakoid membranes of the F54-14 mutant lacking in CP I and ATPase. CP O is enriched in long-wavelength chlorophyll a and contains five polypeptides (27.5, 27, 25, 23 and 19 kDa). Its 77 K fluorescence emission spectrum peaks at 705 nm while CP II have an emission maximum at 682 and 720 nm, respectively. Comparison of the polypeptide pattern of the wild type and AC40 mutant of C. reinhardii shows that the five CP O polypeptides are specifically lacking in the mutant. Although the 77 K emission originating from the Photosystem (PS) I pigments is lower in the mutant than in the wild type, the two spectra show the same peaks at 686, 694 and 717 nm. However, comparison of the 77 K emission spectrum of the F14 mutant lacking in CP I with that of the double mutant AC40-14 lacking in CP I and CP O shows the absence in the latter of the large emission band peaking at 707 nm. The 707 nm emission is thought to arise from some PS I antennae and is quenched in the wild type by the presence of PS I traps located in CP I. We conclude that CP O is a part of the PS I antenna in C. reinhardii which controls the 707 nm fluorescence emission.     

Electron microscopy in structural studies of Photosystem II

Various techniques of electron microscopy (EM) such as ultrathin sectioning, freeze-fracturing, freeze-etching, negative staining and (cryo-)electron crystallography of two-dimensional crystals have been employed, since now, to obtain much of the structural information of the Photosystem II (PS II) pigment–protein complex at both low and high resolution. This review summarizes information about the structure of this membrane complex as well as its arrangement and interactions with the antenna proteins in thylakoid membranes of higher plants and cyanobacteria obtained by means of EM. Results on subunit organization, with the emphasis on the proteins of the oxygen-evolving complex (OEC), are compared with the data obtained by X-ray crystallography of cyanobacterial PS II. This revised version was published online in August 2006 with corrections to the Cover Date.     

Organisation of Photosystem I and Photosystem II in red alga Cyanidium caldarium: encounter of cyanobacterial and higher plant concepts

Structure and organisation of Photosystem I and Photosystem II isolated from red alga Cyanidium caldarium was determined by electron microscopy and single particle image analysis. The overall structure of Photosystem II was found to be similar to that known from cyanobacteria. The location of additional 20 kDa (PsbQ') extrinsic protein that forms part of the oxygen evolving complex was suggested to be in the vicinity of cytochrome c-550 (PsbV) and the 12 kDa (PsbU) protein. Photosystem I was determined as a monomeric unit consisting of PsaA/B core complex with varying amounts of antenna subunits attached. The number of these subunits was seen to be dependent on the light conditions used during cell cultivation. The role of PsaH and PsaG proteins of Photosystem I in trimerisation and antennae complexes binding is discussed.