Two-dimensional crystals of photosystem II: biochemical characterization, cryoelectron microscopy and localization of the D1 and cytochrome b559 polypeptides.

Photosystem II (PS II) is a photosynthetic reaction center found in higher plants which has the unique ability to evolve oxygen from water. Several groups have formed two-dimensional PS II crystals or have isolated PS II complexes and studied them by electron microscopy and image analysis. The majority of these specimens have not been well characterized biochemically and have yielded relatively low resolution two-dimensional projection maps with a variety of unit cell sizes. We report the characterization of the polypeptide and lipid content of tubular crystals of PS II. The crystals contain the reaction center core polypeptides D1, D2, cytochrome b559, as well as the chlorophyll-binding polypeptides (CP) CP47, CP43, CP29, CP26, CP24, and CP22. The lipid composition was similar to the lipids found in the stacked portion of thylakoids. We also report a 2.0-nm resolution projection map determined by electron microscopy and image analysis of frozen, hydrated PS II crystals. This projection map includes information on the portion of the complex buried in the lipid bilayer. The unit cell is a dimer with unit vectors of 17.0 and 11.4 nm separated by an angle of 106.6 degrees. In addition, Fab fragments against D1 and cytochrome b559 were used to localize those two polypeptides, and thus the reaction center, within the PS II complex. The results indicate that D1 and cytochrome b559 are found within one of the heaviest densities of the monomeric unit.

Identification of type 1 and type 2 light-harvesting chlorophyll a/b-binding proteins using monospecific antibodies.

The amino acid sequences of more than 40 apoproteins of the light-harvesting complex associated with Photosystem II (LHC II) of various plants have been deduced by sequencing their corresponding genes. These highly conserved sequences fall into two major categories, type 1 and type 2, that differ mainly in a small number of domains close to the N-terminus. We have made polyclonal, monospecific antibodies against synthetic peptides corresponding to the most unique sequence domains of the N-terminal regions of type 1 and type 2 LHC II apoproteins, using sequences derived from petunia genes. On Western blots our anti-type 1 and 2 antibodies crossreact with light-harvesting proteins of petunia, tomato, spinach and several other plants. By using a new gel-system based on ammediol (2-amino-2-methyl-1,3-propanediol), we are able to resolve up to eight LHC II apoproteins. On petunia, tomato and spinach blots the anti type 1 antibodies bind to two or more of the higher molecular weight LHC II polypeptides, whereas the anti type 2 antibodies recognize very specifically only one or two of the lower molecular weight LHC-proteins. In all plants studied, the type 1 LHC II apoproteins are more numerous and span a greater size range than the type 2 apoproteins. This is consistent with the smaller number of type 2 LHC II CAB genes that have been discovered to date.

D1 protein degradation during photoinhibition of intact leaves. A modification of the D1 protein precedes degradation.

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.

New evidence suggests that the initial photoinduced cleavage of the D1-protein may not occur near the PEST sequence.

When isolated reaction centres of photosystem 2 from pea or wheat are exposed to photoinhibitory illumination in the presence of an electron acceptor, breakdown products of the D1-protein are observed having molecular masses ranging from about 24 to 10 kDa. By using antibodies raised to the C-terminal or N-terminal portions of D1 it was shown that the major breakdown fragment of 24 kDa was derived from the C-terminus. This conclusion was supported by phosphorylation studies and from the digestion pattern obtained by lysine specific endoprotease-induced proteolysis. The complementary N-terminal breakdown fragment was found to have an apparent molecular mass of 10 kDa. The implications of these data are discussed in terms of the possible relationship between the 24 kDa C-terminal fragment and the 23.5 kDa breakdown fragment detected in vivo by Greenberg et al. [1987, EMBO J. 6, 2865-2869] and it is suggested, based on limited proteolysis using papain, that the latter may not be derived from the N-terminus as previously thought but also originates from the C-terminus.

The quenching of photosystem II fluorescence does not protect the D1 protein against light induced degradation in thylakoids.

In spinach thylakoids, the quenching of the singlet excited state in the photosystem II antenna by m-dinitrobenzene does not change the rate of the light induced degradation of the D1 reaction centre protein and offers only limited protection against photoinhibition itself. These results are discussed in terms of the role of non-photochemical quenching as a photoprotective strategy.

pH6 antigen (PsaA protein) of Yersinia pestis, a novel bacterial Fc-receptor.

It was found that recombinant pH6 antigen (rPsaA protein) forming virulence-associated fimbriae on the surface of Yersinia pestis at pH 6.7 in host macrophage phagolysosomes or extracellularly in abscesses such as buboes, is a novel bacterial Fc-receptor. rPsaA protein displays reactivity with human IgG1, IgG2 and IgG3 subclasses but does not react with rabbit, mouse and sheep IgG.

Comparative immunological detection of lipids and carotenoids on peptides of photosystem I from higher plants and cyanobacteria.

Photosystem I preparations were obtained from wild-type tobacco Nicotiana tabacum var. JWB, three chlorophyll-deficient tobacco mutants: Su/su, Su/su var. Aurea and yellow-green leaf patches of the variegated mutant NC 95, Spinacia oleracea and furthermore from the mesophilic cyanobacterium Synechococcus PCC 7942 and the thermophilic cyanobacterium Synechococcus sp.. Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for detection of bound lipids and carotenoids according to the Western blot procedure to nitrocellulose membranes. The PS I preparations from the Nicotiana tabacum species and spinach consist of the core complex and the LHCP I complex, the latter containing, however, traces of the LHCP II polypeptides. The core complex consists of the two core peptides with the apparent molecular mass of 66 kDa each and peptides with molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa. The LHCP I complex contains 4 subunits with molecular masses of 28, 26, 25 and 24 kDa. The PS I preparations of the two mutants Su/su and Su/su var. Aurea contain as impurities traces of the core peptides (D1/D2) and the two chlorophyll-binding peptides (CP43/CP47) of photosystem II. The PS I preparation from the mesophilic and thermophilic cyanobacterium consists of the two core peptides with the apparent molecular mass of 66 kDa and peptides with molecular masses of 16, 14 and 10 kDa. The peptides of the PS I preparations were characterized by specific PS I, CP I and LHCP I antisera. The antiserum to the PS I complex reacts in the Western blot with the homologous peptides of PS I from higher plants, but only with the CP I complex from the two cyanobacteria. In comparative studies with PS II from higher plants the PS I antiserum reacts with the LHCP II complex as expected. The antiserum to the CP I complex reacts only with the 66 kDa peptides of PS I from all objects. There is no cross reaction with the 66 kDa peptides (heterodimer of the D1/D2 peptides) of PS II. The antiserum to the LHCP I complex reacts only with the four LHCP I peptides of PS I from higher plants and as expected with the LHCP II of PS II: Because cyanobacteria do not have LHCP complexes, there is no reaction with the LHCP I antiserum. By means of polyclonal monospecific antisera to lipids it was shown by Western blot procedure that only two lipid species are bound to PS I peptides. The galactolipid monogalactosyldiglyceride is bound to the CP I complex of the Nicotiana tabacum species, spinach and the two cyanobacteria as well as to the LHCP I complex of the higher plants. The phospholipid phosphatidylglycerol is only associated with the CP I complex of the analyzed higher plants and cyanobacteria. With polyclonal monospecific antisera to carotenoids it was demonstrated that beta-carotene, lutein, neoxanthin and zeaxanthin are associated with the CP I complex of the higher plants and the cyanobacteria analyzed. Violaxanthin is also bound to the CP I complex of the two cyanobacteria, whereas it is bound together with neoxanthin to the LHCP I complex of the higher plants.

Cloning and nucleotide sequence of a cDNA encoding a major fucoxanthin-, chlorophyll a/c-containing protein from the chrysophyte Isochrysis galbana: implications for evolution of the cab gene family.

We investigated the primary structure of a cDNA encoding a light-harvesting protein from the marine chrysophyte Isochrysis galbana. Antibodies raised against the major fucoxanthin, chlorophyll a/c-binding light-harvesting protein (FCP) of I. galbana were used to select a cDNA clone encoding one of the FCP apoproteins. The nucleic acid and deduced amino acid sequences reveal conserved regions within the first and third transmembrane spans with Chl a/b-binding proteins and with FCPs of another chromophyte. However, the amino acid identity between I. galbana FCP and other cab genes of FCPs is only ca. 30%. Phylogenetic analyses demonstrated that the FCP genes of both diatoms and chrysophytes sequenced to date are more closely related to cab genes encoding LHC I, CP 29, and CP 24 of higher plants than to cab genes encoding LHC II of chlorophytes. We propose that LHC I, CP 24 and CP 29 and FCP might have originated from a common ancestral chl binding protein and that the major LHC II of Chl a/b-containing organisms arose after the divergence between the chromophytes and the chlorophytes.

Chlorophyll a/b binding (CAB) polypeptides of CP29, the internal chlorophyll a/b complex of PSII: characterization of the tomato gene encoding the 26 kDa (type I) polypeptide, and evidence for a second CP29 polypeptide.

CP29, the core chlorophyll a/b (CAB) antenna complex of Photosystem II (PSII), has two nuclear-encoded polypeptides of approximately 26 and 28 kDa in tomato (Lycopersicon esculentum). Cab9, the gene for the Type I (26 kDa) CP29 polypeptide was cloned by immunoscreening a tomato leaf cDNA library. Its identity was confirmed by sequencing tryptic peptides from the mature protein. Cab9 is a single-copy gene with five introns, the highest number found in a CAB protein. In vitro transcription-translation gave a 31 kDa precursor which was cleaved to about 26 kDa after import into isolated tomato chloroplasts. The Cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides, which define the members of this extended gene family. Outside of the conserved regions, it is only slightly more closely related to other PSII CABs than to PSI CABs. Sequence analysis of tryptic peptides from the Type II (28 kDa) CP29 polypeptide showed that it is also a member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach. All members of the CAB family have absolutely conserved His, Gln and Asn residues which could ligate the Mg atoms of the chlorophylls, and a number of conserved Asp. Glu, Lys and Arg residues which could form H-bonds to the polar groups on the porphyrin rings. The two conserved regions comprise the first and third predicted trans-membrane helices and the stroma-exposed segments preceding them.

Topological study of PSI-A and PSI-B, the large subunits of the photosystem-I reaction center.

The core of the photosystem-I reaction center is formed by polypeptides PSI-A and PSI-B, the products of the homologous psaA and psaB genes. Based on hydropathy analyses, models have been proposed for the folding of these polypeptide chains in the membrane [Fish, L. E., Kück, U. & Bogorad, L. (1985), in Molecular biology of the photosynthetic apparatus, pp. 111-120, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY]. To test these models, we have tried to identify regions of PSI-A that are exposed to the surrounding medium, on the stromal or lumenal surface of the membrane. Immunogold labeling of thylakoid vesicles, with antibodies to synthetic peptides, shows that residues 413-421 of PSI-A are exposed on the stromal surface of the membrane, and that the accessibility of this region is enhanced by NaSCN treatment, which removes extrinsic polypeptides. This treatment also enhances a trypsin-cleavage site which may lie just after residues 413-421. Immunogold labeling also indicates that residues 371-379 and 497-505 are exposed on the lumenal surface. These results establish the conformation of the central portion of the polypeptide. Assuming that the transmembrane regions are correctly predicted by the 11-helix model, the N-terminal domain, as well as the conserved region proposed to bind the iron-sulfur center FX, would be expected to be on the stromal surface.

The atypical chlorophyll a/b/c light-harvesting complex of Mantoniella squamata: molecular cloning and sequence analysis.

cDNA species encoding precursor polypeptides of the chlorophyll a/b/c light-harvesting complex (LHC) of Mantoniella squamata were cloned and sequenced. The precursor polypeptides have molecular weights of 24.2 kDa and are related to the major chlorophyll a/b polypeptides of higher plants. Southern analysis showed that their genes belong to the nuclear encoded Lhc multigene family; the investigated genes most probably do not contain introns. The chlorophyll a/b/c polypeptides contain two highly conserved regions common to all LHC polypeptides and three hydrophobic alpha-helices, which span the thylakoid membrane. The first membrane-spanning helix, however, is not detected by predictive methods: its atypical hydrophilic domains may bind the chlorophyll c molecules within the hydrophobic membrane environment. Homology to LHC II of higher plants and green algae is specifically evident in the C-terminal region comprising helix III and the preceding stroma-exposed domain. The N-terminal region of 29 amino acids resembles the structure of a transit sequence, which shows only minor similarities to those of LHC II sequences. Strikingly, the mature light-harvesting polypeptides of M. squamata lack an N-terminal domain of 30 amino acids, which, in higher plants, contains the phosphorylation site of LHC II and simultaneously mediates membrane stacking. Therefore, the chlorophyll a/b/c polypeptides of M. squamata do not exhibit any light-dependent preference for photosystem I or II. The lack of this domain also indicates that the attractive forces between stacked thylakoids are weak.

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody.

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23-29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector lambda ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosystem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.

Green plant photosystem I binds light-harvesting complex I on one side of the complex.

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.

Identification of the PufQ protein in membranes of Rhodobacter capsulatus.

The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (delta RC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-beta-D-glucopyranoside, a procedure which solubilized the reaction center and light-harvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.

Purification and membrane topology of PSI-D and PSI-E, two subunits of the photosystem I reaction center.

Structural studies have been conducted on polypeptides PSI-D and PSI-E, which are extrinsic but firmly bound to the photosystem I reaction center. These subunits are predicted to be involved in the correct interaction with soluble electron acceptor(s), like ferredoxin. We designed an original method to extract both polypeptides directly from thylakoid membranes and to purify them: a stepwise extraction with NaSCN followed by size fractionation and reverse-phase HPLC. Investigation of the in situ topology of PSI-D and PSI-E was undertaken using monoclonal antibody binding, controlled proteolysis, peptide sequencing and electron microscopy. The precise identification of numerous proteolytic sites indicates that the entire N-terminal regions of PSI-E (up to Glu15) and PSI-D (up to Lys15) are exposed to the medium. Partial mapping of the exposed epitopes was possible using purified fragments of each polypeptide. In the case of PSI-E, this mapping confirmed the accessibility of the N-terminal part, and suggested the need for another exposed sequence, probably located after Met39 in the second half of the protein. For PSI-D, this mapping revealed that the sequence between Met74 and Met140, including the most basic amino acid clusters, is also partly accessible. These experiments provide the first detailed informations, although still partial, on the topology of these polypeptides. They give a preliminary basis for hypotheses concerning the sites of interaction with the soluble counterparts.

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803.

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, FA and FB. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700+ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to FA and FB were absent in the mutant strain, but a reversible Fx signal was present with broad resonances at g = 2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g = 2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of FA- and FB-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700+ [FA/FB]- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to Fx.

Nuclear localization of the Arabidopsis blue light receptor cryptochrome 2.

The cryptochrome blue light photoreceptor family of Arabidopsis thaliana consists of two members, CRY1 and CRY2 (PHH1). CRY2 contains a putative nuclear localization signal (NLS) within its C-terminal region. We examined whether CRY2 is localized in the nucleus and whether the C-terminal region of CRY2 is involved in nuclear targeting. Total cellular and nuclear protein extracts from Arabidopsis were subjected to immunoblot analysis with CRY2-specific antibodies. Strong CRY2 signals were obtained in the nuclear fraction. Fusion proteins consisting of the green fluorescent protein (GFP) and different fragments of CRY2 were expressed in parsley protoplasts and the localization of the fusion proteins was determined by fluorescence and confocal laser scanning microscopy. GFP-fusions containing the entire CRY2 protein or its C-terminal region were found exclusively in the nucleus. We conclude from these results that CRY2 is localized in the nucleus and that nuclear localization is mediated by the C-terminal region of CRY2.