Article

Topography of the Protein Complexes of the Chloroplast Thylakoid Membrane : Studies of Photosystem II using Pronase Digestion and Chemical Labeling

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Abstract

The accessibility of various Photosystem II (PSII)-associated polypeptides to the protease pronase and the chemical modifier trinitrobenzene-sulfonic acid (TNBS) has been investigated. Three polypeptides with apparent molecular weight of 32, 21, and 16 kilodaltons, known to be associated with O(2) evolution, are all resistant to pronase digestion and TNBS labeling in intact thylakoids. All the polypeptides in the isolated PSII preparation were labeled with TNBS while a different pattern of labeling was observed when the PSII complex was isolated from TNBS-modified thylakoids. Attempts to prepare PSII particles from pronase-treated thylakoids using the Triton X-100 solubilization method were unsuccessful. Pronase-treated thylakoids were probed with antisera against the chlorophyll proteins of PSII using immunoblotting techniques. This allowed for a positive identification of proteolytic fragments from the respective proteins. The results are discussed in relation to the transmembrane organization of PSII in spinach thylakoids.

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... The prediction of a large hydrophilic domain of approx. 200 residues between the two last putative helical segments seems to agree with the finding of a stromal exposed region in CP47 and CP43, as shown by pronase digestion, trinitrobenzenesulfonic acid (TNBS) labeling and immunoblotting analysis (Lam and Malkin, 1985), although the dimensions of the exposed regions estimated by these methods are significantly smaller than the large hydrophilic region predicted from the deduced amino acid sequence. The discrepancy may be related to the results of Bricker and Frankel (1987) showing that the extrinsic polypeptides of the oxygen evolving system protect CP47 from tryptic attack and decrease the ability to bind a monoclonal antibody directed against the CP47 apoprotein. ...
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An original immunoenzymatic screening method, based on the use of antigens labeled with the stable enzyme acetylcholinesterase (AChE, EC 3.1.1.7), is described. The high turnover of this enzyme results in a very sensitive detection of antibodies. In this method, monoclonal antibodies from the supernatants of hybridoma cultures are immobilized on a solid phase coated with anti-mouse immunoglobulins and react simultaneously with the appropriate antigen labeled with biotin molecules. In a second step, biotinylated acetylcholinesterase is in turn associated to the system via avidin interactions and subsequently detected by a colorimetric assay. The method appears more sensitive and easier to use than either the corresponding radioimmunological test using a 125I-iodinated antigen or the same type of enzymatic immunoassay performed with biotinylated horseradish peroxidase instead of biotinylated AChE. The combined use of microtiter plates, solid-phase separation, and colorimetric detection allows a high level of automation of the method which makes it very efficient to process a large number of samples. This technique has been successfully applied to the screening of monoclonal antibodies directed against peripheral proteins of the photosystem 1 (PS1) membrane complex in photosynthesis. A complete set of antibodies recognizing these PS1 components was selected. The same technique was also tested in competition immunoassays and appears to be a very precise and useful tool for quantifying PS1 polypeptides in different biological extracts, including sodium dodecyl sulfate-denatured membranes. This can be of special interest for studying the biogenesis of membrane complexes.
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A 2,900 base pair DNA segment of the spinach plastid chromosome which encodes the genes for the 44 kd chlorophyll a apoprotein and a "32 kd"-like protein of the photosystem II reaction center has been subjected to sequence and Northern blot analysis. The genes are located almost centrally in the large single-copy segment of the chromosome adjacent to the two genes for the P700 chlorophyll a apoproteins of the photosystem I reaction center. The DNA sequence reveals two uninterrupted protein-coding regions of 473 (44 kd chlorophyll a apoprotein) and 353 triplets ("32 kd"-like protein). The latter gene is strikingly similar to the gene for the herbicide-binding "32 kd" protein which maps some 30 kbp distant on the plastid chromosome. The two genes overlap by 50 base pairs but are read in different phases. They may be contranscribed and the RNA modified to give several discrete species ranging in size from 1.6 to 4.6 kb. A presumptive promoter site was only identified for the "32 kd"-like protein, while potential ribosome binding and transcription termination sites are found preceding and following both genes, respectively. The polypeptides possess a high content of hydrophobic amino acids, most of which appear to be clustered in transmembrane spans. The molecular weights of 51,785 (44 kd chlorophyll a apoprotein) and 39,465 ("32 kd"-like protein) derived from the deduced amino acid sequences are higher than the experimentally determined protein sizes. Amino acid codon usage for both genes is highly selective. Comparison of the chlorophyll a apoproteins of spinach reveals regions of sequence homology.
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We have determined the sequence of the spinach (Spinacia oleracea) chloroplast genes for the photosystem ∥ proteins, D2 and the 44 kd reaction-centre, chlorophyll a-binding protein, and for tRNASer (UGA).The 3′ end of the D2 gene overlaps the first 50 bp of the 5′ end of the gene for the 44 kd protein. Northern RNA hybridization analysis indicates the two genes are cotranscribed into a single 3.5 kb RNA. The predicted molecular weight of the 353-residue D2 protein is 39536 and that of the 473-residue 44 kd protein is 51816. Both proteins are hydrophobic containing at least five possible membrane-spanning domains. D2 shows significant homology to the 32 kd herbicide-binding protein (Zurawski et al, (1982) Proc. Natl. Acad. Sci. USA 79, 7699–7703), and parts of the 44 kd protein show obvious similarities to parts of the 51 kd reaction-centre, chlorophyll a-binding protein of photosystem ∥ (Morris and, Herrmann (1984) Nucleic Acids Res. 12, 2837–2850). The gene for tRNASer (UGA) which is on the opposite strand to and transcribed towards the photosvstem ∥ genes is 72% homologous with the corresponding Escherichia coli tRNASer
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Dataarepresented whichsuggest theexistence ofalight-harvesting pigment-protein complex which isfunctionally andstructurally associated withphotosystem I(PSI) reaction centers. Theseobservations arebased ontechniques whichaflow isolation ofPSIusing minimal concentrations ofTriton X-100. Properties ofdensity andself aggregation allowed purifi- cation ofa"native" PSIcomplex. Theisolated PSIparticles appear as106A spherical subunits when viewed byfreeze fracture microscopy. Whenincorporated into phosphatidyl choline vesicles, theparticles lose self-aggregation properties anddisperse uniformly within thelipid membrane. Theisolated PSIpreparation contains 110± 10chlorophylls/P700 (Chi a/bratio greater than18); this represents arecovery of27%oftheoriginal chloroplast membrane Chi.Theseparticles wereenriched inChiaforms absorbing at701to710nm.Chifluorescence atroomtemperature ex- hibited amaximumat690nm withapronounced shoulder at710nm.At 77K,peakfluorescence emission wasat736am;inthepresence of dithionite anadditional fluorescence maximumat695nm wasobtained at 77K.Thisdual fluorescence emission peakforthePSIparticles isevidence foratleast twoChlpopulations within thePSImembrane subunit. The fluorescence emission observed at695nmwasidentified asarising from thecoreofPSIwhichcontains 40Chl/P700 (PSI40). Thiscorecomplex, derived fromnative PSIparticles, wasenriched inChiaabsorbing at680 and690nmandfluorescing withmaximal emission at694amat77K.PSI particles consisting ofthePSIcorecomplex plus 20to25Chiantennae (65Chl/P700) could alsobederived fromnative PSIcomplexes. These preparations wereenriched inChla formsabsorbing at697nm and exhibited a77Kfluorescence emission maximumat722am. Acomparison ofnative PSIparticles which contain 110Chl/P700 (PSI- 110) andPSIparticles containing 65Chl/P700 (PSI-65) provides evidence fortheexistence ofaperipheral Chi-protein complex tightly associated in thenative PSIcomplex. Thenative PSIsubunits contain polypeptides of 22,500 to24,500 daltons whicharenotfound inthePSI-65 orPSI-40 subfractions. Itissuggested that these polypeptides function tobind 40to 45Chiperstructural complex, including theChiwhichemits fluorescence at736am. A modelfortheorganization ofChli formsispresented inwhichthe native PSImembrane subunit consists ofareaction center corecomplex plus tworegions ofassociated light-harvesting antennae. Thepresence of energy "sinks" within theantennae isdiscussed.
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Preparations of photosystem II membranes, in which the active site of oxygen evolution is exposed to the bulk aqueous phase, have facilitated biochemical studies of the photosynthetic oxygen-evolving complex (the photosynthetic water oxidase complex). Described here are photosystem II membrane preparations highly active in oxygen evolution and three extrinsic membrane proteins for which the molecular properties and functions in oxygen evolution have been characterized.
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To investigate the transverse bilayer organization of the chlorophyll-proteins of the three intrinsic chlorophyll- protein complexes, the effects of proteolytic enzymes, and an antibody against the light-harvesting complex were compared using right-side-out and inside-out thylakoid vesicles. The vesicles were isolated by aqueous polymer phase partitioning following the fragmentation of spinach thylakoids by passage through a Yeda press. Both vesicle types were agglutinated by an antiserum specific for the light-harvesting complex, although proteo- lytic degradation of the complex occurred only in right-side-out vesicles. In addition, there are different anti- genic sites for the light-harvesting complex on the inner and outer thylakoid surfaces. Polypeptides of the chlorophyll-a-protein complex of photosystem I1 were degraded by proteases at both membrane surfaces. We conclude that both these chlorophyll-protein complexes are membrane spanning and transversely asymmetric, but that the light-harvesting complex polypeptides accessible at the inner thylakoid surface are more resistant to proteolytic attack. In contrast, the main chlorophyll-containing polypeptide (Mr= 64500) of photosystem I complex was resistant to proteolytic attack at both the outer and inner thylakoid surfaces.
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The effect of trypsin treatment on Photosystem-II particles has been investigated by measurements of oxygen evolution, 2,6-dichlorophenolindophenol (DCIP)-reduction and Mn-abundance and by analyzing the peptide pattern. The following results were obtained. (1) Trypsin modifies both the acceptor and donor side of PS II, but striking differences are observed for the pH dependence: whereas the acceptor side is severely attacked between pH 5.5 and 9.0, the destruction of the donor side (oxygen-evolving capacity) by trypsin becomes significant only at pH values higher than 7.25. (2) The pH-dependence of the susceptibility of oxygen evolution to trypsin closely resembles that observed in inside-out thylakoids (Renger, G., Völker, M. and Weiss, W. (1984) Biochim. Biophys. Acta 766, 582–591). (3) The effect of trypsin on the functional integrity of water oxidation cannot be due to an attack on the surface exposed 16 kDa, 24 kDa and 33 kDa polypeptides, because they are digested rapidly even at pH 6.5, where the oxygen-evolving capacity remains almost unaffected. (4) Trypsination of PS-II particles as well as of the isolated 33 kDa protein leads to a 15 kDa fragment. In trypsinized PS-II particles this fragment remains membrane-bound. The amount of the 15 kDa fragment and Mn content are correlated with the oxygen-evolving capcity. These results indicate pH-dependent structural modifications at the donor side of System II which make target proteins accessible to trypsin. The 33 kDa protein is inferred to play a regulatory role in photosynthetic oxygen evolution and this function is realized by only a part of the protein, i.e., the 15 kDa fragment, that remains resistant to mild trypsination.
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An oxygen-evolving Photosystem (PS) II preparation was isolated after Triton X-100 treatment of spinach thylakoids in the presence of Mg2+. The structural and functional components of this preparation have been identified by SDS-polyacrylamide gel electrophoresis and sensitive spectrophotometric analysis. The main findings were: (1) The concentration of the primary acceptor Q of PS II was 1 per 230 chlorophyll molecules. (2) There are 6 to 7 plastoquinone molecules associated with a ‘quinone-pool’ reducible by Q. (3) The only cytochrome present in significant amounts (cytochrome b-559) occurred at a concentration of 1 per 125 chlorophyll molecules. (4) The only kind of photochemical reaction center complex present was identified by fluorescence induction kinetic analysis as PS IIα. (5) An Em = − 10 mV has been measured at pH 7.8 for the primary electron acceptor Qα of PS IIα. (6) With conventional SDS-polyacrylamide gel electrophoresis, the preparation was resolved into 13 prominent polypeptide bands with relative molecular masses of 63, 55, 51, 48, 37, 33, 28, 27, 25, 22, 15, 13 and 10 kDa. The 28 kDa band was identified as the PS II light-harvesting chlorophyll . In the presence of 2 M urea, however, SDS-polyacrylamide gel electrophoresis showed seven prominent polypeptides with molecular masses of 47, 39, 31, 29, 27, 26 and 13 kDa as well as several minor components. CP I under identical conditions had a molecular mass of 60–63 kDa.
Article
The stoichiometry of the proteins of the photosynthetic oxygen evolution system and of the electron transport components in Photosystem II particles prepared with Triton X-100 from spinach chloroplasts were determined. Per about 220 chlorophyll molecules, there were one reaction center II, one molecule each of the 33, 24 and 18 kDa proteins, four Mn atoms, two cytochromes b-559 (one high-potential, the other low-potential), and 3.5 plastoquinone-9 molecules, but practically no cytochrome b-563, cytochrome f, phylloquinone, α-tocopherol or α-tocopherylquinone.
Article
Photosystem II (PS II) particles containing only the two minor chlorophyll a complexes CP a-1 and CP a-2 (Green, B.R., Camm, E. and Van Houten, J. (1982) Biochim. 681, 248-55) were prepared from spinach and barley by a simple procedure involving differential solubilization with octylglucoside and sucrose gradient centrifugation. The gradient fractions with highest PS II activity (light-dependent reduction of 2,6-dichlorophenolindophenol by diphenylcarbazide) contained both CP a-1 and CP a-2, but no other chlorophyll-protein complex. Distribution of PS II activity in spinach was matched more closely by CP a-1 distribution than by CP a-2 distribution, suggesting that the former might carry PS II reaction centre chlorophylls, but a similar role for CP a-2 is also possible.
Article
Analysis of a 0.6 kb fragment of the spinach plastic chromosome adjacent to the 3' end of the apocytochrome f gene has disclosed two uninterrupted reading frames of 83 and 39 triplets, the product of the first one being apocytochrome b-559. The first 27 predicted amino acid residues had been verified by protein sequence analysis and the molecular mass of 9390 Da derived from the amino acid sequence deduced here is close to that of the authentic protein. The two genes are transcribed by a bicistronic RNA in a direction opposite to that of cytochrome f, and their translation stop/potential ribosome binding sites overlap. Features of the two genes resembling those of bacterial genes include putative tetra- or pentanucleotide ShineDalgarno sequences, Pribnow boxes, ‘−35’ promotor consensus sequences and possibly a transcription termination region. Both gene structure and products of DNA- or RNA-programmed cell-free translation preclude that apocytochrome b-559 is made as a precursor. The amino acid sequence includes only one histidine residue located in a predicted secondary structure of strong hydrophobicity which indicates the intriguing possibility that more than one protein chain must cooperate in heme binding of this cytochrome.
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Repeated washes of isolated chloroplasts with dilute sodium pyrophosphate solution results in the removal of carboxydismutase and some minor proteins from the thylakoid membranes. A subsequent treatment of the membranes with hypertonic sucrose solution yields pure coupling factor CF1 in the supernatant. Purity of the protein was demonstrated by disc electrophoresis.The amount of CF1 protein liberated was quantitatively determined. The percentage of CF1 removed by this treatment was calculated from the Ca2+-dependent ATPase activity retained at the thylakoid membranes. From these data the total CF1 content of chloroplasts was calculated. An average value of 0.42 mg CF1 protein/mg chlorophyll was obtained. Based on a molecular weight for CF1 of 326000 (see Farron, F. (1970) Biochemistry 9, 3823–3828), a ratio of 1 mole CF1 per 860 moles chlorophyll was computed.
Article
The isolation of a cytochrome f/b6 complex from spinach chloroplasts, with high yield and purity is reported. The complex consists of five polypeptides with a molecular mass of 34, 33, 23.5, 20 and 17.5 kDa, and contains one cytochrome f, two cytochromes b6 and the Rieske Fe-S center with two non-heme irons. It does not contain plastocyanin and is almost completely devoid of chlorophyll and carotenoids, but lipid and detergent are present. It is lacking cytochrome b-559, although three of the five polypeptides seem to carry heme groups. The preparation has plastoquinol-plastocyanin oxidoreductase activity with plastoquinol-1 and plastoquinol-9, which is sensitive to 2,5-dibromomethylisopropyl-p-benzoquinone, to 2-iodo-6-isopropyl-3-methyl-2',4',4'-trinitrodiphenyl ether, to 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole, and to bathophenanthroline. Characteristics of this activity with respect to substrate concentrations, pH, detergent effect and other parameters are described.
Article
The DNA sequence of 2210 nucleotides including the gene for the “51 kd” chlorophyll α-conjugated thylakoid membrane protein associated with the photosystem II reaction center of spinach has been determined. This protein is functionally identical with the P680 chlorophyll α apoprotein that catalyses the primary light-induced photochemical processes of photosystem II (Camm, E.L. and Green, B.R. (1983) Biochim. Biophys. Acta 724 291–293). The only large open reading frame in the sequence consists of 508 triplets encoding a protein of molecular mass of 56,246 kd. The deduced amino acid sequence shows clustering of hydrophobic residues into seven core regions which probably traverse the membrane, and a large hydrophilic domain of about 200 amino acids interspersed between span VI and VII. Potential transcription promotor and terminator signals flanking the structural gene show prokaryotic-like features. Seven discrete RNA species ranging in size from 2.0 to over 5.0 kilobases display complementarity to apoprotein coding sequences implying that the region can be polycistronically transcribed. The primary transcript includes information for at least two further genes coding for subunits of the cytochrome b/f complex
Article
The gene for the so-called M(r) 32,000 rapidly labeled photosystem II thylakoid membrane protein (here designated psbA) of spinach (Spinacia oleracea) chloroplasts is located on the chloroplast DNA in the large single-copy region immediately adjacent to one of the inverted repeat sequences. In this paper we show that the size of the mRNA for this protein is approximately 1.25 kilobases and that the direction of transcription is towards the inverted repeat unit. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of M(r) 38,950. The nucleotide sequence of psbA from Nicotiana debneyi also has been determined, and comparison of the sequences from the two species shows them to be highly conserved (>95% homology) throughout the entire reading frame. Conservation of the amino acid sequence is absolute, there being no changes in a total of 353 residues. This leads us to conclude that the primary translation product of psbA must be a protein of M(r) 38,950. The protein is characterized by the complete absence of lysine residues and is relatively rich in hydrophobic amino acids, which tend to be clustered. Transcription of spinach psbA starts about 86 base pairs before the first ATG codon. Immediately upstream from this point there is a sequence typical of that found in E. coli promoters. An almost identical sequence occurs in the equivalent region of N. debneyi DNA.
Article
RECONSTRUCTION OF PHOTOSYNTHETIC NONCYCLIC ELECTRON TRANSPORT FROM WATER TO NADP HAS BEEN ACCOMPLISHED BY USING THREE INTEGRAL PROTEIN COMPLEXES ISOLATED FROM CHLOROPLAST THYLAKOID MEMBRANES: photosystems I and II and the cytochrome b(6)-f complex. This system shows an absolute dependence on the presence of all three protein complexes for NADP reduction, in addition to plastocyanin, ferredoxin, and ferredoxin-NADP reductase. The reconstructed system was found to be sensitive to low concentrations of known inhibitors of noncyclic electron transport. Depletion of the Rieske iron-sulfur center and bound plastoquinone from the cytochrome b(6)-f complex resulted in an inhibition of the photoreduction of NADP.
Article
The transverse heterogeneity of the polypeptides associated with the Photosystem I (PSI) complex in spinach thylakoid membranes and in a highly resolved PSI preparation has been studied using the impermeant chemical modifier, 2,4,6-trinitrobenzenesulfonate (TNBS) and the proteolytic enzyme, Pronase E. The present study has shown that the PSI reaction center polypeptide of approximately 62 kilodaltons and the 22 and 20 kilodalton polypeptides of the PSI light-harvesting chlorophyll protein (LHCPI) complex are not labeled by [(14)C]TNBS in unfractionated thylakoids. On the other hand, the 23 kilodalton polypeptide of the PSI LHCP and the 19 and 14 kilodalton polypeptides associated with the PSI primary electron acceptor complex are readily labeled by [(14)C]TNBS and are exposed to the stromal side of the thylakoid. Differences and similarities in the labeling of polypeptides associated with the PSI complex in thylakoids and in the isolated PSI complex are also noted. Treatment of thylakoids with pronase had no effect on the organization of the polypeptides in the LHCPI or the reaction center core complex, as manifested by the separation of these two subcomplexes from pronase-treated membranes. The 62, 19, and 14 kilodalton polypeptides associated with the reaction center core complex and the 23 and 22 kilodalton polypeptides associated with LHCPI are sensitive to pronase treatment while the 20 kilodalton polypeptide of LHCPI was inaccessible to the protease. The proteolysis of the 62 kilodalton polypeptide generated first a single immunodetectable fragment at about 48 kilodaltons, and further proteolytic digestion generated two other fragments at 30 and 17 kilodaltons respectively. These results are discussed in relation to the organization of the PSI complex in spinach thylakoids. A model for the transmembrane topography of the polypeptide constituents of PSI has been developed.
Nucleotide sequence of the gene for apocytochrome b-559 on the spinach plastid chromosome: implications for the structure of the membrane protein Structure of the spinach chloroplast genes for the D2 and 44 kd reaction-center proteins of Photosystem II and for tRNAW (UGA)
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Nucleotide sequences of the gene for Mr 32000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of M, 38950
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ZURAWSKI G, HG BOHNERT, PR WHITFELD, W BOTrOMLEY 1982 Nucleotide sequences of the gene for Mr 32000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of M, 38950. Proc Natl Acad Sci USA 79: 7699-7703
Fractionation of an oxygenevolving Photosystem II preparation: characterization of the light-harvesting antennae and reaction center components
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Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-fcomplex Transmembrane organization of individual polypeptides of Photosystem I complex and the light-harvesting complex of Photosystem II of thylakoid membranes
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Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide
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