Photosystem II of rye. Nucleotide sequence of the psbK gene and regions adjacent to it]

The structure of the rye chloroplast DNA which contains the psbK gene coding for a subunit of photosystem II is determined. The gene psbI encoding an other protein of photosystem II is located 407 bp downstream from the stop codon of this gene. The determination of structure of the intergenic region between the psbI and psbD genes is fully elucidated. The rye BamHI fragment, comprising the psbK gene, is structurally similar to the corresponding fragment of the barley genome.     

Cloning of the psbK gene from Synechocystis sp. PCC 6803 and characterization of photosystem II in mutants lacking PSII-K

We cloned and sequenced the psbK gene, coding for a small photosystem II component (PSII-K), from the transformable cyanobacterium, Synechocystis sp. PCC 6803, and determined the N-terminal sequence of mature PSII-K. The psbK gene product is processed by cleaving off eight amino acid residues from the N terminus. A mutant lacking psbK was constructed; this mutant grew photoautotrophically, but its growth rate was reduced. The number of photosystem II reaction centers on a chlorophyll basis was decreased by less than a factor of 2 in the psbK-deletion mutant. In Synechocystis sp. PCC 6803, the psbK gene is transcribed as a single gene and is not part of an operon. Single-site mutations were introduced into psbK leading to early termination or deletion of the presequence. The phenotype of these mutants strongly resembles that of the psbK deletion mutant, indicating that indeed the change in phenotype in the deletion mutant is directly correlated with PSII-K. PSII-K is not essential for photosystem II assembly or activity but is needed for optimal photosystem II function.     

Characterization and N-terminal sequence of a 5 kDa polypeptide in the photosystem I core complex from spinach

Photosystem I core complexes were isolated from spinach photosystem I particles after heat treatment in the presence of 50% (v/v) ethylene glycol (heat/EG treatment). The core complex from 58 degrees C/EG-treated particles was composed of polypeptides with apparent molecular masses of 63, 60 and 5 kDA; this complex contained the iron sulfur center Fx but lacked center FA and FB. The core complex obtained from the 70 degrees C/EG-treated preparation lacked FX and contained a lesser amount of the 5 kDa polypeptide. The N-terminal amino acid sequence of the 5 kDa polypeptide did not correspond to the sequence derived from any possible reading frame in the chloroplast DNA of liverwort or tobacco. Twelve of the first 29 N-terminal amino acids were hydrophobic, suggesting that this protein is intrinsic to the photosystem I reaction center.     

Identification of photosystem I components from the cyanobacterium, Synechococcus vulcanus by N-terminal sequencing

The photosystem I core complex isolated from a thermophilic cyanobacterium, Synechococcus vulcanus, is composed of eight low-molecular-mass proteins of 18, 14, 12, 9.5, 9, 6.5, 5 and 4.1 kDa in addition to the PS I chlorophyll protein. N-terminal amino acid sequences of all these components were determined and compared with those of higher plants. Clearly, the 9.5 kDa component corresponds to the protein which carries the non-heme iron-sulfur centers A and B. This protein is so poorly visualized by staining that it has probably been overlooked in gel electrophoresis analyses. The 18, 14, 12 and 9 kDa components show appreciable homology with respective subunits of higher plant PS I. In contrast, the 6.5, 5 and 4.1 kDa components do not correspond to any known proteins except that the sequence of the 4.1 kDa component matches an unidentified open reading frame (ORF) 42 (liverwort) or ORF44 (tobacco) of chloroplast DNA.     

N-terminal sequencing of photosystem II low-molecular-mass proteins. 5 and 4.1 kDa components of the O2-evolving core complex from higher plants

High resolution gel electrophoresis in the low-molecular-mass region combined with electroblotting using polyvinylidene difluoride membranes enabled us to sequence the low-molecular-mass proteins of photosystem II membrane fragments from spinach and wheat. The determined N-terminal sequences, all showing considerable homology between the two plants, involved two newly determined sequences for the 4.1 kDa protein and one for the 5 kDa proteins. The sequence of the 4.1 kDa protein did not match any part of the chloroplast DNA sequence from tobacco or liverwort, suggesting that it is encoded by the nuclear genome. In contrast, the sequence of the 5 kDa protein matched ORF38, which is located just downstream of psbE and psbF in the chloroplast DNA and is assumed to be co-transcribed with them. These two components were associated with the O2-evolving core complex. Sequences of other low-molecular-mass proteins confirmed the previous identification as photosystem II components.     

Reality of P-680 chlorophyll protein – Identification of the site of primary photochemistry in oxygenic photosynthesis

Until recently nearly all available experimental evidence seemed to indicate that the largest subunit of about 50 kDa in the photosystem II core complex ( psb B gene product) is the site of primary photochemistry, and thus the name "P-680 apoprotcin" has been given to this subunit. The notion, however, has also been challenged on the basis of deduced amino acid sequence homology between the proteins in the photosystem II and those of the purple bacterial reaction center. The actual preparation of a functionally active photosystem II reaction center completely devoid of the psb B gene product, but consisting of D-1 and D-2 proteins and cytochrome b -559, has now been achieved.     

Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing

The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland. Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559. The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II. N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane. The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants. The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product). These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex.     

The protein responsible for center A/B in spinach photosystem I: isolation with iron-sulfur cluster(s) and complete sequence analysis

The 9 kDa polypeptide from spinach photosystem I (PS I) complex was isolated with iron-sulfur cluster(s) by an n-butanol extraction procedure under anaerobic conditions. The polypeptide was soluble in a saline solution and contained non-heme irons and inorganic sulfides. The absorption spectrum of this iron-sulfur protein was very similar to those of bacterial-type ferredoxins. The amino acid sequence of the polypeptide was determined by using a combination of gas-phase sequencer and conventional procedures. It was composed of 80 amino acid residues giving a molecular weight of 8,894, excluding iron and sulfur atoms. The sequence showed the typical distribution of cysteine residues found in bacterial-type ferredoxins and was highly homologous (91% homology) to that deduced from the chloroplast gene, frxA, of liverwort, Marchantia polymorpha. The 9 kDa polypeptide is considered to be the iron-sulfur protein responsible for the electron transfer reaction in PS I from center X to [2Fe-2S] ferredoxin, namely a polypeptide with center(s) A and/or B in PS I complex. It is noteworthy that the 9 kDa polypeptide was rather hydrophilic and a little basic in terms of the primary structure. A three-dimensional structure was simulated on the basis of the tertiary structure of Peptococcus aerogenes [8Fe-8S] ferredoxin, and the portions in the molecule probably involved in contacting membranes or other polypeptides were indicated. The phylogenetic implications of the structure of the present polypeptide as compared with those of several bacterial-type ferredoxins are discussed.     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

Structure and organization of Marchantia polymorpha chloroplast genome. II. Gene organization of the large single copy region from rps'12 to atpB

The nucleotide sequence (56,410 base-pairs) of the large single-copy region of chloroplast DNA from the liverwort Marchantia polymorpha has been determined. The sequence starts from one end (JLA) of the large single-copy region and encompasses genes for 21 tRNAs, six ATPase subunits (atpA, atpB, atpE, atpF, atpH and atpI), two photosystem I polypeptides (psaA and psaB), four photosystem II polypeptides (psbA, psbC, psbD and psbG), five ribosomal proteins (rps2, rps4, rps7, rps'12 and rps14), and three RNA polymerase subunits (rpoB, rpoC1 and rpoC2). In addition, we detected 18 open reading frames ranging from 29 to 2136 amino acid residues long, four of which share significant amino acid sequence homology to those of an Escherichia coli malK protein (designated mbpX), human mitochondrial ND2 (ndh2) and ND3 (ndh3) of a respiratory chain NADH dehydrogenase, or a bacterial antenna protein of a light-harvesting complex (lhcA). Sequence analysis suggests that four tRNA genes and six protein genes might be split by introns; they are trnG(UCC), trnK(UUU), trnL(UAA), trnV(UAC), atpF, ndh2, rpoC1, rps'12, ORF135 and ORF167. In the large single-copy region described here, the gene organization deduced is highly conserved with respect to that of higher plants, but an inversion of some 30,000 base-pairs flanked by trnL(CAA) and trnD(GUC) was seen between the liverwort and tobacco chloroplast genomes.     

The primary structure of a 4.0-kDa photosystem I polypeptide encoded by the chloroplast psaI gene

Partial amino acid sequences have been determined for a 4.0-kDa photosystem I polypeptide from barley. A comparison with the sequence of the chloroplast genome of Nicotiana tabacum and Marchantia polymorpha identified the polypeptide as chloroplast-encoded. We designate the corresponding gene psaI and the polypeptide PSI-I. The barley chloroplast psaI gene was sequenced. The gene encodes a polypeptide of 36 amino acid residues with a deduced molecular mass of 4008 Da. The 4.0-kDa polypeptide is N-terminally blocked with a formyl-methionine residue. Plasma desorption mass spectrometry established that the polypeptide is not post-translationally processed except for possible conversion of a methionine residue into methionine sulfone. The hydrophobic 4.0-kDa polypeptide is predicted to have one membrane-spanning alpha-helix and is homologous to transmembrane helix E of the D2 reaction center polypeptide of photosystem II.     

The 8 kDa polypeptide in photosystem I is a probable candidate of an iron-sulfur center protein coded by the chloroplast gene frxA

The N-terminal sequence of the 8 kDa polypeptide isolated from spinach photosystem I (PS I) particles was determined by a gas-phase sequencer. The sequence showed the characteristic distribution of cysteine residues in the bacterial-type ferredoxins and was highly homologous to that deduced from the chloroplast gene frxA of liverwort, Marchantia polymorpha. It is strongly suggested that the 8 kDa polypeptide has to be an apoprotein of one of the iron-sulfur center proteins in PS I particles.     

Molecular cloning and sequencing of the psaD gene encoding subunit II of photosystem I from the cyanobacterium, Synechocystis sp. PCC 6803

Photosystem I reaction center was isolated from the cyanobacterium, Synechocystis sp. PCC 6803, in a form which contains seven different polypeptide subunits. One of the subunits, with a molecular mass of about 16 kDa, was isolated, and protein sequence information was obtained for the amino terminus and several tryptic peptides. Oligonucleotide probes, corresponding to these sequences, were used to probe a genomic library, and the gene, designated psaD, encoding subunit II was cloned and sequenced. The gene encodes a polypeptide with a mass 15,644 Da, which exhibits a high degree of similarity to subunit II from tomato, as well as amino acid sequences reported from barley photosystem I. In addition to this gene, three large open reading frames were identified. Two remain unidentified, and the third is highly homologous to anthranilate synthase, component 1 from Escherichia coli and Saccharomyces cerevisiae.     

Photosystem II of rye. Nucleotide sequence of psbD, psbI genes encoding reaction center proteins

Structures of the rye chloroplast DNA regions, containing psbD and psbI genes coding for the components of the reaction centre of photosystem II, D2 protein and I polypeptide, respectively, have been determined. The gene trnS for tRNA(Ser) (GCU) is located 111 bp downstream from the stop codon of the psbI gene on the opposite strand. The high homology between the rye BamHI-fragment comprising these genes and his counterpart from wheat are discussed.     

Structural organization of proteins on the oxidizing side of photosystem II. Two molecules of the 33-kDa manganese-stabilizing proteins per reaction center.

The 33-kDa manganese-stabilizing protein stabilizes the manganese cluster in the oxygen-evolving complex. There has been, however, a considerable amount of controversy concerning the stoichiometry of this photosystem II (PS II) component. In this paper, we have verified the extinction coefficient of the manganese-stabilizing protein by amino acid analysis, determined the manganese content of oxygen-evolving photosystem II membranes and reaction center complex using inductively coupled plasma spectrometry, and determined immunologically the amount of the manganese-stabilizing protein associated with photosystem II. Oxygen-evolving photosystem II membranes and reaction center complex preparations contained 258 +/- 11 and 67 +/- 3 chlorophyll, respectively, per tetranuclear manganese cluster. Immunoquantification of the manganese-stabilizing protein using mouse polyclonal antibodies on "Western blots" demonstrated the presence of 2.1 +/- 0.2 and 2.0 +/- 0.3 molecules of the manganese-stabilizing protein/tetranuclear manganese cluster in oxygen-evolving PS II membranes and highly purified PS II reaction center complex, respectively. Since the manganese-stabilizing protein co-migrated with the D2 protein in our electrophoretic system, accurate immunoquantification required the inclusion of CaCl2-washed PS II membrane proteins or reaction center complex proteins in the manganese-stabilizing protein standards to compensate for the possible masking effect of the D2 protein on the binding of the manganese-stabilizing protein to Immobilon-P membranes. Failure to include these additional protein components in the manganese-stabilizing protein standards leads to a marked underestimation of the amount of the manganese-stabilizing protein associated with these photosystem II preparations.     

Characterization of cDNA clones encoding the extrinsic 23 kDa polypeptide of the oxygen-evolving complex of photosystem II in pea

The 23 kDa polypeptide of the oxygen-evolving complex of photosystem II has been extracted from pea photosystem II particles by washing with 1 M NaCl and purified by anion-exchange chromatography. The N-terminal amino acid sequence has been determined and specific antisera have been raised in rabbits and used to screen a pea-leaf cDNA library in gt11. Determination of the nucleotide sequence of two clones provided the nucleotide sequence for the full 23 kDa polypeptide. The deduced amino acid sequence showed it to code for a mature protein of 186 amino acid residues with an N-terminal presequence of 73 amino acid residues showing a high degree of conservation with previously reported 23 kDa sequences from spinach and Chlamydomonas. Southern blots of genomic DNA from pea probed with the labelled cDNA gave rise to only one band suggesting that the protein is encoded by a single gene. Northern blots of RNA extracted from various organs indicated a message of approximately 1.1 kb, in good agreement with the size of the cDNA, in all chlorophyll-containing tissues. Western blots of protein extracted from the same organs indicated that the 23 kDa polypeptide was present in all major organs of the plant except the roots.Abbreviations bis-Tris bis (2-hydroxyethyl) imino-tris (hydroxymethyl)-methane - pfu plaque-forming units     

Ycf12 is a core subunit in the photosystem II complex

The latest crystallographic model of the cyanobacterial photosystem II (PS II) core complex added one transmembrane low molecular weight (LMW) component to the previous model, suggesting the presence of an unknown transmembrane LMW component in PS II. We have investigated the polypeptide composition in highly purified intact PS II core complexes from Thermosynechococcus elongatus, the species which yielded the PS II crystallographic models described above, to identify the unknown component. Using an electrophoresis system specialized for separation of LMW hydrophobic proteins, a novel protein of approximately 5 kDa was identified as a PS II component. Its N-terminal amino acid sequence was identical to that of Ycf12. The corresponding gene is known as one of the ycf (hypothetical chloroplast reading frame) genes, ycf12, and is widely conserved in chloroplast and cyanobacterial genomes. Nonetheless, the localization and function of the gene product have never been assigned. Our finding shows, for the first time, that ycf12 is actually expressed as a component of the PS II complex in the cell, revealing that a previously unidentified transmembrane protein exists in the PS II core complex.     

Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803

Photosystem I reaction center of the cyanobacterium Synechocystis sp. PCC 6803 contains seven different polypeptide subunits. The subunit with a molecular mass of about 8 kDa was isolated, and the sequence of its amino-terminal residues was determined. Oligonucleotide probes corresponding to this sequence were used to isolate the gene encoding this subunit. The gene, termed as psaE, codes for a polypeptide with a mass of 8075 Da. It is present as a single copy in the genome and is transcribed as a monocistronic messenger. The amino acid sequence of the 8-kDa subunit deduced from the gene sequence shows high homology with the deduced amino acid sequence of subunit IV of photosystem I from spinach. The DNA fragment sequenced in these studies also contains two other unidentified major open reading frames. A stable deletion mutation for the psaE gene was generated by transforming Synechocystis sp. PCC 6803 with a cloned DNA in which the psaE gene for 8-kDa subunit was replaced by a gene conferring resistance to kanamycin. The mutant strain shows minor differences in growth under photoautotrophic conditions and in the photosystem I activity in comparison to the wild type.     

Identities of four low-molecular-mass subunits of the photosystem I complex from Anabaena variabilis ATCC 29413. Evidence for the presence of the psaI gene product in a cyanobacterial complex

Photosystem I (PSI) complex of Anabaena variabilis ATCC 29413 consists of at least 11 subunits, 9 of which are resolved by high resolution gel electrophoresis. N-terminal amino acid sequences of the four subunits with molecular masses of 6.8, 5.2, 4.8 and 3.5 kDa were determined. Based on the sequence homology, the 3.5 kDa subunit was revealed to correspond to PSI-I (the gene product of psaI), which had so far been detected only in higher plant PSI complexes. The 6.8 kDa protein and 4.8 kDa protein were identified as gene products of psaK and psaJ, respectively. The 5.2 kDa protein was homologous to a 4.8 kDa subunit of PSI of the thermophilic cyanobacterium Synechococcus vulcanus, suggesting that this protein is a component of PSI in cyanobacteria.