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 low-molecular-mass components in cyanobacterial photosystem II core complex. Two components correspond to unidentified open reading frames of plant chloroplast DNA

We recently reported the presence of several low-molecular-mass protein components in the PS II O2-evolving core complex from the thermophilic cyanobacterium, Synechococcus vulcanus [(1989) FEBS Lett. 244, 391-396]. Here we have characterized the three components (4.1, 4.7, 5 kDa) of the same cyanobacterial core complex by N-terminal sequencing. There were two components in the 4.7 kDa region, both having a blocked N-terminus. One has a sequence highly homologous to open reading frame 34 of plant chloroplast DNA (tentatively designated psbM), while the other has a sequence partially homologous to open reading frame 43 of chloroplast DNA (designated psbN), although neither of the two gene products has yet been confirmed in chloroplasts. The cyanobacterial 4.1 kDa protein partially corresponds to the 4.1 kDa nuclear-encoded core component of higher plant PS II. The cyanobacterial 5 kDa component, however, shows a sequence that is unrelated to any other known proteins.     

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.     

Nicotiana chloroplast genes for components of the photosynthetic apparatus

In order to understand more fully chloroplast genetic systems, we have determined the complete nucleotide sequence (155, 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA. It contains two copies of an identical 25,339 bp inverted repeat, which are separated by 86, 684 bp and 18,482 bp single-copy regions. The genes for 4 different rRNAs, 30 different tRNAs, 44 different proteins and 9 other predicted protein-coding genes have been located. Fifteen different genes contain introns.Twenty-two genes for components of the photosynthetic apparatus have so far been identified. Most of the genes (except the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) code for thylakoid membrane proteins. Twenty of them are located in the large single-copy region and one gene for a 9-kd polypeptide of photosystem I is located in the small single-copy region. The gene for the 32-kd protein of photosystem II as well as the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase have strong promoters and are transcribed monocistronically while the other genes are transcribed polycistronically. We have found that the predicted amino acid sequences of six DNA sequences resemble those of components of the respiratory-chain NADH dehydrogenase from human mitochondria. As these six sequences are highly transcribed in tobacco chloroplasts, they are probably genes for components of a chloroplast NADH dehydrogenase. These observations suggest the existence of a respiratory-chain in the chloroplast of higher plants.     

Rye photosystem II. Nucleotide sequence of the psbC gene coding 43- kDa chlorophyll(a)-binding proteins

The structure of the rye chloroplast DNA, which contains psbC gene coding for 43-kDa chlorophyll(a)-binding subunit of photosystem II, is determined. The sequence of trnS (UGA) gene encoding tRNA Ser is located at a distance of 140 bp downstream from the stop codon of psbC gene on the opposite DNA strand. The 5'-terminal part of psbC gene, like in other plants, overlaps by 50 bp the 3'-terminal region of psbD gene coding for D2 protein of photosystem II. The amino acid sequence of the psbC gene product reveals common features with the structure of the psbB gene product (CPa-1 protein). The structural similarity of these two proteins seems to reflect their similar functions.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.     

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a-binding protein of photosystem II (psbB) and cytochrome b-563 (petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene (psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N-terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N-terminal region of the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII).     

Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA.     

Chloroplast gene for Mr 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences

The gene for the Mr 32000 herbicide binding polypeptide of photosystem II has previously been mapped to the 5 kbp EcoRI fragment Eco I of Euglena gracilis chloroplast DNA. The nucleotide sequence of 3324 bp of Eco I, containing the psbA locus, has been determined. This locus encodes a polypeptide of 345 amino acids which is co-linear with, and has 86% derived amino acid sequence homology to sequences derived from four higher plants chloroplast psbA loci. The Euglena psbA gene contains four introns of size 435, 443, 434, and 617 bp. The four introns have conserved boundary sequences of the type previously described in the Euglena chloroplast gene (rbcL) for the large subunit of ribulose-1,5-bisphosphate carboxylase (Koller et al., Cell 36, 545-553, 1984).     

Loss of Albino3 leads to the specific depletion of the light-harvesting system

The chloroplast Albino3 (Alb3) protein is a chloroplast homolog of the mitochondrial Oxa1p and YidC proteins of Escherichia coli, which are essential components for integrating membrane proteins. In vitro studies in vascular plants have revealed that Alb3 is required for the integration of the light-harvesting complex protein into the thylakoid membrane. Here, we show that the gene affected in the ac29 mutant of Chlamydomonas reinhardtii is Alb3.1. The availability of the ac29 mutant has allowed us to examine the function of Alb3.1 in vivo. The loss of Alb3.1 has two major effects. First, the amount of light-harvesting complex from photosystem II (LHCII) and photosystem I (LHCI) is reduced >10-fold, and total chlorophyll represents only 30% of wild-type levels. Second, the amount of photosystem II is diminished 2-fold in light-grown cells and nearly 10-fold in dark-grown cells. The accumulation of photosystem I, the cytochrome b(6)f complex, and ATP synthase is not affected in the ac29 mutant. Mild solubilization of thylakoid membranes reveals that Alb3 forms two distinct complexes, a lower molecular mass complex of a size similar to LHC and a high molecular mass complex. A homolog of Alb3.1, Alb3.2, is present in Chlamydomonas, with 37% sequence identity and 57% sequence similarity. Based on the phenotype of ac29, these two genes appear to have mostly nonredundant functions.     

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.     

Is the 9 kDa thylakoid membrane phosphoprotein functionally and structurally analogous to the 'H' subunit of bacterial reaction centres?

Although the amino acid sequence of the 9 kDa (phospho)protein of chloroplasts has been determined, the function of this thylakoid membrane protein in photosynthetic electron transport and the reason for its physiological control remains unclear. In this paper, I briefly review the evidence which indicates that the phosphorylation of the 9 kDa protein results in a partial inhibition of photosynthetic oxygen evolution by increasing the stability of the semiquinone bound to QA the primary, plastoquinone-binding site of photosystem II (PS II). I propose that in its dephosphorylated state, the 9 kDa thylakoid membrane protein may serve PS II to ensure efficient photochemical charge separation by aiding the transfer of reducing equivalents out of the reaction centre to the attendant plastoquinone pool. This function is analogous to that proposed for the H-subunit of the reaction centre of photosynthetic eubacteria. Whether these two proteins have evolved from a common ancestral reaction centre protein is discussed in the light of a comparison of their amino acid sequences and predicted secondary structures.     

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     

Nucleotide sequence of the gene psbD from barley chloroplast DNA coding for protein D2 of the photosystem II

The psbD gene for the membrane polypeptide D2 has been isolated from barley chloroplast DNA and its sequence, along with the flanking regions, has been determined. The 3'-end of the psbD gene is overlapped with a 50 bp stretch of a second open reading frame which belongs to the psbC gene encoding the P6 protein of photosystem II.