David L. Willey

Structure and topology of cytochrome f in pea chloroplast membranes

A transmembrane arrangement of cytochrome f in chloroplast thylakoid membranes, with the N-terminal heme-containing region in the intrathylakoid space and a 15 amino acid C-terminal sequence in the stroma, is suggested by the amino acid sequence deduced from the nucleotide sequence of the pea chloroplast gene. This topology has been confirmed by partial proteolysis of the polypeptide in intact and disrupted thylakoid membranes and in inside-out and right-side-out vesicles of chloroplast membranes.

Location and nucleotide sequence of the gene for cytochrome f in wheat chloroplast DNA

SummaryThe gene for cytochrome f has been located in wheat chloroplast DNA by hybridisation with a 3.3 kbp BglII fragment of pea chloroplast DNA containing the gene for cytochrome f, by in vitro transcription-translation of cloned restriction fragments of wheat chloroplast DNA and by nucleotide sequence analysis. The gene is located 3 kbp from the 3′ end of the gene for the large subunit of ribulose bisphosphate carboxylase and is transcribed from the same DNA strand. Nucleotide sequence analysis reveals an open reading frame of 320 amino acids, of which 285 amino acid residues comprise the mature polypeptide and 35 amino acid residues probably represent an N-terminal signal sequence. The nucleotide sequence of the wheat gene shows 85% homology with the gene for pea cytochrome f.

Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5′-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5′-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3′-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic α-helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning α-helices. Some of these α-helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.

Localization of the gene for cytochrome f in pea chloroplast DNA

SummaryPea chloroplast DNA and cloned restriction fragments of pea chloroplast DNA have been used as templates in a cell-free coupled transcription-translation system from E. coli and have been shown to direct the synthesis of a 39 kD polypeptide related to cytochrome f (37.3 kD). The product was shown to be similar to cytochrome f by immunoprecipitation with specific antibodies and by peptide mapping of the products of limited chymotrypsin digestion. The structural gene for this higher molecular weight form of cytochrome f has been located within a 3.3 kbp BglII fragment situated 18 kbp from the single 16S rRNA gene.

Synthesis and assembly of the cytochrome b-f complex in higher plants

The cytochrome b-f complex is composed of four polypeptide subunits, three of which, cytochrome f, cytochrome b-563 and subunit IV, are encoded in chloroplast DNA and synthesised within the chloroplast, and the fourth, the Rieske FeS protein, is encoded in nuclear DNA and synthesised in the cytoplasm. The assembly of the cytochrome b-f complex therefore requires the interaction of subunits encoded by different genomes. A key role for the nuclear-encoded Rieske FeS protein in the assembly of the complex is suggested by a study of cytochrome b-f complex mutants. The assembly of individual subunits of the complex may be regulated by the availability of prosthetic groups. The genes for the chloroplast-encoded subunits and cDNA clones for the Rieske FeS protein have been isolated and characterised. Cytochrome f and the Rieske FeS protein are synthesised initially with N-terminal presequences required for their correct assembly within the chloroplast. The deduced amino acid sequences of the four subunits have been used to suggest models for the arrangement of the polypeptides in the thylakoid membrane.

Location and nucleotide sequence of the gene for cytochrome b-559 in wheat chloroplast DNA

SummaryThe gene for the apoprotein of cytochrome b-559 has been located in wheat chloroplast DNA by coupled transcription-translation of fragments of chloroplast DNA in a cell-free system from Escherichia coli and by nucleotide sequence analysis. The gene is located 1.5 kbp downstream from the gene for cytochrome f and is transcribed in the opposite direction. Nucleotide sequence determination revealed an open reading frame coding for a hydrophobic protein of 83 amino acid residues (Mr 9434). The deduced amino acid sequence of the first 27 codons corresponds exactly to the determined N-terminal amino acid sequence of spinach cytochrome b-559. Another open reading frame of 39 codons is located 10 bp downstream from the gene for cytochrome b-559. A putative ribosome-binding site, AGGAGG, for this open reading frame overlaps the translation stop codon, UAG, of the cytochrome b-559 gene. The deduced amino acid sequence from this open reading frame shows similarities to the N-terminal sequence of cytochrome b-559. Both sequences contain a histidine residue located in a putative membrane-spanning region of the polypeptide.

Two small open reading frames are co-transcribed with the pea chloroplast genes for the polypeptides of cytochrome b-559

SummaryThe genes encoding the 9 kDa and 4 kDa polypeptides of cytochrome b-559 have been located in pea chloroplast DNA by coupled transcription-translation of cloned restriction fragments of chloroplast DNA in a cell-free extract of Escherichia coli and by nucleotide sequence analysis. The genes (psbE and psbF) are located approximately 1.0 kbp downstream of the gene for Cyochrome f and are transcribed in the opposite direction, similar to the arrangement in the chloroplast genomes of other higher plants. Nucleotide sequence analysis of this region revealed four open reading frames encoding hydrophobic proteins of 83 (psbE), 39 (psbF), 38 and 40 amino acid residues, which are co-transcribed as a single major RNA of 1.1 kb. The 5′ and 3′ ends of this RNA have been located by primer extension and S 1 nuclease mapping. The 5′ end of the RNA is located 140 by upstream of the initiating ATG codon of psbE and is preceded by typical chloroplast promoter sequences. The 3′ end of the RNA is located approximately 515 by downstream of the TAA stop codon of psbF close to a stable stem-loop structure.

An open reading frame encoding a putative haem-binding polypeptide is contranscribed with the pea chloroplast gene for apocytochrome f

The nucleotide sequence of a 1 kbp region of pea chloroplast DNA upstream from the gene petA encoding apocytochrome f has been determined. An open reading frame of 231 condons (ORF231) encoding a putative membrane-spanning polypeptide is separated by 205 bp from the coding region of petA. The open reading frame is homologous to open reading frames located in a similar position with respect to petA in chloroplast DNA from Marchantia polymorpha, tobacco, rice, wheat and Vicia faba. The sequence around a conserved histidine residue in a putative membrane-spanning region of the polypeptide resembles sequences present in cytochrome b from chromaffin granules and neutrophil membranes, suggesting that the open reading frame may encode a haem-binding polypeptide, possibly a b-type cytochrome. Northern hybridisation analysis indicates the presence in pea chloroplasts of a complex pattern of transcripts containing ORF231. Large transcripts of 5.5 kb, 4.3 kb, 3.4 kb and 2.7 kb encode both ORF231 and apocytochrome f, indicating that ORF231 and petA are co-transcribed.

Pea chloroplast genes encoding a 4kDa polypeptide of photosystem I and a putative enzyme of C1 metabolism

SummaryThe nucleotide sequence of 3.2 kbp of pea chloroplast DNA located upstream from the petA gene for cytochrome f, and previously reported to contain the gene for a photosystem I polypeptide, has been determined. Three open reading frames of 587, 40 and 157 codons have been identified. Orf40 encodes a highly conserved, hydrophobic, membrane-spanning polypeptide, and is identified as the gene psaI for the 4 kDa subunit of photosystem I. Orf587 is an extended version of the gene zfpA previously identified as encoding a conserved putative zinc-finger protein. The product of orf587 shows extensive homology to an unidentified open reading frame cotranscribed with a gene for folate metabolism in Escherichia coli and local homology to a region of the β subunit of rat mitochondrial propionyl-CoA carboxylase. It is suggested that the product of orf587 is an enzyme of C1 metabolism and is unlikely to be a regulatory DNA-binding protein. Orf157 potentially encodes an unidentified basic protein, but the protein sequence is not conserved in other plants.

Genes and Polypeptides of Photosystem II

Photosystem II catalyses the light-driven transfer of electrons from water to plastoquinone, producing oxygen and generating a proton gradient across the thylakoid membrane. The complex may be regarded as made up of three assemblies of polypeptides: a light-harvesting complex (LHCH), a core complex containing the reaction centre and two antenna chlorophyll proteins, and an extrinsic complex concerned with oxygen evolution. Photosystem II is structually the most complex of the supramolecular assemblies of the thylakoid membrane and is currently recognised to be composed of at least 20 different polypeptides.1