Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M _r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.     

Nucleotide sequence of cDNAs encoding the entire precursor polypeptide for thioredoxin m from spinach chloroplasts

Using the expression vector gt11 and immunochemical detection, six cDNA clones that encode the entire precursor polypeptides for spinach thioredoxin m were isolated and characterized. The ca. 1.0 kb cDNA sequence of the largest clone hybridizes to an RNA species of 1.1 kb. In each instance the cDNA sequences display single open reading frames encoding polypeptides of 181 amino acid residues corresponding to a molecular mass of 19.8 kDa. The sequences of the independently selected cDNAs fall into two classes that are indicative of at least two (closely related) genes for this protein. The amino acid sequences deduced from the cDNA sequences differ to some extent from the amino acid sequence published for spinach thioredoxin m. The sequences predict identical mature proteins of 112–114 amino acids corresponding to a polypeptide molecular mass of ca. 12.4–12.6 kDa, and include stroma-targeting N-terminal transit peptides of 67 residues which are removed during or after import into the organelle. Precursor protein was made in vitro from each of the different cDNA clones and imported into isolated intact chloroplasts. Independent of the cDNA clone used, two isoforms were detected in the chloroplasts after import in each instance. They comigrated with authentic thioredoxin mb and mc. These results indicate that the size variants observed for this protein in vivo result from post-translational modification and do not originate in different genes.     

Cloning and sequencing of spinach cDNA clones encoding the 20 kDa PS I polypeptide

Apart from the 8 kDa subunit, which is of chloroplast origin, most of the small polypeptides of the PS I reaction center from higher plants are encoded in nuclear genes. We describe here the first nucleotide sequence of a nuclear component of this photosystem, the precursor of the 20 kDa protein. The deduced sequence of the large transit peptide (55-60 amino acids) is rich in serine/threonine residues and has a net positive charge, which are classical features of these precursors. The sequence itself is mainly hydrophilic, with no possibility of classical membrane-spanning alpha-helices; it exhibits an interesting stretch of five basic amino acids in close vicinity: Thr-Arg-Leu-Arg-Ser-Lys-Tyr-Lys-Ile-Lys-Tyr.     

A cDNA clone encoding the precursor for a 10.2 kDa photosystem I polypeptide of barley

Two cDNA clones for the barley photosystem I polypeptide which migrates with an apparent molecular mass of 9.5 kDa on SDS-polyacrylamide gels have been isolated using antibodies and an oligonucleotide probe. The determined N-terminal amino acid sequence for the mature polypeptide confirms the identification of the clones. The 644 base-pair sequence of one of the clones contains one large open reading frame coding for a 14 882 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 193 Da. The hydropathy plot of the polypeptide shows one membrane-spanning region with a predicted -helix secondary structure. The gene for the 9.5 kDa polypeptide has been designated PsaH.     

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     

A cDNA clone from barley encoding the precursor from the photosystem I polypeptide PSI-G: Sequence similarity to PSI-K

A cDNA clone encoding the photosystem I subunit, PSI-G was isolated from barley using an oligonucleotide specifying a partial amino acid sequence from a 9 kDa polypeptide of barley photosystem I. The 724 bp sequence contains an open reading frame encoding a precursor polypeptide of 15 107 kDa. Import studies using the in vitro expressed barley PsaG cDNA clone demonstrate that PSI-G migrates with an apparent molecular mass of 9 kDa on SDS-polyacrylamide gels together with PSI-C (subunit-VII). The previous assignment of the gene product of PsaG from spinach as subunit V (Steppuhn J, Hermans J, Nechushtai R, Ljungberg U, Thümmler F, Lottspeich F, Herrmann RG, FEBS Lett 237: 218–224, 1988) needs to be re-examined. The expression of the psaG gene is light-induced similar to other barley photosystem I genes. A significant sequence similarity to PSI-K from Chlamydomonas reinhardtii was discovered when a gene database was searched with the barley PSI-G amino acid sequence. Extensive sequence similarity between the nuclear-encoded photosystem I subunits has not previously been found. The observed sequence similarity between PSI-G and PSI-K suggests a symmetric location of these subunits in the photosystem I complex. The hydropathy plot of the barley PSI-G polypeptide indicates two membrane-spanning regions which are also found at the corresponding locations in the PSI-K polypeptide. PSI-G and PSI-K probably have evolved from a gene duplication of an ancestral gene.     

Isolation and characterization of cDNA clones encoding a 18.8 kDa polypeptide,the product of the gene psaL,associated with photosystem I reaction center from spinach

Several cDNA clones encoding subunit XI of photosystem I reaction center (PSI-L) have been isolated from two gt11 expression libraries based on polyadenylated RNA of spinach seedlings illuminated for 4 and 16 h, respectively. The precursor polypeptide made from these recombinant DNAs in vitro can be efficiently imported into isolated spinach chloroplasts. It is correctly processed to the size of the authentic polypeptide and integrates into the photosystem I assembly. The 834 nucleotide sequence of the longest cDNA insert encodes a precursor polypeptide of 24 kDa (216 residues) and a mature protein of probably 18.8 kDa (169 residues). Hydropathy analysis suggests that the polypeptide contains two transmembrane segments. The protein appears to originate in a single-copy gene in spinach and to be decoded from RNA species of ca. 900 bases.     

Pressure-exploration of the 33-kDa protein from the spinach photosystem II particle.

The 33-kDa protein isolated from the spinach photosystem II particle is an ideal model to explore high-pressure protein-unfolding. The protein has a very low free energy as previously reported by chemical unfolding studies, suggesting that it must be easy to modulate its unfolding transition by rather mild pressure. Moreover, the protein molecule consists of only one tryptophan residue (Trp241) and eight tyrosine residues, which can be conveniently used to probe the protein conformation and structural changes under pressure using either fluorescence spectroscopy or fourth derivative UV absorbance spectroscopy. The different experimental methods used in the present study indicate that at 20 degrees C and pH 6, the 33-kDa protein shows a reversible two-state unfolding transition from atmospheric pressure to about 180 MPa. This value is much lower than those found for the unfolding of most proteins studied so far. The unfolding transition induces a large red shift of the maximum fluorescence emission of 34 nm (from 316 nm to 350 nm). The change in standard free energy (DeltaGo) and in volume (DeltaV) for the transition at pH 6.0 and 20 degrees C are -14.6 kJ.mol-1 and -120 mL.mol-1, respectively, in which the DeltaGo value is consistent with that obtained by chemical denaturation. We found that pressure-induced protein unfolding is promoted by elevated temperatures, which seem largely attributed to the decrease in the absolute value of DeltaGo (only a minor variation was observed for the DeltaV value). However, the promotion of the unfolding by alkaline pH seems mainly related to the increase in DeltaV without any significant changes in DeltaGo. It was also found that NaCl significantly protects the protein from pressure-induced unfolding. In the presence of 1 M NaCl, the pressure needed to induce the half-unfold of the protein is shifted to a higher value (shift of 75 MPa) in comparison with that observed without NaCl. Interestingly, in the presence of NaCl, the value of DeltaV is significantly reduced whilst that of DeltaGo remains as before. The unfolding-refolding kinetics of the protein has also been studied by pressure-jump, in which it was revealed that both reactions are a two-state transition process with a relatively slow relaxation time of about 102 s.     

Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem II.

An intrinsic 22 kDa polypeptide is found associated with the oxygen-evolving photosystem II (PSII) core complex in all green plants and cyanobacteria so far examined, although it does not appear to be required for oxygen evolution. Amino acid sequence information obtained from the purified 22 kDa protein was used to construct a probe that was employed to isolate a full-length cDNA clone encoding the 274-residue precursor of the 22 kDa protein. Hydropathy plot analysis predicts the existence of four membrane-spanning helices in the mature protein. The two halves of the approximately 200-residue mature protein show high sequence similarity to each other, suggesting that the psbS gene arose from an internal gene duplication. The 22 kDa protein has some sequence similarity to chlorophyll a/b-binding proteins.