Purification and binding features of a pea fructose-1,6-bisphosphatase domain involved in the interaction with thioredoxin f

We previously demonstrated that a cluster in the available 150 Asn-¹⁷⁰Glu region of pea chloroplast fructose-1,6-bisphosphatase (FBPase) could be involved in its interaction with the physiological modulator thioredoxin (Trx). Using as template a cDNA coding for pea chloroplast FBPase, a DNA insert coding for a 19 amino acid fragment ( 149 Pro-¹⁶⁷Gly) was amplified by PCR. After insertion in the pGEX-4T vector-1, it was expressed in Escherichia coli as a fusion protein (GST-19) with the vector-coded glutathione transferase (GST). This protein appears in the supernatant of cell lysates, and was purified to homogeneity. After thrombin digestion, the 19 amino acid insert was isolated as a polypeptide which displayed a positive reaction against pea chloroplast FBPase antibodies. GST-19 linked to glutathione-Sepharose beads, but not the GST, strongly interacts with pea Trx f , suggesting that this binding depends on the 19 amino acid insert. ELISA and Western blot experiments also demonstrate the existence of a GST-19-Trx f interaction, as well as a negligible quantity of Trx f bound by the vector-coded GST. Putative competitive inhibition assays of FBPase activity carried out in the presence of increasing concentrations of the 19 amino acid insert do not demonstrate any enzyme inhibition. On the contrary, this protein fragment enhances the enzyme activity proportionally to its concentration in the assay mixture. This indicates that the FBPase-Trx f binding promotes some type of structural modification of the Trx molecule, or of the FBPase-Trx docking site, thus facilitating the reductive modulation of FBPase.     

Cloning,structure and expression of a pea cDNA clone coding for a photosynthetic fructose-1,6-bisphosphatase with some features different from those of the leaf chloroplast enzyme

A positive clone against pea (Pisum sativum L.) chloroplast fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) antibodies was obtained from a copy DNA (cDNA) library in λgt11. The insert was 1261 nucleotides long, and had an open reading frame of 1143 base pairs with coding capability for the whole FBPase subunit and a fragment of a putative processing peptide. An additional 115 base pairs corresponding to a 3′-untranslated region coding for an mRNA poly(A)⁺ tail were also found in the clone. The deduced sequence for the FBPase subunit was a 357-amino-acid protein of molecular mass 39253 daltons (Da), showing 82–88% absolute homology with four chloroplastic FBPases sequenced earlier. The 3.1-kilobase (kb)KpnI-SacI fragment of the λgt11 derivative was subcloned between theKpnI-SacI restriction sites of pTZ18R to yield plasmid pAMC100. Lysates ofEscherichia coli (pAMC100) showed FBPase activity; this was purified as a 170-kDa protein which, upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, displayed a 44-kDa band. As occurs with native FBPases, this indicates a homotetrameric structure for the expressed FBPase. When assayed under excess Mg²⁺ (10 mM), the expressed enzyme had a higher affinity for the substrate than the native pea leaf FBPase; this parameter appears to be substantiated by a tenfold higher specific activity than that of the native enzyme. However, when activated with dithiothreitol plus saturating concentrations of pea thioredoxin (Td) f, both FBPase had similar activities, with a 4:1 Td f-FBPase stoichiometry. In contrast to the native pea chloroplast FBPase, theE. coli-expressed enzyme did not react with the monoclonal antibody GR-PB5. It also had a higher heat sensitivity, with 42% residual activity after heating for 30 min at 60°C, conditions which preserved the native enzyme in a fully active state. These results show the existence of some difference(s) in the conformation of the two FBPases; this could be a consequence of a different expression of the genomic and cDNA clones, or be due to the need for some factor for the correct assembly of the oligomeric structure of the native chloroplast enzyme. Accession number for pea chloroplast FBPase coding sequence: X68826 in the European Molecular Biology Laboratory (EMBL)     

Purification and properties of pea (Pisum sativum L.) thioredoxin f,a plant thioredoxin with unique features in the activation of chloroplast fructose-1,6-bisphosphatase

Thioredoxin (Td) f from pea (Pisum sativum L.) leaves was purified by a simple method, which provided a high yield of homogeneous Td f. Purified Td f had an isoelectric point of 5.4 and a relative molecular mass (M_r) of 12 kilodaltons (kDa) when determined by filtration through Superose 12, but an M_r of 15.8 kDa when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protein remained fully active for several months when conserved frozen at — 20° C. The pea protein was able to activate fructose1,6-bisphosphatase (FBPase; EC 3.1.3.11), but in contrast to other higher-plant Td f proteins, was not functional in the modulation of NADP⁺-malate dehydrogenase activity. In spite of the absence of immunological cross-reactions of pea and spinach Td f proteins with the corresponding antibodies, pea Td f activated not only the homologous FBPase, but also the spinach enzyme. The saturation curves for pea FBPase, either with fructose-1,6-bisphosphate in the presence of different concentrations of homologous Td f, or with pea Td f in the presence of excess substrate, showed sigmoid kinetics; this can be explained on the basis of a random distribution of fructose-1,6-bisphosphate, and of the oxidized and reduced forms of the activator, among the four Td f- and substrate-binding sites of this tetrameric enzyme. From the saturation curves of pea and spinach Td f proteins against pea FBPase, a 4:1 stoichiometry was determined for the Td f-enzyme binding. This is in contrast to the 2:1 stoichiometry found for the spinach FBPase. The UV spectrum of pea Td f had a maximum at 277 nm, which shifted to 281 nm after reduction with dithiothreitol (s at 280 nm for 15.8-kDa M_r = 6324 M^–1 · cm^–1). The fluorescence emission spectrum after 280-nm excitation had a maximum at 334 nm, related to tyrosine residues; after denaturation with guanidine isothiocyanate an additional maximum appeared at 350 nm, which is concerned with tryptophan groups. Neither the native nor the denatured form showed a significant increase in fluorescence after reduction by dithiothreitol, which means that the tyrosine and tryptophan groups in the reduced Td f are similarly exposed. Pea Td f appears to have one cysteine residue more than the three cysteines earlier described for spinach and Scenedesmus Td f proteins.Abbreviations DDT dithiothreitol - ELISA enzyme-linked immunosorbent assay - FBPase fructose- 1,6-bisphosphatase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - Td thioredoxin The authors are grateful to Mrs. Francisca Castro and Mr. Narciso Algaba for skilful technical assistance. This work was supported by grant PB87-0431 of Dirección General de Investigación Cientifica y Técnica (DGICYT, Spain).     

Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase

The amino acid sequence of the spinach chloroplast fructose-1,6-bisphosphatase (FBPase) subunit has been determined. Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage. The sequence of spinach chloroplast FBPase shows clear homology (ca. 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose-1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme. The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity. This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines. The recent X-ray crystallographic resolution of pig kidney FBPase (H. Ke, C. M. Thorpe, B. A. Seaton, F. Marcus, and W. N. Lipscomb, 1989, Proc. Natl. Acad. Sci. USA 86, 1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms. It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are conserved in the chloroplast enzyme. The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxin-linked mechanism can be accommodated in the surface of the FBPase molecule.     

Binding features of chloroplast fructose-1,6-bisphosphatase-thioredoxin interaction

It has been proposed that a hydrophobic groove surrounded by positively charged amino acids on thioredoxin (Trx) serves as the recognition and docking site for the interaction of Trx with target proteins. This model for Trx-protein interactions fits well with the Trx-mediated fructose-1,6-bisphosphatase (FBPase) activation, where a protruding negatively charged loop of FBPase would bind to this Trx groove, in a process involving both electrostatic and hydrophobic interactions. This model facilitates the prediction of Trx amino acid residues likely to be involved in enzyme binding. Site-directed mutagenesis of some of these amino acids, in conjunction with measurements of the FBPase activation capacity of the wild type and mutated Trxs, was used to check the model and provided evidence that lysine-70 and arginine-74 of pea Trx m play an essential role in FBPase binding. The binding parameters for the interaction between chloroplast FBPase and the wild type pea Trxs f and m, as well as mutated pea Trx m, determined by equilibrium dialysis in accordance with the Koshland-Nemethy-Filmer model of saturation kinetics, provided additional support for the role of these basic Trx residues in the interaction with FBPase. These data, in conjunction with the midpoint redox potential (E(m)) determinations of Trxs, support the hydrophobic groove model for the interaction between chloroplast FBPase and Trx. This model predicts that differences in the FBPase activation capacity of Trxs arise from their different binding abilities.     

High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation.

A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schürmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.     

Hybrids from pea chloroplast thioredoxins f and m: physicochemical and kinetic characteristics

Two hybrid thioredoxins (Trx) have been constructed from cDNA clones coding for pea chloroplast Trxs m and f. The splitting point was the AvaII site situated between the two cysteines of the regulatory cluster. One hybrid, Trx m/f, was purified from Escherichia coli-expressed cell lysates as a high yielding 12 kDa protein. Western blot analysis showed a positive reaction with antibodies against pea Trxs m and f and, like the parenteral pea Trx m, displayed an acidic pI (5.0) and a high thermal stability. In contrast, the opposite hybrid Trx f/m appeared in E. coli lysates as inclusion bodies, where it was detected by Western blot against pea Trx f antibodies as a 40 kDa protein. Trx f/m was very unstable, sensitive to heat denaturation, and could not be purified. Trx m/f showed a higher affinity for pea chloroplast fructose-1,6-bisphosphatase (FBPase) and a smaller Trx/FBPase saturation ratio than both parenterals; however, the FBPase catalytic rate was lower than that with Trxs m and f. Surprisingly, the hybrid Trx m/f appeared to be incompetent in the activation of pea NADP-malate dehydrogenase. Computer-assisted models of pea Trxs m and f, and of the chimeric Trx m/f, showed a change in the orientation of the α₄-helix in the hybrid, which could explain the kinetic modifications with respect to Trxs m and f. We conclude that the stability of Trxs lies on the N-side of the regulatory cluster, and is associated with the acidic character of this fragment and, as a consequence, with the acidic pI of the whole molecule. In contrast, the ability of FBPase binding and enzyme catalysis depends on the structure on the C-side of the regulatory cysteines.     

Antigenic relationships between chloroplast and cytosolic fructose-1,6-bisphosphatases.

Cytosolic fructose-1,6-biphosphatases (FBPase, EC 3.1.3.11) from pea (Pisum sativum L. cv Lincoln) and spinach (Spinacia oleracea L. cv Winter Giant) did not cross-react by double immunodiffusion and western blotting with either of the antisera raised against the chloroplast enzyme of both species; similarly, pea and spinach chloroplast FBPases did not react with the spinach cytosolic FBPase antiserum. On the other hand, spinach and pea chloroplast FBPases showed strong cross-reactions against the antisera to chloroplast FBPases, in the same way that the pea and spinach cytosolic enzymes displayed good cross-reactions against the antiserum to spinach cytosolic FBPase. Crude extracts from spinach and pea leaves, as well as the corresponding purified chloroplast enzymes, showed by western blotting only one band (44 and 43 kD, respectively) in reaction with either of the antisera against the chloroplast enzymes. A unique fraction of molecular mass 38 kD appeared when either of the crude extracts or the purified spinach cytosolic FBPase were analyzed against the spinach cytosolic FBPase antiserum. These molecular sizes are in accordance with those reported for the subunits of the photosynthetic and gluconeogenic FBPases. Chloroplast and cytosolic FBPases underwent increasing inactivation when increasing concentrations of chloroplast or cytosolic anti-FBPase immunoglobulin G (IgG), respectively, were added to the reaction mixture. However, inactivations were not observed when the photosynthetic enzyme was incubated with the IgG to cytosolic FBPase, or vice versa. Quantitative results obtained by enzyme-linked immunosorbent assays (ELISA) showed 77% common antigenic determinants between the two chloroplast enzymes when tested against the spinach photosynthetic FBPase antiserum, which shifted to 64% when assayed against the pea antiserum. In contrast, common antigenic determinats between the spinach cytosolic FBPase and the two chloroplast enzymes were less than 10% when the ELISA test was carried out with either of the photosynthetic FBPase antisera, and only 5% when the assay was performed with the antiserum to the spinach cytosolic FBPase. These results were supported by sequencing data: the deduced amino acid sequence of a chloroplast FBPase clone isolated from a pea cDNA library indicated a 39,253 molecular weight protein, with a homology of 85% with the spinach chloroplast FBPase but only 48.5% with the cytosolic enzyme from spinach.     

Two light-responsive elements of pea chloroplastic fructose-1,6-bisphosphatase gene involved in the red-light-specific gene expression in transgenic tobaccos

The pea chloroplast fructose-1,6-bisphosphatase (FBPase) gene was cloned from a pea genomic library and sequenced. The gene contained three introns and four exons. Both in vitro and in vivo analyses of the promoter region of the gene were carried out simultaneously to elucidate the mechanisms of light-mediated gene expression. Two light-responsive elements were identified in gel mobility shift assays: a GT-1-like sequence for the binding of a GT-1-like factor (termed pea factor 1; PF1) and a binding site for a dark-specific factor (termed pea factor 2; PF2). The binding affinity of PF1 was higher in light-grown peas than in dark-grown peas and was affected by phosphorylation. The binding site was located at nucleotides (nt) -326 to -341. PF2 binding was dark-specific and the binding region was located upstream of the PF1-binding site (nt -492 to -412). In vivo experiments with transgenic tobacco plants suggested that the region between nt -411 and -272 contained a PF1-binding site that promoted light-mediated expression of the pea chloroplast FBPase. In contrast, the 81-bp region between nt -492 and -412, which is located further upstream than the PF1-binding site, negatively regulated light-mediated expression of FBPase. Moreover, activation of gene expression by the region (nt -411 to -272) contained a PF1-binding site that was sensitive to red-light irradiation, suggesting that the expression of the chloroplast FBPase was regulated by the phytochrome system. Interestingly, the binding region for the dark-specific factor (PF2; nt -492 to -412) not only repressed gene expression in the dark, but also acted as a light-dependent activating element of the chloroplast FBPase gene.     

Purification and characterization of pea thioredoxin f expressed in Escherichia coli

The recently cloned cDNA for pea chloroplast thioredoxin f was used to produce, by PCR, a fragment coding for a protein lacking the transit peptide. This cDNA fragment was subcloned into a pET expression vector and used to transform E. coli cells. After induction with IPTG the transformed cells produce the protein, mainly in the soluble fraction of the broken cells. The recombinant thioredoxin f has been purified and used to raise antibodies and analysed for activity. The antibodies appear to be specific towards thioredoxin f and do not recognize other types of thioredoxin. The recombinant protein could activate two chloroplastic enzymes, namely NADP-dependent malate dehydrogenase (NADP-MDH) and fructose 1,6-bisphosphatase (FBPase), both using dithiothreitol as a chemical reductant and in a light-reconstituted/thylakoid assay. Recombinant pea thioredoxin f turned out to be an excellent catalyst for NADP-MDH activation, being the more efficient than a recombinant m-type thioredoxin of Chlamydomonas reinhardtii and the thioredoxin of E. coli. At the concentrations of thioredoxin used in the target enzyme activation assays only the recombinant thioredoxin f activated the FBPase.     

Energetic aspects of the light activation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase

The light energy requirements for photoactivation of two chloroplast enzymes: fructose-1,6-bisphosphatase and NADP-malate dehydrogenase were studied in a reconstituted chloroplast system. This system comprised isolated pea thylakoids, ferredoxin (Fd), ferredoxin-thioredoxin reductase (FTR) thioredoxin_m and _f (Td_m, Td_f) and the photoactivatable enzyme. Light-saturation curves of the photoactivation process were established with once washed thylakoids which did not require the addition of Td for light activation. They exhibited a plateau at 10 W·m^–2 under nitrogen and 50 W·m^–2 under air, while NADP photoreduction was saturated at 240 W·m^–2. Cyclic and pseudocyclic phosphorylations saturated at identical levels as enzyme photoactivations. All these observations suggested that the shift of the light saturation plateau towards higher values under air was due to competing oxygen-dependent reactions. With twice washed thylakoids, which required Td for enzyme light-activation, photophosphorylation was stimulated under N₂ by the addition of the components of the photoactivation system. Its rate increased with increasing Td concentrations, just as did the enzyme photoactivation rate, while varying the target enzyme concentration had only a weak effect. Considering that Td concentrations were in a large excess over target enzyme concentrations, it may be assumed that the observed ATP synthesis was essentially dependent on the rate of Td reduction.Under air, Fd-dependent pseudo-cyclic photophosphorylation was not stimulated by the addition of the other enzyme photoactivation components, suggesting that an important site of action of O₂ was located at the level of Fd.Abbreviations Fd ferredoxin - FBPase fructose-1,6-bisphosphatase - FTR ferredoxin-thioredoxin reductase - LEM light effect mediator - NADP-MDH NADP-malate dehydrogenase - Td thioredoxin     

Binding and activation of pea chloroplast fructose-1,6-bisphosphatase by homologous thioredoxins m and f

Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) binds its putative physiological activator thioredoxin f (Trx f ) at pH 7.9, the pH in the stroma of the illuminated chloroplast. Since Trx m , described as specific in NADP⁺-malate dehydrogenase (NADPMDH) activation, appears in pea (Pisum sativum L.) also to be functional in FBPase modulation, we have here analyzed the effect of pH and the redox status of the chloroplast stroma in the pea FBPase binding of homologous Trx f and m . Both pea Trx were strongly bound by purified FBPase when they were preincubated at pH 7.9 with 2.5 m M dithiothreitol (DTT), but not when the reductant was omitted. As occurs with Trx f the Trx m /FBPase ratio of the complex was 4, but this was only observed with a Trx m /FBPase concentration ratio > 10 in the preincubation mixture. The FBPase-Trx m binding disappeared in the presence of 100 m M NaCl, even with 2.5 m M DTT at pH 7.9, with a concomitant appearance of different aggregation states of the FBPase subunit. A similar FBPase-Trx m complex was detected in the stromal solution when pea chloroplasts were lysed at pH 7.9 in the presence of DTT. No interaction was observed between NADP-MDH and Trx f or m , either in the presence or in the absence of DTT. Pea FBPase showed sigmoidal activation kinetics with pea Trx m , and an S_0.5 of 133 n M versus 6.6 n M with pea Trx f . About 10-fold higher concentration of the former than that of the latter was required for obtaining maximum activity; however, the V_max with Trx f was only 2-fold higher than that with Trx m . We conclude that pea FBPase binds and is activated by the homologous Trx m , even though to a lesser extent than with Trx f . We also deduce that in the light the conditions in the chloroplast stroma are optimal for forming an FBPase-Trx complex.     

Immunogold localization of photosynthetic fructose-1,6-bisphosphatase in pea leaf tissue

An enriched IgG serum fraction obtained from rabbits immunized against pea chloroplast fructose-1,6-bisphosphatase (FBPase) was used, coupled to colloidal gold (15 nanometer particles) goat anti-rabbit IgG, to analyze by electron microscopy the location of photosynthetic FBPase in pea (Pisum sativum L.) leaf ultrathin sections. In accordance with earlier biochemical studies on distribution of FBPase activity, the enzyme was visualized both in the stromal space and bound to the chloroplast membranes. Some gold particles also appear in the cytoplasm, which can be related to the presence in the cytosol of a high molecular weight precursor of this nuclear coded enzyme.     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.     

Synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein in the chloroplast membranes of peas and Chlamydomonas reinhardi

The synthesis, transport and localization of a nuclear coded 22-kd heat-shock protein (HSP) in the chloroplast membranes was studied in pea plants and Chlamydomonas reinhardi. HSPs were detected in both systems by in vivo labeling and in vitro translation of poly(A)⁺RNA, using the wheat-germ and reticulocyte lysate systems. Heat-shock treatment of pea plants for 2 h at 42-45°C induces the expression of ˜10 nuclear coded proteins, among which several (18 kd, 19 kd, 22 kd) are predominant. A 22-kd protein is synthesized as a 26-kd precursor protein and is localized in a chloroplast membrane fraction in vivo. Following post-translational transport into intact chloroplasts in vitro of the 26-kd precursor, the protein is processed but the resulting 22-kd mature protein is localized in the chloroplast stroma. If, however, the in vitro transport is carried out with chloroplasts from heat-shocked plants, the 22-kd protein is preferentially transported to the chloroplast membrane fraction. In C. reinhardi the synthesis of poly(A)⁺RNAs coding for several HSPs is progressively and sequentially induced when raising the temperature for 1.5 h from 36°C to 42°C, while that of several preexisting RNAs is reduced. Various pre-existing poly(A)⁺RNAs endure in the cells at 42°C up to 5 h but are no longer translated in vivo, whereas some poly(A)⁻RNAs persist and are translated. As in pea, a poly(A)⁺RNA coded 22-kd HSP is localized in the chloroplast membranes in vivo, although it is translated as a 22-kd protein in vitro. The in vitro translated protein is not transported in isolated pea chloroplast which, however, processes and transports other nuclear coded chloroplast proteins of Chlamydomonas. The poly(A)⁺RNA coding for the 22-kd HSP appears after 1 h at 36°C. Its synthesis increases with the temperature of incubation up to 42°C, although it decreases after ˜2 h of heat treatment and the already synthesized RNA is rapidly degraded. The degradation is faster upon return of the cells to 26°C. None of the heat-induced proteins is identical to the light-inducible proteins of the chloroplast membranes.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.