Cloning and characterization of the calreticulin gene from Ricinus communis L.

A full-length cDNA encoding a calreticulin-like protein was isolated by immune-screening a germinating castor bean endosperm cDNA library with antisera raised to the total lumenal fraction of purified plant endoplasmic reticulum. The calcium-binding properties of the recombinant protein were characterized and shown to be essentially identical to those reported for the mammalian calreticulin. Calcium overlays and immune blot analysis confirmed the endoplasmic lumenal identity of this reticuloplasmin. Probing protein blots of endoplasmic reticulum subfractions with radio-iodinated calreticulin showed specific associations with various polypeptides including one identified as the abundant reticuloplasmin protein disulfide isomerase. Characterization of the corresponding genomic clones revealed that calreticulin is encoded by a single gene of 3 kb in castor. The full genomic sequence reveals the presence of 12 introns, 12 translated exons, and one exon containing the last three amino acids of the translated sequence and the 3'-untranslated region of the gene. Northern blot analysis of RNA isolated from various organ tissues showed a basal constitutive level of expression throughout the plant, but more abundant mRNA being detected in tissues active in secretion. This was confirmed by analysis of transgenic tobacco plants containing 1.8 kb of 5'-untranslated genomic sequence fused to the beta-glucuronidase reporter gene (GUS) showed a more localized pattern of expression. Activity being localized to the vasculature (phloem, root hairs and root tip) in vegetative tissue, and being strongly expressed in the floral organs including the developing and germinating seed.

Characterization of a structurally and functionally diverged acyl-acyl carrier protein desaturase from milkweed seed.

A cDNA for a structurally variant acyl-acyl carrier protein (ACP) desaturase was isolated from milkweed (Asclepias syriaca) seed, a tissue enriched in palmitoleic (16:1delta9)* and cis-vaccenic (18:1delta11) acids. Extracts of Escherichia coli that express the milkweed cDNA catalyzed delta9 desaturation of acyl-ACP substrates, and the recombinant enzyme exhibited seven- to ten-fold greater specificity for palmitoyl (16:0)-ACP and 30-fold greater specificity for myristoyl (14:0)-ACP than did known delta9-stearoyl (18:0)-ACP desaturases. Like other variant acyl-ACP desaturases reported to date, the milkweed enzyme contains fewer amino acids near its N-terminus compared to previously characterized delta9-18:0-ACP desaturases. Based on the activity of an N-terminal deletion mutant of a delta9-18:0-ACP desaturase, this structural feature likely does not account for differences in substrate specificities.

Structure and analysis of phospholipase D gene from Ricinus communis L.

Phospholipase D (PLD; EC 3.1.4.4) has been proposed to play a pivotal role in various cellular processes, but molecular understanding of this enzyme is rather limited. This report describes the nucleotide sequence, structure, and genomic organization of a PLD gene from castor bean (Ricinus communis L. cv. Hale). The PLD gene was isolated from a castor bean genomic library using the PLD cDNA as a hybridization probe. Sequence comparison with the PLD cDNA revealed that the PLD gene consisted of four exons and three introns, one of which interrupts the 5'-untranslated region. Southern blot analysis indicated that the cloned PLD gene was present as a single-copy gene, and yet there were other PLD or PLD-related sequences in the castor bean genome.

Molecular characterisation of plant endoplasmic reticulum. Identification of protein disulfide-isomerase as the major reticuloplasmin.

Purified endoplasmic reticulum devoid of contaminating endomembranes has been isolated from both germinating and developing castor bean endosperm by a modified two-step centrifugation procedure. These membranes have been characterised for protein and lipid composition, subfractionated into lumenal and integral membrane protein fractions, and antisera raised to these two components. A cDNA clone encoding a major lumenal protein of 55 kDa was cloned using affinity-purified antisera and shown to encode a protein with strong sequence similarity to the endoplasmic reticulum lumenal chaperone protein disulfide-isomerase. Northern and Southern blot analysis showed that the mRNA from a single-copy gene was constitutively expressed in all tissues investigated, but was preferentially expressed in developing seed where it was the most abundant lumenal protein. Expression of the recombinant protein in Escherichia coli yielded a homodimer with a molecular mass of 110 kDa with protein disulfide-isomerase catalytic activity, thus confirming identity of this protein.

Ovarian carcinoma-associated TaqI restriction fragment length polymorphism in intron G of the progesterone receptor gene is due to an Alu sequence insertion.

Alu sequences, short, repetitive transposable DNA elements, are factors in a number of genetic diseases. We previously identified a germline TaqI RFLP, located in intron G of the human progesterone receptor gene, that showed an association with the incidence of sporadic ovarian carcinoma. Furthermore, the polymorphism was characterized as a small (approximately 300-bp) insertion that was inherited in a Mendelian fashion. Because of its insertional character, we named this polymorphism PROGINS. We report the identification of PROGINS as a 306-bp Alu element of the PV or HS-1 Alu subfamily.

Spinach thylakoid polyphenol oxidase: cloning, characterization, and relation to a putative protein kinase.

A 64-kDa protein was purified from an octyl glucoside/cholate extract of spinach thylakoids. N-Terminal analysis yielded 23 residues of sequence, of which the first 15 were identical to a sequence reported [Gal, A., Herrmann, R. G., Lottspeich, F., & Ohad, I. (1992) FEBS Lett. 298, 33-35] for a protein kinase with specificity toward the photosystem II light-harvesting complex (LHC-II). We report the complete sequence of this 64-kDa protein, deduced from cDNA clones. The transit peptide has a chloroplast import signal at the N-terminus and a C-terminal hydrophobic span bounded by basic amino acids that predicts localization of the protein to the thylakoid lumen. The mature protein sequence is about 50% identical to several polyphenol oxidases (PPOs). Canonical protein kinase motifs are absent, as are sequences characteristic of ATP-binding sites. The mature protein resembles arthropodan hemocyanin (Hc), possessing three major domains. The N-terminal domain is rich in cysteine residues and predicted alpha-helices. The central domain has a conserved motif, N-terminal to a presumptive Cu-A site, that is not found in tyrosinases or Hc and is proposed as the provider of a third imidazole ligand to Cu-A. An unusual 13-residue, glutamine-rich link begins a C-terminal domain containing 7 predicted beta-strands which, by analogy with Hc, may form an antiparallel beta-barrel. We conclude that this 64-kDa polypeptide is a lumenal PPO and the precursor of a 42.5-kDa PPO form described previously [Golbeck, J. H., & Cammarata, K. V. (1981) Plant Physiol. 67, 977-984].(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of an alkaline phosphatase from pea thylakoids.

Endogenous dephosphorylation of the light-harvesting chlorophyll-protein complex of photosystem II in pea (Pisum sativum, L. cv Progress 9) thylakoids drives the state 2 to state 1 transition; the responsible enzyme is a thylakoid-bound, fluoride-sensitive phosphatase with a pH optimum of 8.0 (Bennett J [1980] Eur J Biochem 104: 85-89). An enzyme with these characteristics was isolated from well-washed thylakoids. Its molecular mass was estimated at 51.5 kD, and this monomer was catalytically active, although the activity was labile. The active site could be labeled with orthophosphate at pH 5.0. High levels of alkaline phosphatase activity were obtained with the assay substrate, 4-methylumbelliferyl phosphate (350 micromoles per minute per milligram purified enzyme). The isolated enzyme functioned as a phosphoprotein phosphatase toward phosphorylated histone III-S and phosphorylated, photosystem II-enriched particles from pea, with typical activities in the range of 200 to 600 picomoles per minute per milligram enzyme. These activities all had a pH optimum of 8.0 and were fluoride sensitive. The enzyme required magnesium ion for maximal activity but was not dependent on this ion. Evidence supporting a putative function for this phosphatase in dephosphorylation of thylakoid proteins came from the inhibition of this process by a polyclonal antibody preparation raised against the partially purified enzyme.

Reactive blue 2 is a potent inhibitor of a thylakoid protein kinase.

The anthraquinone dye reactive blue 2 was found to be a potent inhibitor of a protein kinase isolated and purified from thylakoids. This enzyme was also inhibited in situ, with corresponding inhibition of ATP-dependent quenching of the chlorophyll fluorescence. The mode of inhibition was noncompetitive, with a Ki of 8 microM for the membrane-bound kinase, and 6 microM for the purified kinase. The inhibitor did not modify the substrate preference of the endogenous kinase and could be removed from the membrane by washing. Unlike reactive blue 2, the enzyme did not partition into detergent micelles and is therefore presumably not a hydrophobic, intrinsic membrane protein.

Purification of membrane-bound ferredoxin: NADP(+) oxidoreductase and of plastocyanin from a detergent extract of washed thylakoids.

A method is described for the isolation and purification of ferredoxin-NADP(+) oxidoreductase (FNR, E.C. 1.18.1.2) and plastocyanin from spinach thylakoids. FNR is recovered from pools which are loosely and tightly bound to the membrane, with minimal disruption of pigment-protein complexes; yields can thus be higher than from procedures which extract only the loosely bound enzyme.Washed thylakoid membranes were incubated with the dipolar ionic detergent CHAPS (3-(3-cholamidopropyl-dimethylammonio)-1-propane-sulfonate). This provided an extract containing FNR and PC as its principal protein components, which could be rapidly separated from one another by chromatography on an anion-exchange column. FNR was purified to homogeneity (as judged from sodium dodecyl sulfate gel electrophoresis and the ratio between protein and flavin absorption maxima), using chromatography on phosphocellulose followed by batchwise adsorption to, and elution from hydroxylapatite. Plastocyanin was further purified on a Sephadex G-75 molecular sieve column.A typical yield, obtained in 3-4 days from 1 kg of deveined spinach leaves, was 7 mg of pure FNR (a single protein of Mr=37,000) and 3.5 mg of plastocyanin.

Protein kinases of the thylakoid membrane.

The claim of Racker and co-workers (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156 and Lucero, H. A., Lin, Z. F., and Racker, E. (1982) J. Biol. Chem. 257, 12157-12160) that two protein kinases, designated CPK1 (25 kDa) and CPK2 (38 kDa), are present in spinach thylakoid membranes was investigated in light of results from this laboratory (Coughlan, S. J., and Hind, G. (1986) J. Biol. Chem. 261, 11378-11385) showing that 75-80% of the measurable protein kinase activity of isolated thylakoids is attributable to a protein kinase of 64 kDa apparent molecular mass. Extraction of thylakoid membranes with octyl glucoside/cholate according to the procedure of Lin et al. (Lin, Z. F., Lucero, H. A., and Racker, E. (1982) J. Biol. Chem. 257, 12153-12156) released proteins assignable to CPK1 and CPK2 on the basis of photoaffinity labeling with 8-azido-[32P]ATP. The 64-kDa protein kinase was present in this extract and accounted for greater than 80% of the total phosphotransferase activity toward lysine-rich histone as substrate; it was not labeled by the photoaffinity reagent. The three presumptive kinases were purified by ammonium sulfate precipitation, sucrose density gradient centrifugation, hydroxylapatite chromatography, and affinity chromatography. CPK1 was specifically eluted from Cibacron blue-Sepharose by 10 mM ATP; it electrophoresed on denaturing polyacrylamide gels as a single band with apparent molecular mass of 25 kDa. Its specific activity toward lysine-rich histone as substrate was approximately 250 pmol of phosphate transferred (mg protein)-1 min-1. The 64-kDa protein kinase was eluted from the affinity column by 1% (w/v) lithium dodecyl sulfate or from a histone IIIs-Sepharose affinity column by 0.25 M NaCl. Its specific activity towards lysine-rich histone was 100-200 times greater than that of CPK1. CPK2 eluted from the Cibacron blue affinity column in 10 mM NADP+; it had an apparent molecular mass of 38 kDa, possessed NADPH-dependent diaphorase activity (specific activity: 225 nmol of ferricyanide reduced (mg protein)-1 min-1), and cross-reacted with immunoglobulin raised against purified ferredoxin:NADP+ oxidoreductase, with which it was thus identified. Kinase activity was not detectable in CPK2 or in reductase isolated by conventional procedures.

Removal of ferredoxin:NADP+ oxidoreductase from thylakoid membranes, rebinding to depleted membranes, and identification of the binding site.

Ferredoxin-NADP+ oxidoreductase associates with thylakoid membranes into two pools of different binding strength that are experimentally distinguished on the basis of resistance to removal by washes in low ionic strength media. The nondenaturing zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid is uniquely able to remove the more tightly bound pool of enzyme, without solubilization of major membrane proteins. The reconstitution of reductase onto depleted thylakoid membranes requires available membrane binding sites and cations, in order of effectiveness trivalent greater than divalent greater than monovalent. The hetero/bifunctional 125I-iodinated Denny-Jaffe cross-linking reagent yields a 54-kDa, covalently cross-linked adduct between ferredoxin-NADP+ oxidoreductase and a component of the thylakoid membrane. Our results show that the more tightly bound pool of enzyme is associated with the 17.5-kDa reductase-binding protein (Vallejos, R. H., Ceccarelli, E., and Chan, R. (1984) J. Biol. Chem. 259, 8048-8051).

Purification and characterization of a membrane-bound protein kinase from spinach thylakoids.

A protein kinase was isolated from spinach thylakoid membranes by solubilization with octyl glucoside and cholate. The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, and sucrose density centrifugation, followed by affinity chromatography on either Affi-Gel blue (yielding denatured enzyme) or on histone cross-linked to Sepharose (yielding active enzyme). Electrophoresis on denaturing polyacrylamide gels, followed by staining with silver, revealed the kinase as a single band corresponding to an apparent molecular mass of 64 kDa. The active enzyme underwent autophosphorylation and could be detected by autoradiography following incubation with [gamma-32P]ATP and Mg2+ ion. The specific phosphotransferase activity of purified kinase was approximately 30 nmol of phosphate min-1 (mg protein)-1 with lysine-rich histone (III-S or V-S) as substrate; casein was phosphorylated at approximately 30% of this rate. The physiological substrate for the kinase is presumed to be light-harvesting chlorophyll a/b protein complex. In solubilized form, this was phosphorylated at approximately 10% of the rate observed with histone III-S as substrate, or 10-100 times slower than the estimated rate of phosphorylation of the light-harvesting complex in situ. Possible reasons for this shortfall are considered. The kinase is proposed as the principal effector of thylakoid protein phosphorylation and associated State transition phenomena.

The role of glycinebetaine in the protection of spinach thylakoids against freezing stress.

The quaternary ammonium compound glycinebetaine has been tested for cryoprotective properties, using isolated spinach thylakoids as a model membrane system. The effect of a 3-h,-20°C freezing regime on different photosynthetic parameters was measured. These parameters were the light-stimulated ΔpH formation and dark ΔpH decay, light-stimulated proton uptake, electron flow through photosystem II, photosystem I and total linear electron flow, and pyocyanine-mediated cyclic photophosphorylation. It was shown that below 100 mM glycinebetaine was superior as a cryoprotectant to sucrose on a molar, a molal and an activity basis. At higher concentrations, glycinebetaine was less efficient in preventing inactivation of thylakoids during freezing than sucrose. These observations are discussed in relation to the permeability of biomembranes to glycinebetaine and the colligative theory of cryoprotection. It is concluded that colligative protection is modified by direct interaction between cryoprotectant and membranes.

Glycinebetaine biosynthesis and its control in detached secondary leaves of spinach.

In secondary leaves from spinach plants pretreated in vermiculite for 24 h with 300 mM NaCl, glycinebetaine accumulated at a rate of circa 0.16 µmol 100 µg(-1) Chl d(-1) (2 µmol g(-1) FW d(-1)), about three times the rate of control plants. The soluble carbohydrate and free amino acid contents did not increase significantly following salinisation until after 4 d when the relative growth rate also decreased. Leaf proline levels remained very low throughout the experimental period. K(+) on a tissue water basis remained constant at 200 mM while Cl(-) and Na(+) levels increased linearly to reach 175 and 100 mM respectively after 5 d of saline treatment. The osmotic pressure of leaf tissue also increased from 300 to 500 mosmol kg(-1). These experimental conditions were considered suitable to study glycinebetaine biosynthesis and its induction by salinity in the absence of marked growth inhibition or metabolic disturbance. Radioactive labelled [(14)C]serine, ethanolamine and choline (all 1 µmol, 13.3 MBq in 10 µl) were fed to detached secondary leaves via the petiole 24 h after the exposure of plants to salt. The rate of isotope incorporation into water soluble products, lipids and residue was measured over a further 24 h. The major metabolic fate of exogenous [(14)C]choline and [(14)C]ethanolamine was incorporation into glycinebetaine while less (14)C-label was found in phosphatidyl choline and phosphatidyl ethanolamine. Incorporation rates were identical in control and salinised leaves and were adequate to account for observed values of glycinebetaine accumulation previously reported in spinach. In contrast the labelling of glycinebetaine from [(14)C]serine was twice as great in salinated plants as in the controls. These results, together with short term labelling experiment with [(14)C]ethanolamine using leaf slices, were consistent with the formation of glycinebetaine via serine, ethanolamine and its methylated derivatives to choline with some control being exerted at the serine level. However a flux through the phosphorylated intermediates is not excluded.From a consideration of these results and the published data on barley subjected to water stress (Hanson and Scott, 1980 Plant Physiol. 66, 342-348) there appear to be significant differences in the biosynthetic pathways in spinach and barley.

The role of Golgi bodies in polysaccharide sulphation in Fucuszygotes.

The cell wall of 24-h zygotes of Fucus serratus is composed of 3 layers--an inner fibrillar layer (sulphated fucan), an outer fibrillar layer (alginic aicd/cellulose) and an exterior amorphous layer (sulphated fucan, alginic acid). The 2 layers containing sulphated fucan are preferentially thickened at the rhizoid pole. Light- and electron-microscope autoradiographic pulse-chase experiments on 22-h zygotes using 35SO2-(4) show the Golgi bodies to be the sites of fucan sulphation. The isolation and characterization of isolated Golgi-rich fractions from 22-h zygotes shows that the first detectable labelled macromolecule is associated with these fractions 2 min after addition of 35SO2-(4). The sulphate acceptor molecule has been partially characterized. 35S-APS and 35S-paps are detectable in the soluble fraction 0.5 min after addition of 35SO2-(4). The results are discussed in relation to other published work on the differentiation of Fucus embryos and on polysaccharide sulphation.