Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported beta-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts

The large subunit of ribulose bisphosphate carboxylase from Anacystis nidulans 6301, and the β subunit of chloroplast ATP synthase from maize, were fused to the transit peptide of the small subunit of ribulose bisphosphate carboxylase from soybean. These proteins were assayed for post-translational import into isolated pea chloroplasts. Both proteins were imported into chloroplasts. Imported large subunits were associated with two distinct macromolecular structures. The smaller of these structures was a hybrid ribulose bisphosphate carboxylase holoenzyme, and the larger was the binding protein oligomer. Time-course experiments following import of the large subunit revealed that the amount of large subunit associated with the binding protein oligomer decreased over time, and that the amount of large subunit present in the assembled holoenzyme increased. We also observed that imported small subunits of ribulose bisphosphate carboxylase, although predominantly present in the holoenzyme, were also found associated with the binding protein oligomer. In contrast, the imported β subunit of chloroplast ATP synthase did not assemble into a thylakoid-bound coupling factor complex.     

Is ribulose bisphosphate carboxylase present in guard cell chloroplasts

Evidence for and against the presence of ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in guard cell chloroplasts is presented. Methods to investigate this question, including immunocytochemistry, are compared.     

Localization of ribulose bisphosphate carboxylase in the guard cells by an indirect, immunofluorescence technique

Ribulose bisphosphate carboxylase, a key enzyme in the photosynthetic carboxylation process, has been localized through an indirect immunofluorescent technique in the guard cells of some of the 41 species of plants examined. This sample includes 17 families of both dicotyledons and monocotyledons, one gymnosperm, and one pteridophyte. Plants were selected to represent all of the three major photosynthetic categories, namely C₃, C₄, and Crassulacean acid metabolism. Antibodies raised against tobacco (Nicotiana tabacum L.) ribulose bisphosphate carboxylase were used for this immunofluorescent study. A good degree of fluorescence was observed in the guard cells of seven out of 21 species exhibiting Crassulacean acid metabolism. C₃ plants exhibited a very low degree (almost negligible) of fluorescence, while the C₄ species did not exhibit any fluorescence.     

Genome Expression during Normal Leaf Development : 2. Direct Correlation between Ribulose Bisphosphate Carboxylase Content and Nuclear Ploidy in a Polyploid Series of Wheat

The quantitative relationships between ribulose bisphosphate carboxylase, nuclear ploidy, and plastid DNA content were examined in the nonisogenic polyploid series Triticum monococcum (2×), Triticum dicoccum (4×), and Triticum aestivum (6×). Ribulose bisphosphate carboxylase per mesophyll cell increased in step with each increase in nuclear ploidy so the ratios of ribulose bisphosphate carboxylase per mesophyll cell (picograms) to nuclear DNA per mesophyll cell (picograms) were almost identical in the three species. Ribulose bisphosphate carboxylase per plastid was 14.1, 14.7, and 16.8 picograms in the 2×, 4×, and 6× ploidy levels, respectively. Plastid area in these three species decreased with increasing nuclear ploidy so the concentration of ribulose bisphosphate carboxylase in the plastoids was 60% higher in the hexaploid compared to the diploid species. DNA levels per plastid were 64 and 67 femtograms for the diploid and tetraploid species, respectively, but were 40% less in the plastids of the hexaploid species. These relationships are discussed in terms of cellular and plastid control of ribulose bisphosphate carboxylase content.     

Ribulose bisphosphate carboxylase activity in halophilic Archaebacteria

Among the several strains of halobacteria grown heterotrophically, ribulose bisphosphate carboxylase activity was detected in those which accumulate poly (-hydroxybutyrate), viz. Haloferax mediterranei, Haloferax volcanii and Halobacterium marismortui. In H. mediterranei, the activity was present in cell extracts prepared after growth on a variety of carbohydrates. The ribulose bisphosphate carboxylase activity in H. mediterranei was inhibited by carboxyarabinitol bisphosphate, and the enzyme cross-reacted with antibodies raised against the spinach enzyme. CO₂ fixation by cell extract was stimulated by the addition of ATP and NADH. Preliminary data suggested that hydrogen could be a possible reductant.Abbreviations RuBP ribulose bisphosphate - Ru5P ribulose 5-phosphate - R5P ribose 5-phosphate - CABP carboxyarabinitol bisphosphate - PHB poly (-hydroxybutyrate) - DTT dithiothreitol     

Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts

Ribulose bisphosphate carboxylase consists of cytoplasmically synthesized "small" subunits and chloroplast-synthesized "large" subunits. Large subunits of ribulose bisphosphate carboxylase synthesized in vivo or in organello can be recovered from intact chloroplasts in the form of two different complexes with sedimentation coefficients of 7S and 29S. About one-third to one-half of the large subunits synthesized in isolated chloroplasts are found in the 7S complex, the remainder being found in the 29S complex. Upon prolonged illumination of the chloroplasts, newly synthesized large subunits accumulate in the 18S ribulose bisphosphate carboxylase molecule and disappear from both the 7S and the 29S large subunit complexes. The 29S complex undergoes an in vitro dissociation reaction and is not as stable as ribulose bisphosphate carboxylase. The data indicate that (a) the 7S large subunit complex is a chloroplast product, the (b) the 29S large subunit complex is labeled in vivo, that (c) each of these two complexes can account quantitatively for all the large subunits assembled into RuBPCase in organello, and that (d) excess large subunits are degraded in chloroplasts.     

Ribulose bisphosphate carboxylase from a mutant strain of Chlamydomonas reinhardii deficient in chloroplast ribosomes

The ac-20 mutant strain of the unicellular green alga, Chlamydomonas reinhardii, lacks both chloroplast ribosomes and ribulose bisphosphate carboxylase activity when grown on organic medium. Under these conditions, the cells do not posses pools of either the large or small subunit of this enzyme. When transferred to inorganic medium, the carboxylase activity recovers. During this recovery, de novo synthesis of both subunits occurs. Synthesis of both subunits is inhibited by chloramphenicol even when possible free subunit pools rather than just the subunits incorporated into whole enzyme are examined.Abbreviations RubP ribulose bisphosphate - CAP D-threochloramphenicol - CHI cycloheximide - PPO 2,5-diphenyloxazole - POPOP 1,4-bis[2(5-phenyloxazolyl)]-benzene - SDS sodium dodecyl sulfate     

Ribulose 1,5-bisphosphate and activation of the carboxylase in the chloroplast

Ribulose 1,5-bisphosphate in the chloroplast has been suggested to regulate the activity of the ribulose bisphosphate carboxylase/oxygenase. To generate high levels of ribulose bisphosphate, isolated and intact spinach chloroplasts were illuminated in the absence of CO₂. Under these conditions, chloroplasts generate internally up to 300 nanomoles ribulose 1,5-bisphosphate per milligram chlorophyll if O₂ is also absent. This is equivalent to 12 millimolar ribulose bisphosphate, while the enzyme, ribulose bisphosphate carboxylase, offers up to 3.0 millimolar binding sites for the bisphosphate in the chloroplast stroma. During illumination, the ribulose bisphosphate carboxylase is deactivated, due mostly to the absence of CO₂ required for activation. The rate of deactivation of the ribulose bisphosphate carboxylase was not affected by the chloroplast ribulose bisphosphate levels. Upon addition of CO₂, the carboxylase in the chloroplast was completely reactivated. Of interest, addition of 3-phosphoglycerate stopped deactivation of the carboxylase in the chloroplast while ribulose bisphosphate accumulated. With intact chloroplasts in light, no correlation between deactivation of the carboxylase and ribulose bisphosphate levels could be shown.     

Proteolysis of endogenous substrates in senescing oat leaves : I. Specific degradation of ribulose bisphosphate carboxylase

Proteolysis of ribulose bisphosphate carboxylase (RuBPCase) during senescence was monitored using oat leaf segments (Avena sativa cv Victory), kept in the dark. We here report the development of a novel approach for measuring protein degradation of endogenous substrates both in situ and in vitro in crude extracts using specific antibodies against highly purified polypeptides. The proteolytic products were separated on sodium dodecyl sulfate-gels. They were then electrotransferred onto nitrocellulose paper and identified with specific antibodies to both the large and small subunits of RuBPCase. We could show differences in pH optima between two proteases degrading the subunits of RuBPCase. While both subunits were best hydrolyzed in acid and basic pH, they degraded differently at neutral pH. Furthermore, the large subunit displayed a different pattern of degradative products at the different pH levels. Older leaf segments, which were incubated in darkness, underwent enhanced proteolysis, as compared with young ones. These results show the advantages of the assay in demonstrating: (a) in situ proteolysis of specific substrates in crude extracts without further purification; (b) in vitro differential proteolysis of endogenous substrates during senescence.     

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

The biosynthesis of ribulose bisphosphate carboxylase. Uncoupling of the synthesis of the large and small subunits in isolated soybean leaf cells

Isolated leaf cells from soybean (Glycine max) incorporate [35S]methionine into protein at a linear rate for at least 5h. Analysis of the products of incorporation by one-dimensional and two-dimensional polyacrylamide gel electrophoresis shows that major products are the large and small subunits of the chloroplast enzyme, ribulose bisphosphate carboxylase. The large subunit is synthesized by chloroplast ribosomes and the small subunit by cytoplasmic ribosomes. Addition of chloramphenicol to the cells reduces incorporation into the large subunit without affecting incorporation into the products of cytoplasmic ribosomes. Addition of cycloheximide or 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide stops incorporation into the small subunit, but large subunit continues to be made for at least 4 h. For accurate estimates of incorporation into the large subunit, it is essential to use two-dimensional gel electrophoresis, because the large subunit region on one-dimensional gels is contaminated with the products of cytoplasmic ribosomes. Newly synthesized large subunits continue to enter complete molecules of ribulose bisphosphate carboxylase in the absence of small subunit synthesis. These results suggest that, in contrast to the situation in algal cells, the synthesis of the two subunits of ribulose bisphosphate carboxylase in the different subcellular compartments of higher plant cells is not tightly coupled over short time periods, and that a pool of small subunits exists in these cells. The results are disucssed in relation to possible mechanisms for the integration of the synthesis of the large and small subunits of ribulose bisphosphate carboxylase.     

Protein degradation in lemna with particular reference to ribulose bisphosphate carboxylase: I. The effect of light and dark

Ribulose bisphosphate carboxylase from Lemna minor resembles the structure reported for the enzyme from other plants. When grown in the light, the enzyme appears to undergo little or no degradation, as measured by a double-isotope method. This situation is similar to that reported for wheat and barley, but is unlike that reported for maize, where the enzyme degrades at the same rate as total protein. Prolonged periods of darkness usually induce leaf senescence, characterized by the rapid degradation of chlorophyll and protein, with ribulose bisphosphate carboxylase undergoing preferential degradation. In L. minor there is selective protein degradation in the dark, but chlorophyll and ribulose bisphosphate carboxylase are stable when fronds are kept in the darkness for up to 8 days. It appears that Lemna is not programmed to senesce, or at least that darkness does not induce senescence in Lemna. Although there is no evidence for in vivo degradation or modification of ribulose bisphosphate carboxylase during prolonged periods of darkness, extracts from fronds which have been kept in the dark for periods in excess of 24 hours convert ribulose bisphosphate carboxylase to a more acidic form. The properties of the dark-induced system which acts on ribulose bisphosphate carboxylase, suggest that it may be a mixed function oxidase. The proposition that the selectivity of protein degradation is genetically determined, so that the rate at which a protein is degraded is determined by its charge or size, was tested for fronds grown in the light or maintained in the dark. There was no significant correlation between protein degradation and either charge or size, in light or dark.     

High temperature effects on RuBP carboxylase and carbonic anhydrase activity in two tomato cultivars

The effects of temperature on ribulose bisphosphate carboxylase activity were studied in two tomato ( Lycopersicon esculentum Mill.) cultivars which differed in sensitivity to high temperatures. The heat tolerant cultivar, Saladette, had a smaller reduction in photosynthesis and a smaller increase in mesophyll resistance then the sensitive cultivar Roma VF, after 24 h at 35 to 40°C. One hour in vitro treatments at 50°C decreased the activity of ribulose bisphosphate carboxylase extracted from Roma VF by 75%, while Saladette was not affected. Heat stress to the entire plant caused greater inhibition of ribulose bisphosphate carboxylase in the heat sensitive cultivar. Ribulose bisphosphate carboxylase activity in both cultivars decreased with heat treatment but recovered under normal temperatures. Ribulose bisphosphate oxygenase activity decreased similarly in both cultivars under 37/18°C day/night temperatures, which resulted in an apparent change in the relative carboxylase/oxygenase activity of the two cultivars. Carbonic anhydrase activity was slightly greater in Saladette than in Roma VF but no significant decrease in activity was observed in plants exposed to high temperatures.     

Subunit dissociation and reconstitution of ribulose-1,5-bisphosphate carboxylase from Chromatium vinosum

The large and small subunits of ribulose bisphosphate carboxylase from Chromatium vinosum were dissociated and separated at pH 9.6 by sucrose density gradient centrifugation. After further purification by gel filtration, the small subunit fraction contained no carboxylase activity. The large subunit fraction was highly depleted of small subunit based on analysis by denaturing polyacrylamide gel electrophoresis. Carboxylase activity of the large subunit fraction was approximately 1% of the untreated native enzyme. Addition of purified small subunit to the large subunit fraction yielded increases of up to 67-fold in carboxylase activity, further indicating that both subunit types are required for catalysis by this enzyme. The isolated large subunit was fully capable of high-affinity activator 14CO2 binding in the presence of Mg2+ and 2-carboxyarabinitol bisphosphate, indicating that the activator and catalytic sites were not grossly denatured by the depletion of small subunit. Kinetic constants of the native C. vinosum enzyme defined a new class of ribulose bisphosphate carboxylase, which permits the detection of possible kinetic differences if the large and small subunits can be favorably reassembled with those of another kinetic class. From experiments with the enzymes from tobacco and spinach leaves it is concluded that the enzyme from higher plant sources is not suitable for such dissociation/reconstitution-type experiments.     

A model for the kinetics of activation and catalysis of ribulose 1,5-bisphosphate carboxylase.

Further evidence for time-dependent interconversions between active and inactive states of ribulose 1,5-bisphosphate carboxylase is presented. It was found that ribulose bisphosphate oxygenase and ribulose bisphosphate carboxylase could be totally inactivated by excluding CO2 and Mg2+ during dialysis of the enzyme at 4 degrees C. When initially inactive enzyme was assayed, the rate of reaction continually increased with time, and the rate was inversely related to the ribulose bisphosphare concentration. The initial rate of fully activated enzyme showed normal Michaelis-Menten kinetics with respect to ribulose bisphosphate (Km = 10muM). Activation was shown to depend on both CO2 and Mg2+ concentrations, with equilibrium constants for activation of about 100muM and 1 mM respectively. In contrast with activation, catalysis appeared to be independent of Mg2+ concentration, but dependent on CO2 concentration, with a Km(CO2) of about 10muM. By studying activation and de-activation of ribulose bisphosphate carboxylase as a function of CO2 and Mg2+ concentrations, the values of the kinetic constants for these actions have been determined. We propose a model for activation and catalysis of ribulose bisphosphate carboxylase: (see book) where E represents free inactive enzyme; complex in parentheses, activated enzyme; R, ribulose bisphosphate; M, Mg2+; C, CO2; P, the product. We propose that ribulose bisphosphate can bind to both the active and inactive forms of the enzyme, and slow inter-conversion between the two states occurs.     

The Regulation of Photosynthesis in Leaves of Field-Grown Spring Wheat (Triticum aestivum L., cv Albis) at Different Levels of Ozone in Ambient Air

Wheat (Triticum aestivum L. cv Albis) was grown in open-top chambers in the field and fumigated daily with charcoal-filtered air (0.015 microliters per liter O₃), nonfiltered air (0.03 microliters per liter O₃), and air enriched with either 0.07 or 0.10 microliters per liter ozone (seasonal 8 hour/day [9 am-5 pm] mean ozone concentration from June 1 until July 10, 1987). Photosynthetic ¹⁴CO₂ uptake was measured in situ. Net photosynthesis, dark respiration, and CO₂ compensation concentration at 2 and 21% O₂ were measured in the laboratory. Leaf segments were freeze-clamped in situ for the determination of the steady state levels of ribulose 1,5-bisphosphate, 3-phosphoglycerate, triose-phosphate, ATP, ADP, AMP, and activity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. Photosynthesis of flag leaves was highest in filtered air and decreased in response to increasing mean ozone concentration. CO₂ compensation concentration and the ratio of dark respiration to net photosynthesis increased with ozone concentration. The decrease in photosynthesis was associated with a decrease in chlorophyll, soluble protein, ribulose bisphosphate carboxylase/oxygenase activity, ribulose bisphosphate, and adenylates. No decrease was found for triose-phosphate and 3-phosphoglycerate. The ratio of ATP to ADP and of triosephosphate to 3-phosphoglycerate were increased suggesting that photosynthesis was limited by pentose phosphate reductive cycle activity. No limitation occurred due to decreased access of CO₂ to photosynthetic cells since the decrease in stomatal conductance with increasing ozone concentration did not account for the decrease in photosynthesis. Ozonestressed leaves showed an increased degree of activation of ribulose bisphosphate carboxylase/oxygenase and a decreased ratio of ribulose bisphosphate to initial activity of ribulose bisphosphate carboxylase/oxygenase. Nevertheless, it is suggested that photosynthesis in ozone stressed leaves is limited by ribulose bisphosphate carboxylation possibly due to an effect of ozone on the catalysis by ribulose bisphosphate carboxylase/oxygenase.     

The relative catalytic specificities of the large subunit core of Synechococcus ribulose bisphosphate carboxylase/oxygenase

The relative specificities of the carboxylase and oxygenase reactions catalyzed by the recombinant large subunit core (L8) of Synechococcus ribulose 1,5-bisphosphate carboxylase have been determined. The L8 core still retained the ability to catalyze both reactions but at a much reduced turnover rate, about 0.6% of the holoenzyme. The fate of ribulose 1,5-bisphosphate during carboxylation and oxygenation by L8 was compared with the Synechococcus holoenzyme (reconstituted from L8 and recombinant small subunits), the carboxylase from Rhodospirullum rubrum, and that of spinach. The absence of small subunits had no significant effect on the partitioning of the bisphosphate substrate between the two reactions. Thus the course of the two competing reactions is a characteristic of the structural elements that compose the L-subunits, whereas the S-subunits exert their effect on factors common to both reactions such as the specificity of the bisphosphate substrate.     

Influence of Inorganic Phosphate on Photosynthesis of Wheat Chloroplasts: II. RIBULOSE BISPHOSPHATE CARBOXYLASE ACTIVITY

Isolated wheat chloroplasts were pre-incubated in the dark inthe presence of various concentrations of inorganic phosphatewith or without carbon dioxide, oxaloacetate, glycerate, and3-phosphoglycerate. The effect of subsequent illumination onphotosynthetic oxygen evolution, ribulose bisphosphate carboxylaseactivity, ATP content, and ribulose bisphosphate content wasinvestigated. Inorganic phosphate had little effect on ribulosebisphosphate carboxylase activity in darkness or during theinitial phase of illumination, but it prevented the declinein activity that occurred during later stages of illumination,when photoreduction of CO₂ was decreasing in rate. Additionof inorganic phosphate to chloroplasts illuminated without phosphaterestored the ribulose bisphosphate carboxylase activity, increasedthe ATP, and decreased the ribulose bisphosphate in the organelles.The responses to CO₂, oxaloacetate, glycerate, and 3-phosphoglyceratesuggest that the decreased activity of ribulose bisphosphatecarboxylase during photosynthesis results from ATP consumption. Purified ribulose bisphosphate carboxylase was activated byinorganic phosphate, but this activation did not occur in thepresence of ATP. ATP inhibited ribulose bisphosphate carboxylasewhen it was present in combination with various photosyntheticmetabolites. Inactivation of ribulose bisphosphate carboxylase in chloroplasts,illuminated in the absence of inorganic phosphate, is not dueto lack of activation by inorganic phosphate or ATP. It mayresult from decreased stromal pH. Key words: Ribulose bisphosphate carboxylase, Chloroplasts, Wheat, Phosphate, ATP     

Examination of subunit interactions at the active site of ribulose 1,5-bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum by hybridization of site-directed mutants

The Protein Journal - The two active sites of homodimeric ribulose bisphosphate carboxylase/oxygenase fromRhodospirillum rubrum are constituted by interacting domains of adjacent subunits, in which...     

Immunological evidence for a ribulose bisphosphate carboxylase-like protein in the symbiotic dinoflagellate Symbiodinium sp.

Whereas previously there has been no convincing evidence for ribulose bisphosphate carboxylase in dinoflagellates, a strong and highly specific reaction was observed when antibodies to the denatured large subunit of the (silver beet) protein were used to probe Western blots of whole soluble fractions of various Symbiodinium isolates. No reaction was observed using extracts from Symbiodinium isolated from a host which had been maintained under low light intensity. The results imply extensive sequence homology between the large subunit of ribulose bisphosphate carboxylase and a dinoflagellate protein of M , approximately 35 000.