Purification of ribulose 1,5-bisphosphate carboxylase/oxygenase and of carboxysomes from Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa (Montagu)

The bacterial symbionts of many marine invertebrates contain ribulose 1,5-bisphosphate (RuBP) carboxylase but apparently no carboxysomes, polyhedral bodies containing RuBP carboxylase. In the few cases where polyhedral bodies have been observed they have not been characterised enzymatically. Polyhedral bodies, 50–90 nm in diameter, were observed in thin cell sections of Thiobacillus thyasiris the putative symbiont of Thyasira flexuosa and RuBP carboxylase activity was detected in both soluble and particulate fractions after centrifugation of cell-free extracts. RuBP carboxylase purified 90-fold from the soluble fraction was of high molecular weight and consisted of large and small subunits, with molecular weights of 53,110 and 11,100 respectively. Particulate RuBP carboxylase activity was associated with polyhedral bodies 50–100 nm in diameter, as revealed by density gradient centrifugation and electron microscopy. Therefore, the polyhedral bodies were inferred to be carboxysomes. Native electrophoresis of isolated carboxysomes demonstrated a major band which comigrated with the purified RuBP carboxylase and three minor bands of lower molecular weight. Sodium dodecyl-sulphate (SDS) gel electrophoresis of SDS-dissociated carboxysomes demonstrated nine major polypeptides two of which were the large and small subunits of RuBP carboxylase. The RuBP carboxylase subunits represented 21% of the total carboxysomal protein. The most abundant polypeptide had a molecular weight of 40,500. Knowledge of carboxysome composition is necessary to provide an understanding of carboxysome function.Abbreviations FPLC fast performance liquid chromatography - IB isolation buffer - PAGE polyacrylamide gel electrophoresis - RuBP carboxylase - ribulose 1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl-sulphate     

Protein transport in intact, purified pea etioplasts

We have developed a method to isolate intact, purified pea etioplasts. These etioplasts were capable of recognizing, transporting, and processing the precursor form of the small subunit of the ribulose-1,5-bisphosphate carboxylase, a protein which is not detectable at this developmental stage. Transport of proteins was completely dependent on ATP and could not be substituted for or stimulated by light. The transported precursor protein was processed to its proper molecular weight. The mature form of the small subunit was assembled with the large subunit of the ribulose-1,5-bisphosphate carboxylase already present at this stage to form an oligomer. Protein transport was completely abolished using the phosphatase inhibitor sodium fluoride. This is the first time protein transport has been demonstrated in isolated, purified etioplasts.     

A point mutation in the N-terminus of ribulose-1,5-bisphosphate carboxylase affects ribulose-1,5-bisphosphate binding

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans was used to generate novel enzymes. Two conserved residues, threonine 4 and lysine 11 in the N-terminus were changed. The substitution of threonine 4 with serine or valine had little effect on the kinetic parameters. The substitution of lysine 11 with leucine, which is non-polar, increased the K _m for ribulose-1,5-bisphosphate from 82 to 190 M but its replacement with glutamine, which has polar properties, had no appreciable effect.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - LSU large sub-unit of Rubisco - SSU small subunit of Rubisco We thank Dr. S. Gutteridge (DuPont, Wilmington, USA) for structural information and for his comments on the results described. The technical assistance of Mr. A. Cowland and Mr. I. Major was invaluable.     

香蕉rbcS基因启动子的克隆及序列分析

以巴西香蕉为材料,根据已经获得的香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的全长cDNA序列设计1对专一引物,通过PCR扩增得到了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基的基因组全长,序列长811 bp,含有2个内含子。根据其基因组序列设计引物,采用SEFA-PCR方法,以总DNA为模板克隆了香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的启动子序列,长1 681 bp。用PLACE软件分析发现该序列具有启动子的基本元件TATA-box、CAAT-box,包含多个胁迫诱导元件,如光诱导元件、赤霉素、低温诱导元件、昼夜节律调控元件等。该序列的克隆与分析为进一步研究香蕉1,5-二磷酸核酮糖羧化/加氧酶小亚基基因的表达调控奠定了基础。     

羧酶体结构及其CO_2浓缩机制研究进展

羧酶体(Carboxysome)是高效的固碳微体,在CO2浓缩机制(CO2-concentrating mechanism,CCM)中发挥重要作用。在蓝藻及某些化能自养菌中,羧酶体作为类细胞器包裹1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)和碳酸酐酶(Carbonic anhydrase,CA),它与无机碳转运蛋白共同在胞质中积累HCO3–,通过增加RubisCO周围的CO2浓度来提高固碳效率。随着羧酶体结构和功能的阐明,异源表达羧酶体已成功实现,并且已鉴定出编码羧酶体壳蛋白及内部组分的基因。首先简要介绍羧酶体的发现和种类,然后系统分析其结构及在CCM机制中的作用,并对其在代谢工程上的广阔应用前景进行了展望。     

Diurnal and circadian rhythmicity in the expression of light-induced plant nuclear messenger RNAs

The levels of nuclear mRNAs for three light-inducible proteins (light-harvesting chlorophyll a/b protein, small subunit of ribulose-1,5-bisphosphate carboxylase and early light-induced protein) have been analyzed under light-dark and constant light conditions. The levels of all three mRNAs have been found to vary considerably during the day, both under ligh-dark and under constant light conditions, demonstrating the existence of diurnal and circadian rhythmicity in the expressionoof these nuclear-coded plant proteins. The levels of two of these mRNAs have been found to be enhanced 2 h before the beginning of illumination when active phytochrome levels are still low.Abbreviations ELIP early light-inducible protein - LHCP light-harvesting chlorophyll alb protein; poly(A)RNA=polyadenylated RNA - (ss)RuBPCase (small subunit) ribulose-1,5-bisphosphate carboxylase     

Intact carboxysomes in a cyanobacterial cell visualized by hilbert differential contrast transmission electron microscopy

Carboxysomes in rapidly frozen ice-embedded whole cells of the cyanobacterium Synechococcus sp. strain PCC 7942 were visualized by the recently developed Hilbert differential contrast transmission electron microscope. Structural details of carboxysomes were especially clearly visualized in the ruptured cells. The novel electron microscopy exhibited the paracrystalline arrays of molecules of the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase in the carboxysomes in much better contrast than conventional transmission electron microscopy with ultrathin sections of cells. The carboxysome was surrounded by a 5- to 6-nm-thick monolayer shell which consisted of orderly arrays of globular particles.     

Comparison of Properties of the Proteolytic Degradation of Unassembled Nuclear-encoded Subunits of Ribulose-1,5-bisphosphate Carboxylase and of the Coupling Factor of Photophosphorylation CF1*

The proteolytic degradation of unassembled small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase and of the δ-subunit of the coupling factor of photophosphorylation CF₁ were analyzed and compared in vitro in the presence of stroma or membrane preparations from ribosome-deficient plastids isolated from 32°C-grown rye leaves (Secale cereale L.). Extracts obtained from 70S ribosome-deficient rye leaves after radioactive labeling were used as substrate source for the unassembled polypeptides. Soluble stroma as well as membrane preparations from isolated plastids contained proteolytic activities catalyzing the degradation of both the small subunits of ribulose-1,5-bisphosphate carboxylase and CF₁-δ in vitro. Maximal in vitro degradation was observed at pH 2–3 for the unassembled small subunits, but at pH 6–7 for the purified holoprotein of ribulose-1,5-bisphosphate carboxylase, and at pH 6.0 for unassembled CF₁-δ. Degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase at pH 3.0 was stimulated by Cu²⁺ but not by Ca²⁺, Mg²⁺ or ATP. At pH 3.0 the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase was not inhibited by various protease inhibitors but was even stimulated. At pH 7.0 its degradation was inhibited by HgCl₂ and diazoacetyl nor-leucine methyl ester + Cu-acetate. The degradation of CF₁-δ was markedly inhibited by phenylmethylsulphonyl fluoride (PMSF) and to a lesser extent by 1,10-phenanthroline. According to present results different proteolytic systems appear to be involved in the degradation of unassembled small subunits of ribulose-1,5-bisphosphate carboxylase and of unassembled CF₁-δ.     

Electron microscopic studies of carboxysomes of Thiobacillus neapolitanus

The outer part of the carboxysomes of Thiobacillus neapolitanus was examined by electron microscopy using negatively stained, cryo-treated, frozen hydrated and freeze dried specimens. From stereo-micrographs of freeze dried and fixated carboxysomes the three dimensional structure of the carboxysomes was elucidated. The carboxysomes always appear as hexagonal bodies, which possess twelve pentameric planes. This indicates that carboxysomes have the form of a pentagonal dodecahedron. Inside the carboxysomes the ribulose-1,5-bisphosphate carboxylase molecules are arranged in rows and concentric rings. Negatively stained and cryo-treated carboxysomes do not differ significantly in size. The mean size of these carboxysomes is 117.3±6.9 nm (n=782)     

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10⁸ years, underlining the strong conservatism of the large subunit.Abbreviations A Acetabularia mediterranea - Ac. Acicularia schenckii - HPLC high-performance liquid chromatography - LSU large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - PAGE polyacrylamide gel electrophoresis - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SDS sodium dodecyl sulfate     

Organization, Structure, and Assembly of α-Carboxysomes Determined by Electron Cryotomography of Intact Cells

Carboxysomes are polyhedral inclusion bodies that play a key role in autotrophic metabolism in many bacteria. Using electron cryotomography, we examined carboxysomes in their native states within intact cells of three chemolithoautotrophic bacteria. We found that carboxysomes generally cluster into distinct groups within the cytoplasm, often in the immediate vicinity of polyphosphate granules, and a regular lattice of density frequently connects granules to nearby carboxysomes. Small granular bodies were also seen within carboxysomes. These observations suggest a functional relationship between carboxysomes and polyphosphate granules. Carboxysomes exhibited greater size, shape, and compositional variability in cells than in purified preparations. Finally, we observed carboxysomes in various stages of assembly, as well as filamentous structures that we attribute to misassembled shell protein. Surprisingly, no more than one partial carboxysome was ever observed per cell. Based on these observations, we propose a model for carboxysome assembly in which the shell and the internal RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) lattice form simultaneously, likely guided by specific interactions between shell proteins and RuBisCOs.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

Comparative analysis of carboxysome shell proteins

Carboxysomes are metabolic modules for CO(2) fixation that are found in all cyanobacteria and some chemoautotrophic bacteria. They comprise a semi-permeable proteinaceous shell that encapsulates ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase. Structural studies are revealing the integral role of the shell protein paralogs to carboxysome form and function. The shell proteins are composed of two domain classes: those with the bacterial microcompartment (BMC; Pfam00936) domain, which oligomerize to form (pseudo)hexamers, and those with the CcmL/EutN (Pfam03319) domain which form pentamers in carboxysomes. These two shell protein types are proposed to be the basis for the carboxysome's icosahedral geometry. The shell proteins are also thought to allow the flux of metabolites across the shell through the presence of the small pore formed by their hexameric/pentameric symmetry axes. In this review, we describe bioinformatic and structural analyses that highlight the important primary, tertiary, and quaternary structural features of these conserved shell subunits. In the future, further understanding of these molecular building blocks may provide the basis for enhancing CO(2) fixation in other organisms or creating novel biological nanostructures.     

Ultrastructural detection of ribulose-1,5-bisphosphate carboxylase protein and its subunit mRNAs in wild-type and holoenzyme-deficient Nicotiana using immuno-gold and in-situ-hybridization techniques

In-situ-localization techniques have been adapted to the ultrastructural detection of the holoenzyme ribulose-1,5-bisphosphate carboxylase (RuBPCase) and its composite large- and smallsubunit mRNAs in wild-type and mutant RuBPCase deficient plantlets of Nicotiana tabacum L. Immuno-gold techniques which show the distribution of target proteins have confirmed visually the presence of the holoenzyme in the wild-type plastids and its total absence in the enzyme-less mutant. Using in-situ hybridization coupled with electron microscopy and biotinylated probes for the two subunits, we have directly visualized specific small-subunit mRNAs located in the cytoplasm and large-subunit mRNAs confined to plastids in the enzyme-deficient mutant, and with apparent distributions comparable to those visualized in the wild-type counterpart. These results show that (i) gene products can be visualized in situ by electronmicroscopy techniques under conditions where the respective cellular compartments are readily recognizable and (ii) that an accumulation of mRNAs corresponding to the composite subunits can occur without translation and-or assembly of the protein.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - SSU RuBPCase small subunit - LSU RubBPCase large subunit     

Comparative analysis of the action of cytokinin and light on the formation of ribulosebisphosphate carboxylase and plastid biogenesis

The role of cytokinin in plastid biogenesis was investigated in etiolated rye leaves (Secale cereale L.) and compared with the effect of white light. Cytokinin deficiency of the leaves was induced by early excision of the seedling roots and reversed by the application of kinetin. The cytokinin supply had a much greater influence on plastid biogenesis than on leaf growth in general. The activities of several chloroplastic enzymes were increased 200%–400% after kinetin treatment of cytokinin-depleted leaves. The activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and the amount of fraction-I protein even showed a sevenfold increase. In cytokinin-depleted leaves the development of ribulose-1,5-bisphosphate carboxylase and NADP-glyceraldehydephosphate dehydrogenase was specifically, and markedly inhibited by actinomycin D. The inhibition was partially or even completely overcome after treatment with kinetin. However, under all conditions, RNA synthesis of the leaves, was only partially inhibited by actinomycin D. According to immunologic studies, all dark-grown leaves, in addition to the complete enzyme, contained an excess of free small subunit of ribulose-1,5-bisphosphate carboxylase that was absent in mature light-grown leaves. The most striking accumulation of free small subunit, protein occurred in cytokinin-depleted dark-grown leaves, indicating a deficiency of the plastidic synthesis of the large subunit. The capacity as well as the activity of plastidic protein synthesis was preferentially increased by cytokinin and light. Cytokinin increased, the amount of plastidic ribosomes per leaf and relative to the amount of cytoplasmic ribosomes. While the percentage of cytoplasmic ribosomes bound as polyribosomes was little affected by the cytokinin supply, the proportion of plastidic polyribosomes was increased from 11% to 18% after kinetin treatment of cytokinin-depleted leaves. In the light, the proportion of plastidic polyribosomes reached 39% of the total plastidic ribosomes.Abbreviations RuBP carboxylase ribulose-1,5-bisphosphate carboxylase - NADP-GAP dehydrogenase NADP-dependent glyceraldehyde-3-phosphate dehydrogenase     

The Association of d-Ribulose- 1,5-Bisphosphate Carboxylase/Oxygenase with Phosphoribulokinase

When Ribulose- 1,5-bisphosphate carboxylase/oxygenase was purified from spinach leaves (Spinacia oleracea) using precipitation with polyethylene glycol and MgCl₂ followed by DEAE cellulose chromatography, 75% of phosphoribulokinase and 7% of phosphoriboisomerase activities copurified with ribulose- 1,5-bisphosphate carboxylase/oxygenase. This enzyme preparation showed ribose-5-phosphate and ribulose-5-phosphate dependent carboxylase and oxygenase activities which were nearly equivalent to its corresponding ribulose- 1,5-bisphosphate dependent activity. The ribose-5-phosphate and ribulose-5-phosphate dependent reaction rates were stable and linear for much longer time periods than the ribulose- 1,5-bisphosphate dependent rates. When sucrose gradients were used to purify ribulose- 1,5-bisphosphate carboxylase/oxygenase from crude stromal extracts, phosphoribulokinase was found to cosediment with ribulose- 1,5-bisphosphate carboxylase. Under these conditions most of the phosphoriboisomerase activity remained with the slower sedimenting proteins. Ammonium sulfate precipitation resulted in separation of the ribulose- 1,5-bisphosphate carboxylase peak from phosphoribulokinase peak. Crude extracts of peas Pisum sativum and spinach contained 0.725 to 0.730 milligram of phosphoribulokinase per milligram of chlorophyll, respectively, based on an enzyme-linked immunosorbent assay.     

Biosynthesis of ribulose-1.5-bisphosphate carboxylase in greening cells of Euglena gracilis

Light induction of chloroplast development in Euglena leads to quantitative changes in the protein composition of the soluble cell part. One major part of these is the observed accumulation of ribulose-1.5-bisphosphate carboxylase/oxygenase (RuBPCase) enzyme (EC 4.1.1.39). As measured by immunoelectrophoresis, a small amount of RuBPCase (about 10^-6 pmol) is present in a dark-grown cell, whereas a greening cell (72h) contains 10–20 pmol enzyme. Both the cytoplasmic and chloroplastic translation inhibitors, cycloheximide and spectinomycin, have a strong inhibitory effect on the synthesis of the enzyme throughout the greening process of Euglena cells. Electrophoretic and immunological analyses of the soluble phase prepared from etiolated or greening cells do not show the presence of free subunits of the enzyme. For each antibiotic-treated greening cell, the syntheses of both subunits are blocked. Our data indicate that tight reciprocal control between the syntheses of the two classes of subunits occurs in Euglena. In particular, the RuBPCase small subunit synthesis in greening Euglena seems more dependent on the protein synthesis activity of the chloroplast than the syntheses of other stromal proteins from cytoplasmic origin.Abbreviations LSU large subunit of ribulose-1.5-bisphosphate carboxylase - RuBP ribulose-1.5-bisphosphate - RuBP-Case ribulose-1.5-bisphosphate carboxylase - SSU small subunit of ribulose-1.5-bisphosphate carboxylase     

The N-terminal hydrophobic region of the mature phosphate translocator is sufficient for targeting to the chloroplast inner envelope membrane.

To locate the sequence required for directing the phosphate translocator to the chloroplast inner envelope membrane, a series of chimeric proteins constituting parts of the phosphate translocator and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, which is normally located in the stroma, has been produced. Reciprocal exchanges of the presequences and mature sequences of the phosphate translocator and the small subunit indicated that the phosphate translocator presequence contains stromal targeting information and that the mature protein is responsible for inner envelope membrane targeting. Chimeric proteins containing the N-terminal 46 amino acid residues of the phosphate translocator were directed to the inner envelope membrane. Subdivision of this region into its composite hydrophilic and hydrophobic regions showed that the hydrophobic region alone, which consists of amino acid residues 24 to 45, was able to direct the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase to the inner envelope membrane.     

Suitable conditions for characterization,identification, and isolation of the mRNA of the small subunit of ribulose-1,5-bisphosphate carboxylase from Nicotiana sylvestris

The products synthesized in vitro by messenger RNA (mRNA) extracted from Nicotiana sylvestris were analyzed by electrophoresis on polyacrylamide slab gels. Only three of the major polypeptides synthesized are considered here: P₅₅, P₃₂, and P₂₀. P₅₅ and P₃₂ were translated from chloroplast mRNA. P₅₅ corresponds to the large subunit of ribulose-1,5-bisphosphate (RuP₂) carboxylase; P₃₂ is probably a chloroplast membrane protein. P₂₀, the polypeptide synthesized from cytoplasmic poly(A)⁺ RNA, is the precursor of the small subunit of RuP₂ carboxylase. The balance between P₂₀ and P₃₂, in which their relative proportions varied inversely, was regulated by the age of the leaves and the time of illumination; we took advantage of this phenomenon to isolate the mRNA from the small subunit in relatively large amounts. This mRNA has a molecular weight of 350,000.Abbreviations RuP₂ ribulose-1,5-bisphosphate - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Mutations in loop six of the large subunit of ribulose-1,5-bisphosphate carboxylase affect substrate specificity

Mutagenesis in vitro of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase (EC 4.1.1.39) from Anacystis nidulans Synechococcus PCC 6301) was used to generate novel enzymes in Escherichia coli. Residues in C-terminal loop 6 of the / barrel structure of the large subunit were changed. Replacement of valine 331 with alanine caused a 90% reduction in V _max but did not alter the enzyme's relative specificity towards either of its gaseous substrates, CO₂ and O₂. However replacement of alanine 340 with glutamate decreased the enzyme's specificity for CO₂ but had no significant effect on either the K _m for ribulose-1,5-bisphosphate or CO₂ or on V _max. In contrast replacing a small cassette of residues 338-341 produced a small increase in the specificity factor.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - CABP 2-carbox-yarabinitol-1,5-bisphosphate We thank Karen Moore for the statistical analysis of the specificity factors. We acknowledge helpful discussions with Jim Pitts and Richard Pickersgill. This work was aided by the invaluable technical assistance of Iain Major.