Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.     

Assembly of in Vitro-Synthesized Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase Is Sensitive to CI-, Requires ATP,and Does Not Proceed When Large Subunits Are Synthesized at Temperatures [greater than or equal to]32[deg]C

In higher plants, ribulose bisphosphate carboxylase/oxygenase (Rubisco) consists of eight large "L" subunits, synthesized in chloroplasts, and eight small "S" subunits, synthesized as precursors in the cytosol. Assembly of these into holoenzyme occurs in the chloroplast stroma after import and processing of the S subunits. A chloroplast chaperonin interacts with the L subunits, which dissociate from the chaperonin before they assemble into holoenzyme. Our laboratory has reported L subunit assembly into Rubisco in chloroplast extracts after protein synthesis in leaves, intact chloroplasts, and most recently in membrane-free chloroplast extracts. We report here that the incorporation of in vitro-synthesized L subunits into holoenzyme depends on the conditions of L subunit synthesis. Rubisco assembly did not occur after L subunit synthesis at 160 mM KCI. When L subunit synthesis occurred at approximately 70 mM KCI, assembly depended on the temperature at which L subunit synthesis took place. These phenomena were the result of postsynthetic events taking place during incubation for protein synthesis. We separated these events from protein synthesis by lowering the temperature during protein synthesis. Lower temperatures supported the synthesis of full-length Rubisco L subunits. The assembly of these completed L subunits into Rubisco required intervening incubation with ATP, before addition of S subunits. ATP treatment mobilized L subunits from a complex with the chloroplast chaperonin 60 oligomer. Addition of 130 mM KCI at the beginning of the intervening incubation with ATP blocked the incorporation of L subunits into Rubisco. The inhibitory effect of high KCI was due to CI- and came after association of newly synthesized L subunits with chaperonin 60, but before S subunit addition. It is interesting that L subunits synthesized at [greater than or equal to]32[deg]C failed to assemble into Rubisco under any conditions. These results agree with previous results obtained in this laboratory using newly synthesized L subunits made in intact chloroplasts. They also show that assembly of in vitro-synthesized L subunits into Rubisco requires ATP, that CI- inhibits Rubisco assembly, and that synthesis temperature affects subsequent assembly competence of L subunits.     

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.     

Synthesis and assembly of large subunits into ribulose bisphosphate carboxylase/oxygenase in chloroplast extracts

We have developed a new system for the in vitro synthesis of large subunits and their assembly into ribulose bisphosphate carboxylase oxygenase (Rubisco) holoenzyme in extracts of higher plant chloroplasts. This differs from previously described Rubisco assembly systems because the translation of the large subunits occurs in chloroplast extracts as opposed to isolated intact chloroplasts, and the subsequent assembly of large subunits into holoenzyme is completely dependent upon added small subunits. Amino acid incorporation in this system displayed the characteristics previously reported for chloroplast-based translation systems. Incorporation was sensitive to chloramphenicol or RNase but resistant to cycloheximide, required magnesium, and was stimulated by nucleotides. The primary product of this system was the large subunit of Rubisco. However, several lower molecular weight polypeptides were formed. These were structurally related to the Rubisco large subunit. The initiation inhibitor aurintricarboxylic acid (ATA) decreased the amount of lower molecular weight products accumulated. The accumulation of completed large subunits was only marginally reduced in the presence of ATA. The incorporation of newly synthesized large subunits into Rubisco holoenzyme occurred under conditions previously identified as optimal for the assembly of in organello-synthesized large subunits and required the addition of purified small subunits.     

Synthesis and assembly of bacterial and higher plant Rubisco subunits in Escherichia coli

The synthesis in Escherichia coli of both the large and small subunits of cereal ribulose bisphosphate carboxylase/oxygenase has been obtained using expression plasmids and bacteriophages. The level and order of synthesis of the large and small subunits were regulated using different promoters, resulting in different subunit pool sizes and ratios that could be controlled in attempts to optimize the conditions for assembly. Neither assembly nor enzyme activity were observed for the higher plant enzyme. In contrast, cyanobacterial large and small subunits can assemble to give an active holoenzyme in Escherichia coli. By the use of deletion plasmids, followed by infection with appropriate phages, it can be demonstrated that the small subunit is essential for catalysis. However, the small subunit is not required for the assembly of a large subunit octomer core in the case of the Synechococcus enzyme; self-assembly of the octomer will occur in an rbcS deletion strain. The cyanobacterial small subunits can be replaced by wheat small subunits to give an active enzyme in Escherichia coli. The hybrid cyanobacterial large/wheat small subunit enzyme has only about 10% of the level of activity of the wild-type enzyme, reflecting the incomplete saturation of the small subunit binding sites on the large subunit octomer, and possibly a mismatch in the subunit interactions of those small subunits that do bind, giving rise to a lower rate of turnover at the active sites.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - L large subunit - Rubisco ribulose bisphosphate carboxylase/oxygenase - S small subunit     

Construction of a tobacco master line to improve Rubisco engineering in chloroplasts

The inability to assemble Rubisco from any photosynthetic eukaryote within Escherichia coli has hampered structure-function studies of higher plant Rubisco. Precise genetic manipulation of the tobacco chloroplast genome (plastome) by homologous recombination has facilitated the successful production of transplastomic lines that have either mutated the Rubisco large subunit (L) gene, rbcL, or replaced it with foreign variants. Here the capacity of a new tobacco transplastomic line, (cm)trL, to augment future Rubisco engineering studies is demonstrated. Initially the rbcL was replaced with the selectable marker gene, aadA, and an artificial codon-modified (cm)rbcM gene that codes for the structurally novel Rubisco dimer (L(2), approximately 100 kDa) from Rhodosprillum rubrum. To obtain (cm)trL, the aadA was excised by transiently introducing a T-DNA encoding CRE recombinase biolistically. Selection using aadA enabled transplantation of mutated and wild-type tobacco Rubisco genes into the (cm)trL plastome with an efficiency that was 3- to 10-fold higher than comparable transformations into wild-type tobacco. Transformants producing the re-introduced form I tobacco Rubisco variants (hexadecamers comprising eight L and eight small subunits, approximately 520 kDa) were identified by non-denaturing PAGE with fully segregated homoplasmic lines (where no L(2) Rubisco was produced) obtained within 6-9 weeks after transformation which enabled their Rubisco kinetics to be quickly examined. Here the usefulness of (cm)trL in more readily examining the production, folding, and assembly capabilities of both mutated tobacco and foreign form I Rubisco subunits in tobacco plastids is discussed, and the feasibility of quickly assessing the kinetic properties of those that functionally assemble is demonstrated.     

Linked Rubisco subunits can assemble into functional oligomers without impeding catalytic performance

Although transgenic manipulation in higher plants of the catalytic large subunit (L) of the photosynthetic CO2-fixing enzyme ribulose 1,5-bisphospahte carboxylase/oxygenase (Rubisco) is now possible, the manipulation of its cognate small subunit (S) is frustrated by the nuclear location of its multiple gene copies. To examine whether L and S can be engineered simultaneously by fusing them together, the subunits from Synechococcus PCC6301 Rubisco were tethered together by different linker sequences, producing variant fusion peptides. In Escherichia coli the variant PCC6301 LS fusions assembled into catalytically functional octameric ([LS]8) and hexadecameric ([[LS]8]2) quaternary structures that excluded the integration of co-expressed unfused S. Assembly of the LS fusions into Rubisco complexes was impaired 50-90% relative to the assembly of unlinked L and S into L8S8 enzyme. Assembly in E. coli was not emulated using tobacco SL fusions that accumulated entirely as insoluble protein. Catalytic measurements showed the CO2/O2 specificity, carboxylation rate, and Michaelis constants for CO2 and ribulose 1,5-bisphosphate for the cyanobacterial Rubisco complexes comprising fusions where the S was linked to the N terminus of L closely matched those of the wild-type L8S8 enzyme. In contrast, the substrate affinities and carboxylation rate of the Rubisco complexes comprising fusions where L was fused to the N terminus of S or a six-histidine tag was appended to the C terminus of L were compromised. Overall this work provides a framework for implementing an alternative strategy for exploring simultaneous engineering of modified, or foreign, Rubisco L and S subunits in higher plant plastids.     

Co-expression of plastid chaperonin genes and a synthetic plant Rubisco operon in Escherichia coli

It has been suggested that lack of specialized molecular chaperone function(s) in Escherichia coli may account for the fact that although E. coli cells transformed with plant Rubisco genes synthesize the Rubisco subunit polypeptides, the active enzyme fails to assemble. If so, co-expression of plant chaperone and Rubisco genes might permit plant Rubisco assembly in E. coli. Introduction of genes encoding plant chaperonin polypeptides has been shown to enhance the capacity of E. coli to assemble active cyanobacterial Rubisco. We now report that co-expression of plant Rubisco and chaperonin genes affected the solubility and stability of Rubisco large subunit polypeptides, however, neither the assembled oligomeric protein nor Rubisco enzyme activity was detected.     

Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli

A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide. Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus. A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes. When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min. Its size on sucrose gradients indicates a monomeric or dimeric form. When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur. The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits. This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.     

In vitro reassembly of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase from fully denatured subunits

It has been generally proved impossible to reassemble ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from fully denatured subunits in vitro in higher plant,because large subunit of fullydenatured Rubisco is liable to precipitate when the denaturant is removed by common methods of directdilution and one-step dialysis.In our experiment,the problem of precipitation was resolved by an improvedgradual dialysis method,which gradually decreased the concentration of denaturant.However,fully denaturedRubisco subunits still could not be reassembled into holoenzyme using gradual dialysis unless chaperonin 60was added.The restored activity of reassembled Rubisco was approximately 8% of natural enzyme.Thequantity of reassembled Rubisco increased greatly when heat shock protein 70 was present in the reassemblyprocess.ATP and Mg~(2 ) were unnecessary for in vitro reassembly of Rubisco,and Mg~(2 ) inhibited the reassemblyprocess.The reassembly was weakened when ATP,Mg~(2 ) and K~ existed together in the reassembly process.     

Binding of a Transition State Analog to Newly Synthesized Rubisco

Radioactive amino acids, when added to isolated pea chloroplasts or chloroplast extracts engaged in protein synthesis, are incorporated into Rubisco large subunits that co-migrate with native Rubisco during nondenaturing electrophoresis. We have added the transition state analog 2′-carboxyarabinitol bisphosphate (CABP) to chloroplast extracts after in organello or in vitro incorporation of radioactive amino acids into Rubisco large subunits. Upon addition of CABP the radioactive bands co-migrating with native Rubisco undergo a readily detected shift in electrophoretic mobility just as the native enzyme, thus demonstrating the ability of the newly assembled molecules to interact with this transition state analog.     

Several proteins imported into chloroplasts form stable complexes with the GroEL-related chloroplast molecular chaperone.

Nine different proteins were imported into isolated pea chloroplasts in vitro. For seven of these [the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), beta-subunit of ATP synthase, glutamine synthetase, the light-harvesting chlorophyll a/b binding protein, chloramphenicol acetyltransferase, and pre-beta-lactamase], a fraction was found to migrate as a stable high-molecular-weight complex during nondenaturing gel electrophoresis. This complex contained the mature forms of the imported proteins and the groEL-related chloroplast chaperonin 60 (previously known as Rubisco subunit binding protein). Thus, the stable association of imported proteins with this molecular chaperone is widespread and not necessarily restricted to Rubisco subunits or to chloroplast proteins. With two of the imported proteins (ferredoxin and superoxide dismutase), such complexes were not observed. It seems likely that, in addition to its proposed role in assembly of Rubisco, the chloroplast chaperonin 60 is involved in the assembly or folding of a wide range of proteins in chloroplasts.     

The Rubisco subunit binding protein

Chloroplasts contain an abundant soluble protein that binds non-covalently newly synthesized large and small subunits of the enzyme ribulose bisphosphate carboxylase-oxygenase. This binding protein has been purified from Pisum sativum and Hordeum vulgare in the form of a dodecamer consisting of equal amounts of two types of subunit. These subunits are synthesized as higher molecular mass precursors by cytoplasmic ribosomes before import into the chloroplast. Antibodies raised against the purified binding protein from Pisum sativum detect polypeptides not only in extracts of plastids from several plant species but also in cell extracts of several bacterial species. The oligomeric binding protein dissociates reversibly into monomeric subunits in the presence of 1–5 mmol/liter MgATP. For one type of subunit the cDNA sequence has been isolated and determined and reveals homology with certain bacterial proteins.These observations are discussed in relation to the idea that the binding protein is an example of a general class of proteins termed "molecular chaperones" which are required for the correct assembly of certain oligomeric proteins such as the carboxylase from their subunits.Abbreviations BP Binding protein - Rubisco Ribulose bisphosphate carboxylase-oxygenase     

Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco

After folding, many proteins must assemble into oligomeric complexes to become biologically active. Here we describe the role of RbcX as an assembly chaperone of ribulose-bisphosphate carboxylase/oxygenase (Rubisco), the enzyme responsible for the fixation of atmospheric carbon dioxide. In cyanobacteria and plants, Rubisco is an approximately 520 kDa complex composed of eight large subunits (RbcL) and eight small subunits (RbcS). We found that cyanobacterial RbcX functions downstream of chaperonin-mediated RbcL folding in promoting the formation of RbcL(8) core complexes. Structural analysis revealed that the 15 kDa RbcX forms a homodimer with two cooperating RbcL-binding regions. A central cleft specifically binds the exposed C-terminal peptide of RbcL subunits, enabling a peripheral surface of RbcX to mediate RbcL(8) assembly. Due to the dynamic nature of these interactions, RbcX is readily displaced from RbcL(8) complexes by RbcS, producing the active enzyme. The strategies employed by RbcX in achieving substrate specificity and efficient product release may be generally relevant in assisted assembly reactions.     

Structural Analysis of the Rubisco-Assembly Chaperone RbcX-II from Chlamydomonas reinhardtii

The most prevalent form of the Rubisco enzyme is a complex of eight catalytic large subunits (RbcL) and eight regulatory small subunits (RbcS). Rubisco biogenesis depends on the assistance by specific molecular chaperones. The assembly chaperone RbcX stabilizes the RbcL subunits after folding by chaperonin and mediates their assembly to the RbcL₈ core complex, from which RbcX is displaced by RbcS to form active holoenzyme. Two isoforms of RbcX are found in eukaryotes, RbcX-I, which is more closely related to cyanobacterial RbcX, and the more distant RbcX-II. The green algae Chlamydomonas reinhardtii contains only RbcX-II isoforms, CrRbcX-IIa and CrRbcX-IIb. Here we solved the crystal structure of CrRbcX-IIa and show that it forms an arc-shaped dimer with a central hydrophobic cleft for binding the C-terminal sequence of RbcL. Like other RbcX proteins, CrRbcX-IIa supports the assembly of cyanobacterial Rubisco in vitro, albeit with reduced activity relative to cyanobacterial RbcX-I. Structural analysis of a fusion protein of CrRbcX-IIa and the C-terminal peptide of RbcL suggests that the peptide binding mode of RbcX-II may differ from that of cyanobacterial RbcX. RbcX homologs appear to have adapted to their cognate Rubisco clients as a result of co-evolution.     

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis

The incorporation of newly synthesized large subunits into ribulose bisphosphate carboxylase/oxygenase (RuBisCO) in pea chloroplast extracts occurs at the expense of intermediate forms of the large subunit which are complexed with a binding protein. Most subunits of this binding protein are found in dodecameric complexes in chloroplast extracts. Addition of small subunits to these extracts results in approximately 40 to 60% increased incorporation of newly made large subunits into RuBisCO at low or zero concentrations of ATP, but is without significant effect at high concentrations of ATP, a condition in which the dodecameric binding protein complex is dissociated into subunits. Overall, these data support the assumption that the incorporation of large subunits into RuBisCO in chloroplast extracts reflects de novo assembly rather than `mere' exchange of subunits. The in vitro assembly of large subunits into RuBisCO is a function of the conditions under which the large subunits are synthesized in organello. When the large subunits are made in chloroplasts suspended in 188 millimolar sorbitol, they are approximately 2- to 3-fold better able to assemble into RuBisCO when subsequently incubated in vitro than when they are synthesized in chloroplasts suspended in 375 millimolar sorbitol. This observation indicates that mere synthesis of large subunits is not sufficient to confer maximal assembly competence on large subunits.     

The rbcX gene product promotes the production and assembly of ribulose-1,5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli

The operon encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the cyanobacterium Synechococcus sp. PCC7002 contains three rbc genes, rbcL, rbcX and rbcS, in this order. Introduction of translational frameshift into the rbcX gene resulted in a significant decrease in the production of large (RbcL) and small (RbcS) subunits of the Rubisco protein in Synechococcus sp. PCC7002 and in Escherichia coli. To investigate the function of the rbcX gene product (RbcX), we constructed the expression plasmid for the rbcX gene and examined the effects of RbcX on the recombinant Rubisco production in Escherichia coli. The coexpression experiments revealed that RbcX had marked effects on the production of large and small subunits of Rubisco without any significant influence on the mRNA level of rbc genes and/or the post-translational assembly of the Rubisco protein. The present rbcX coexpression system provides a novel and useful method for investigating the Rubisco maturation pathway.     

ATP-released large subunits participate in the assembly of RuBP carboxylase

Preincubation of 35S-methionine-labeled chloroplast extracts with ATP at 0 degree C potentiates the subsequent assembly of labeled large subunits into RuBPCase . This is correlated with the dissociation of newly synthesized large subunits from the 29S large subunit binding protein complex. These released large subunits then assemble into RuBPCase in a second, nucleotide-stimulated reaction. The data demonstrate that the 29S complex can play an active role in the assembly of RuBPCase .