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.     

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.     

A point mutation in the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase affects holoenzyme assembly in Nicotiana tabacum.

In photosynthetic eukaryotes, the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is composed of eight large and eight small subunits. Chloroplast-coded large subunits are found in association with chaperonins (binding proteins) of 60-61 kd to form a high mol. wt pre-assembly complex (B-complex). We have isolated a heterotrophic, maternally-inherited mutant from Nicotiana tabacum var. Xanthi which accumulates the B-complex but contains no Rubisco holoenzyme. The B-complex of the mutant dissociates in the presence of ATP, as does that of the wild-type. Processing of the nuclear-coded small subunit takes place in the mutant and neither large nor small subunits accumulate. The large subunit gene from mutant and wild-type plants was cloned and sequenced. A single nucleotide difference was found between them predicting an amino acid change of serine to phenylalanine at position 112 in the mutant. Based on the resolved structure of N.tabacum Rubisco, it is argued that the alteration at position 112 prevents holoenzyme assembly by interfering with large subunit assembly.     

A mutation in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that reduces the rate of its incorporation into holoenzyme

A mutant of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), in which Arg53 is replaced by Glu, was synthesized and imported into isolated chloroplasts. The mutant protein was efficiently imported into the chloroplast and correctly processed to the mature size. Like the wild type protein, it was stable over a period of at least 2 h. Unlike the wilk-type protein however, most of the mutant protein was not assembled with holo-Rubisco at the end of a 10-min import reaction. It migrated instead as a diffused band on a non-denaturing gel, slower than the precursor protein, but faster than the holoenzyme. The level of the unassembled mutant protein in the stroma decreased with time, while its level in the assembled fraction has increased, indicating that this protein is a slowly-assembled, rather than a non-assembled, mutant of the small suubunit of Rubisco. Accumulation of the mutant protein in the holoenzyme fraction was dependent on ATP and light. The transient species, migrating faster than the holoenzyme but slower than the precursor protein, may represent an intermediate in the assembly process of the small subunit of RubiscoAbbreviations LSU large subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of Rubisco     

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.     

Chimeric small subunits influence catalysis without causing global conformational changes in the crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase

Comparison of subunit sequences and X-ray crystal structures of ribulose-1,5-bisphosphate carboxylase/oxygenase indicates that the loop between beta-strands A and B of the small subunit is one of the most variable regions of the holoenzyme. In prokaryotes and nongreen algae, the loop contains 10 residues. In land plants and green algae, the loop is comprised of approximately 22 and 28 residues, respectively. Previous studies indicated that the longer betaA-betaB loop was required for the assembly of cyanobacterial small subunits with plant large subunits in isolated chloroplasts. In the present study, chimeric small subunits were constructed by replacing the loop of the green alga Chlamydomonas reinhardtii with the sequences of Synechococcus or spinach. When these engineered genes were transformed into a Chlamydomonas mutant that lacks small-subunit genes, photosynthesis-competent colonies were recovered, indicating that loop size is not essential for holoenzyme assembly. Whereas the Synechococcus loop causes decreases in carboxylation V(max), K(m)(O(2)), and CO(2)/O(2) specificity, the spinach loop causes complementary decreases in carboxylation V(max), K(m)(O(2)), and K(m)(CO(2)) without a change in specificity. X-ray crystal structures of the engineered proteins reveal remarkable similarity between the introduced betaA-betaB loops and the respective loops in the Synechococcus and spinach enzymes. The side chains of several large-subunit residues are altered in regions previously shown by directed mutagenesis to influence CO(2)/O(2) specificity. Differences in the catalytic properties of divergent Rubisco enzymes may arise from differences in the small-subunit betaA-betaB loop. This loop may be a worthwhile target for genetic engineering aimed at improving photosynthetic CO(2) fixation.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

The gene for the ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit relocated to the plastid genome of tobacco directs the synthesis of small subunits that assemble into Rubisco

To assess the extent to which a nuclear gene for a chloroplast protein retained the ability to be expressed in its presumed preendosymbiotic location, we relocated the RbcS gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the tobacco plastid genome. Plastid RbcS transgenes, both with and without the transit presequence, were equipped with 3' hepta-histidine-encoding sequences and psbA promoter and terminator elements. Both transgenes were transcribed abundantly, and their products were translated into small subunit polypeptides that folded correctly and assembled into the Rubisco hexadecamer. When present, either the transit presequence was not translated or the transit peptide was cleaved completely. After assembly into Rubisco, transplastomic small subunits were relatively stable. The hepta-histidine sequence fused to the C terminus of a single small subunit was sufficient for isolation of the whole Rubisco hexadecamer by Ni(2)+ chelation. Small subunits produced by the plastid transgenes were not abundant, never exceeding approximately 1% of the total small subunits, and they differed from cytoplasmically synthesized small subunits in their N-terminal modifications. The scarcity of transplastomic small subunits might be caused by inefficient translation or assembly.     

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.     

不同生态型芦苇Rubisco免疫学特性差异

以河西走廊荒漠地区不同生态型芦苇为研究材料,提取并纯化得Rubisco蛋白,经SDS-PAGE凝胶电泳将Rubisco大、小亚基分离,用Rubisco全酶蛋白及其大、小亚基分别注射昆明系雄性小白鼠制备抗体,经Western-blotting鉴定结果表明:(1)水芦Rubisco全酶抗体可与水芦、沙芦及菠菜Rubisco大亚基发生反应,而与小亚基均未见显色反应,且水芦显色最深,沙芦略浅,菠菜最浅;(2)水芦、沙芦Rubisco大亚基抗体可与水芦、沙芦、菠菜大亚基发生抗原交叉反应,且均不与小亚基发生反应,并且其与菠菜Rubisco大亚基的反应程度明显低于水芦和沙芦;(3)用与Rubisco大亚基抗体同样的制备方法,均未检测到水芦、沙芦Rubisco小亚基抗体的产生;(4)菠菜Rubisco全酶抗体可与菠菜、水芦、沙芦、水稻Rubisco大亚基均发生抗原交叉反应,但仅与其自身小亚基反应,且与菠菜Rubisco大亚基显色反应最深,水稻略浅,沙芦、水芦稍有反应.由此说明,水芦、沙芦Rubisco全酶蛋白及其大亚基免疫学特性差异较小,而与双子叶植物菠菜相比差异较大;水芦、沙芦Rubisco蛋白免疫化学决定簇的差异主要决定于小亚基上,且其小亚基不具有抗原活性或抗原活性较弱.     

Analysis of Two Rubisco-Deficient Tobacco Mutants, H7 and Sp25; Evidence for the Production of Rubisco Large Subunits in the Sp25 Mutant that Form Clusters and are Inactive

The basis for the lesions in the Sp25 and H7 ribulose-l,5-bisphosphatecarboxylase/oxygenase (Rubisco) mutants of tobacco was studiedin detail because these plants may be suitable hosts for transformationwith the genes for Rubisco enzymes of various origins that havedifferent substrate specificities. We show that the Sp25 mutantlacks active holoenzyme, but contains the large and small subunitpolypeptides, Rubisco activase and the chloroplast chaperonin,Cpn 60. The large subunit polypeptides were not distributeduniformly in the stroma in the Sp25 mutant as they were in thewild-type plants, but had an anomalous distribution being presentonly in aggregated clusters notably in chloroplasts with largestarch grains. Furthermore, these clusters were not uniformlydistributed throughout the photosynthetic cells but were localizedlargely in the mesophyll cells surrounding the vascular tissue.In contrast to the Sp25 mutant, the H7 mutant contained theRubisco holoenzyme, but in this case the enzyme was inactive.It is clear that in both these mutants the Rubisco holoenzymefails to assemble correctly. In the Sp25 mutant assembly islost completely while in the H7 mutant the holoenzyme is formed,but the assembly process fails to produce an active enzyme.We suggest that the flaw in assembly in the Sp25 mutant resultsfrom a defect in chloroplast encoded proteins. Key words: Rubisco, assembly, tobacco, mutants     

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.     

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.     

Formation of Disulphide Cross-Linked Aggregates of Large Subunit from Higher Plant Ribulose-1, 5-Bisphosphate Carboxylase-Oxygenase

The properties of the large and small subunit polypeptides ofpurified wheat ribulose-1, 5-bisphosphate carboxylase-oxygenase(Rubisco, E.C. 4.1.1.39 [EC] ) were studied. The protein was dissociatedinto subunits by extreme pH or detergent treatment. The separatedsubunits were unable to reassemble into functional holoproteinwhen the starting conditions were restored. Some of the separatedsmall subunit polypeptides retained their ability to form functionalheterologous Rubisco when mixed with large subunits from a cyanobacterialRubisco. The separated large subunits of wheat Rubisco formednon-functional, high-molecular-weight aggregates. Treatmentwith both sodium dodecyl sulphate and thiol reductant was necessaryto disrupt the aggregated structures, which indicates that thelarge subunits had been cross-linked by disulphide bridges.Since added thiol reductant did not prevent aggregation of theseparated subunits during attempted reconstitution, oxidationof the sulphydryl groups apparently took place on contact facessheltered by the secondary and tertiary structures of the polypeptides.High concentration of large subunits or freezing and thawingof the solution stimulated the formation of disulphide cross-linksbetween the large subunits. The presence of small subunits didnot prevent aggregation of large subunits. The results suggestthat large subunits have a tendency to cross-link with disulphidebridges thus preventing proper assembly with small subunits. Key words: Rubisco, aggregates of large subunit, disulphide cross-linkage, assembly of Rubisco     

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     

Subunit interface dynamics in hexadecameric rubisco

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) plays an important role in the global carbon cycle as a hub for biomass. Rubisco catalyzes not only the carboxylation of RuBP with carbon dioxide but also a competing oxygenation reaction of RuBP with a negative impact on photosynthetic yield. The functional active site is built from two large (L) subunits that form a dimer. The octameric core of four L₂ dimers is held at each end by a cluster of four small (S) subunits, forming a hexadecamer. Each large subunit contacts more than one S subunit. These interactions exploit the dynamic flexibility of Rubisco, which we address in this study. Here, we describe seven different types of interfaces of hexadecameric Rubisco. We have analyzed these interfaces with respect to the size of the interface area and the number of polar interactions, including salt bridges and hydrogen bonds in a variety of Rubisco enzymes from different organisms and different kingdoms of life, including the Rubisco-like proteins. We have also performed molecular dynamics simulations of Rubisco from Chlamydomonas reinhardtii and mutants thereof. From our computational analyses, we propose structural checkpoints of the S subunit to ensure the functionality and/or assembly of the Rubisco holoenzyme. These checkpoints appear to fine-tune the dynamics of the enzyme in a way that could influence enzyme performance.     

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     

Role of the small subunit in ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes.