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.     

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.     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.     

Cotranscription, deduced primary structure, and expression of the chloroplast-encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain N1.

The primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase from the marine diatom Cylindrotheca sp. strain N1 has been determined. Unlike higher plants and green algae, the genes encoding the large and the small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase are chloroplast-encoded and closely associated (Hwang and Tabita, 1989). The rbcL and rbcS genes in strain N1 are cotranscribed and are separated by an intergenic region of 46 nucleotide base pairs. Ribosome binding sites and a potential promoter sequence were highly homologous to previously determined chloroplast sequences. Comparison of the deduced primary structure of the diatom large and small subunits indicated significant homology to previously determined sequences from bacteria; there was much less homology to large and small subunits from cyanobacteria, green algae, and higher plants. Although high levels of recombinant diatom large subunits could be expressed in Escherichia coli, the protein synthesized was primarily insoluble and incapable of forming an active hexadecameric enzyme. Edman degradation studies indicated that the amino terminus of the large subunit isolated from strain N1 was blocked, suggesting that the mechanism responsible for processing and subsequent assembly of large and small subunits resembles the situation found with other eucaryotic ribulose-1,5-bisphosphate carboxylase/oxygenase proteins, despite the distinctive procaryotic gene arrangement and sequence homology.     

Artificially evolved Synechococcus PCC6301 Rubisco variants exhibit improvements in folding and catalytic efficiency

The photosynthetic CO2-fixing enzyme, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), is responsible for most of the world's biomass, but is a slow non-specific catalyst. We seek to identify and overcome the chemical and biological constraints that limit the evolutionary potential of Rubisco in Nature. Recently, the horizontal transfer of Calvin cycle genes (rbcL, rbcS and prkA) from cyanobacteria (Synechococcus PCC6301) to gamma-proteobacteria (Escherichia coli) was emulated in the laboratory. Three unique Rubisco variants containing single (M259T) and double (M259T/A8S, M259T/F342S) amino acid substitutions in the L (large) subunit were identified after three rounds of random mutagenesis and selection in E. coli. Here we show that the M259T mutation did not increase steady-state levels of rbcL mRNA or L protein. It instead improved the yield of properly folded L subunit in E. coli 4-9-fold by decreasing its natural propensity to misfold in vivo and/or by enhancing its interaction with the GroES-GroEL chaperonins. The addition of osmolites to the growth media enhanced productive folding of the M259T L subunit relative to the wild-type L subunit, while overexpression of the trigger factor and DnaK/DnaJ/GrpE chaperones impeded Rubisco assembly. The evolved enzymes showed improvement in their kinetic properties with the M259T variant showing a 12% increase in carboxylation turnover rate (k(c)cat), a 15% improvement in its K(M) for CO2 and no change in its K(M) for ribulose-1,5-bisphosphate or its CO2/O2 selectivity. The results of the present study show that the directed evolution of the Synechococcus Rubisco in E. coli can elicit improvements in folding and catalytic efficiency.     

Oxidative stress causes rapid membrane translocation and in vivo degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase.

We have studied the turnover of an abundant chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase/oxygenase), in plants (Spirodela oligorrhiza and Triticum aestivum L.) and algae (Chlamydomonas reinhardtii and C. moewusii) induced to senesce under oxidative conditions. Rbu-P2 carboxylase/oxygenase activity and stability in vivo were found to be highly susceptible to oxidative stress, resulting in intermolecular cross-linking of large subunits by disulfide bonds within the holoenzyme, rapid and specific translocation of the soluble enzyme complex to the chloroplast membranes, and finally protein degradation. The redox state of Cys-247 in Rbu-P2 carboxylase/oxygenase large subunit seems involved in the sensitivity of the holoenzyme to oxidative inactivation and cross-linking. However, this process did not drive membrane attachment or degradation of Rbu-P2 carboxylase/oxygenase in vivo. Translocation of oxidized Rbu-P2 carboxylase/oxygenase to chloroplast membranes may be a necessary step in its turnover, particularly during leaf senescence. Thus, processes that regulate the redox state of plant cells seem closely intertwined with cellular switches shifting the leaf from growth and maturation to senescence and death.     

Degradation of Rubisco SSU during oxidative stress triggers aggregation of Rubisco particles in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Oxidative stress in plants and green algae has multiple damaging effects, and leads to the degradation of Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco). We recently showed for the green algae Chlamydomonas reinhardtii that in response to a photo-oxidative stress, nascent synthesis of its chloroplast encoded large subunit (LSU) stops. In parallel, newly synthesized small subunits (SSU) that are encoded by the nucleus are rapidly degraded, thus assembly of new holoenzyme particles is inhibited. Here we show that under extreme oxidizing conditions, the steady-state level of the SSU is also reduced. Cleavage of the LSU under oxidizing conditions is well established, and we show, using sucrose gradients, that the resulting fragments of the LSU co-exist as parts of the holoenzyme. In parallel, we demonstrate the selective in-vivo formation of high-density aggregates of Rubisco particles, in response to oxidative stress. Given the known tendency of unassembled LSUs to aggregate, we propose that the rapid elimination of the SSU during oxidative stress along with the fragmentation of the LSU and formation of intra-protein disulfide bridges, leads to the observed aggregation of Rubisco particles. Indeed, we note here a substantially decreased ratio of SSU in the aggregated Rubisco particles. We also observed that this aggregation marks the viability threshold of C. reinhardtii cells exposed to oxidative stress.     

Crystal structure of a novel-type archaeal rubisco with pentagonal symmetry.

BACKGROUND: Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the key enzyme of the Calvin-Benson cycle and catalyzes the primary reaction of CO2 fixation in plants, algae, and bacteria. Rubiscos have been so far classified into two types. Type I is composed of eight large subunits (L subunits) and eight small subunits (S subunits) with tetragonal symmetry (L8S8), but type II is usually composed only of two L subunits (L2). Recently, some genuinely active Rubiscos of unknown physiological function have been reported from archaea. RESULTS: The crystal structure of Rubisco from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Rubisco) was determined at 2.8 A resolution. The enzyme is composed only of L subunits and showed a novel (L2)5 decameric structure. Compared to previously known type I enzymes, each L2 dimer is inclined approximately 16 degrees to form a toroid-shaped decamer with its unique L2-L2 interfaces. Differential scanning calorimetry (DSC), circular dichroism (CD), and gel permeation chromatography (GPC) showed that Tk-Rubisco maintains its secondary structure and decameric assembly even at high temperatures. CONCLUSIONS: The present study provides the first structure of an archaeal Rubisco, an unprecedented (L2)5 decamer. Biochemical studies indicate that Tk-Rubisco maintains its decameric structure at high temperatures. The structure is distinct from type I and type II Rubiscos and strongly supports that Tk-Rubisco should be classified as a novel type III Rubisco.     

水稻和烟草核酮糖1，5－二磷酸羧化酶／加氧酶大小亚基之间的分子杂交

利用固定化Ｒｕｂｉｓｃｏ大小亚基解离重组技术,进行水稻和烟草Ｒｕｂｉｓｃｏ大小亚基之间的分子杂交,实验表明,无论同源或异源的小亚基重组到固定化的大亚基上去后,其羧化酶活性没有明显的变化,但对加氧酶活性却有明显的影响。当水稻Ｒｕｂｉｓｃｏ的大亚基同烟草小亚基杂交重组后,其加氧酶活性同固定化水稻Ｒｕｂｉｓｃｏ相比有明显的增高,因而其羧化／氧化比值下降,并且接近于对照的固定化烟草Ｒｕｂｉｓｃｏ。反之,当烟草Ｒｕｂｉｓｃｏ的大亚基与水稻小亚基杂交重组后,其加氧酶活性同固定化烟草Ｒｕｂｉｓｃｏ相比有明显降低,因而其羧化／氧化比值升高,并接近于对照的固定化水稻Ｒｕｂｉｓｃｏ。由此推测,高等植物Ｒｕｂｉｓｃｏ的小亚基对酶的羧化／氧化比值有一定的影响。     

C172S substitution in the chloroplast-encoded large subunit affects stability and stress-induced turnover of ribulose-1,5-bisphosphate carboxylase/oxygenase.

Previous work has indicated that the turnover of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1. 39) may be controlled by the redox state of certain cysteine residues. To test this hypothesis, directed mutagenesis and chloroplast transformation were employed to create a C172S substitution in the Rubisco large subunit of the green alga Chlamydomonas reinhardtii. The C172S mutant strain was not substantially different from the wild type with respect to growth rate, and the purified mutant enzyme had a normal circular dichroism spectrum. However, the mutant enzyme was inactivated faster than the wild-type enzyme at 40 and 50 degrees C. In contrast, C172S mutant Rubisco was more resistant to sodium arsenite, which reacts with vicinal dithiols. The effect of arsenite may be directed to the cysteine 172/192 pair that is present in the wild-type enzyme, but absent in the mutant enzyme. The mutant enzyme was also more resistant to proteinase K in vitro at low redox potential. Furthermore, oxidative (hydrogen peroxide) or osmotic (mannitol) stress-induced degradation of Rubisco in vivo was delayed in C172S mutant cells relative to wild-type cells. Thus, cysteine residues could play a role in regulating the degradation of Rubisco under in vivo stress conditions.     

Electron microscopy of the complexes of ribulose-1,5-bisphosphate carboxylase (Rubisco) and Rubisco subunit-binding protein from pea leaves

The structure of ribulose-1,5-bisphosphate carboxylase (Rubisco) subunit-binding protein and its interaction with pea leaf chloroplast Rubisco were studied by electron microscopy and image analysis. Electron-microscopic evidence for the association of Rubisco subunit-binding protein, consisting of 14 subunits arranged with 72 point group symmetry, and oligomeric (L8S8) Rubisco was obtained.     

Immunogold localization of ribulose-1,5-bisphosphate carborylsae/oxygenase in chloroplasts of Chlorella]

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae.     

小球藻Rubisco在叶绿体中的免疫金标定位

应用免疫技术对Rubisco在中国小球藻(Chlorellaspp.640909)叶绿体中进行了分子定位及Native-PAGE电泳、SDS-PAGE电泳及其Westen印迹分析,并对小球藻淀粉核(Pyrenoid)超微结构进行了观察.结果显示Native-PAGE电泳图谱主要为一条主带,Westen印迹反应证明该条带即为Rubisco酶,SDS-PAGE电泳及其Western印迹图谱显示Rubisco大亚基分子量大约为55kD.中国小球藻淀粉核为椭圆形,被淀粉鞘所包围,中央有一条由2个类囊体组成的纵向通道,并在蛋白核内段处稍膨胀.淀粉核与叶绿体基质存在多处联系.免疫分子定位显示Rubisco大亚基和全酶分子主要分布于叶绿体的淀粉核上,且Rubisco在淀粉鞘部位也有少量分布,极少部分分布在叶绿体基质中,表明叶绿体淀粉核与光合作用关系密切.Rubisco聚集于淀粉核可能有利于藻类对CO2固定.     

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO₂ fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.     

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.     

Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/oxygenase

A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.     

Regulation of Rubisco activity in vivo

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is not able to achieve and maintain adequate CO₂ and Mg²⁺ activation under physiological conditions. Higher plants and green algae contain Rubisco activase, a soluble protein which not only facilitates Rubisco activation in situ but also regulates enzyme activity in response to irradiance and other factors. Regulation of Rubisco activity by modulation of activation state coordinates the rate of CO₂ fixation with the rate of substrate regeneration. This regulation may be required to ensure that the levels of photosynthetic metabolites in the chloroplast are optimal for photosynthesis under a variety of environrmental conditions. Some plant species also appear to regulate Rubisco activity by synthesizing 2-carboxyarabinitol 1-phosphate, an inhibitor of Rubisco in the dark. This inhibitor may function primarily as a regulator of metabolite binding in the dark rather than as a modulator of Rubisco activity in the light.     

Analysis of Chromophytic and Rhodophytic Ribulose-1,5-Bisphosphate Carboxylase Indicates Extensive Structural and Functional Similarities among Evolutionarily Diverse Algae

Ribulose-1,5-bisphosphate carboxylase (Rubisco) from the algae Olisthodiscus luteus (chromophyte) and Griffithsia pacifica (rhodophyte) are remarkably similar to each other. However, both enzymes differ significantly in the structure and function when compared to Rubisco from green algae and land plants. Analysis of purified Rubisco from O. luteus and G. pacifica indicates that the size of the holoenzyme and stoichiometry of the 55 and 15 kilodalton subunit polypeptides are approximately 550 kilodaltons and eight:eight for both algae. Antigenic determinants are highly conserved between the O. luteus and G. pacifica enzymes and differ from those of the spinach subunit polypeptides. Sequence similarity between the two algal large subunits has been further confirmed by one-dimensional peptide mapping. Substrate ribulose bisphosphate has no effect on the rate of CO₂/Mg²⁺ activation of O. luteus and G. pacifica enzymes which contrasts to the extensive inhibition of spinach Rubisco activation at similar concentrations of this compound. In addition, the Michaelis constant for CO₂ and the inhibition constant for 6-phosphogluconate are similar for the O. luteus and G. pacifica catalyzed carboxylation reaction. Both values are intermediate to those observed for the tight binding spinach enzyme and weak binding prokaryotic (Rhodospirillum rubrum) enzyme. The biochemical similarities documented between O. luteus and G. pacifica may be due to a common evolutionary origin on the chromophytic and rhodophytic chloroplast but could also result from the fact that both subunit polypeptides are chloroplast DNA encoded in these algal taxa.     

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.