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.     

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.     

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.     

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     

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     

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.     

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Lipid-protein particles bearing the 55-kD ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (EC 4.1.1.39) large subunit (RLSU) and no detectable corresponding Rubisco small subunit (RSSU) were isolated from the stroma of intact chloroplasts by flotation centrifugation. Stromal RLSU-bearing particles appear to originate from thylakoids because they can also be generated in vitro by illumination of isolated thylakoids. Their formation in vitro is largely heat denaturable and is facilitated by light or ATP. RLSU-containing lipid-protein particles range from 0.05 to 0.10 [mu]m in radius, contain the same fatty acids as thylakoids, but have a 10- to 15-fold higher free-to-esterified fatty acid ratio than thylakoids. RLSU-bearing lipid-protein particles with no detectable RSSU were also immunopurified from the populations of both stromal lipid-protein particles and those generated in vitro from illuminated thylakoids. Protease shaving indicated that the RLSU is embedded in the lipid-protein particles and that there is also a protease-protected RLSU in thylakoids. These observations collectively indicate that the RLSU associated with thylakoids is released into the stroma by light-facilitated blebbing of lipid-protein particles. The release of RLSU-containing particles may in turn be coordinated with the assembly of Rubisco holoenzyme because chaperonin 60 is also associated with lipid-protein particles isolated from stroma.     

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.     

Assembly of Two Subunits of the Cyanobacterial Photosystem I on the n-Side of Thylakoid Membranes

Photosystem I contains several peripheral membrane proteins that are located on either positive (luminal) or negative (stromal or cytoplasmic) sides of thylakoid membranes of chloroplasts or cyanobacteria. Incorporation of two peripheral subunits into photosystem I of the cyanobacterium Synechocystis species PCC 6803 was studied using a reconstitution system in which radiolabeled subunits II (PsaD) and IV (PsaE) were synthesized in vitro and incubated with the isolated thylakoid membranes. After such incubation, the subunits were found in the membranes and were resistant to digestion with proteases and removal by 2 molar NaBr. All of the radioactive proteins incorporated in the membrane were found in the photosystem I complex. The subunit II was assembled specifically into cyanobacterial thylakoid membranes and not into Escherichia coli cell membranes or thylakoid membranes isolated from spinach. The assembly process did not require ATP or proton motive force, and it was not stimulated by ATP. The assembly of subunits II and IV into thylakoid membranes isolated from the strain AEK2, which lacks the gene psaE, was increased two- to threefold. The incorporation of subunit II was 15 to 17 times higher in the thylakoids obtained from the strain ADK3 in which the gene psaD has been inactivated. However, assembly of subunit IV in the same thylakoids was reduced by 65%, demonstrating that the presence of subunit II is required for the stable assembly of subunit IV. Large deletions in subunit II prevented its incorporation into thylakoids and assembly into photosystem I, suggesting that the overall conformation of the protein rather than a specific targeting sequence is required for its assembly into photosystem I.     

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 .     

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.     

Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota

In early biological evolution anoxygenic photosynthetic bacteria may have been established through the acquisition of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The establishment of cyanobacteria may have followed and led to the production of atmospheric oxygen. It has been postulated that a unicellular cyanobacterium evolved to cyanelles which were evolutionary precursors of chloroplasts of both green and non-green algae. The latter probably diverged from ancestors of green algae as evidenced by the occurrence of large (L) and small (S) subunit genes for Rubisco in the chloroplast genome of the chromophytic algae Olisthodiscus luteus. In contrast, the gene for the S subunit was integrated into the nucleus in the evolution of green algae and higher plants. The evolutionary advantages of this integration are uncertain because the function of S subunits is unknown. Recently, two forms of Rubisco (L8 and L8S8) of almost equivalent carboxylase and oxygenase activity have been isolated from the photosynthetic bacterium Chromatium vinosum. This observation perpetuates the enigma of S subunit function. Current breakthroughs are imminent, however, in our understanding of the function of catalytic L subunits because of the application of deoxyoligonucleotide-directed mutagenesis. Especially interesting mutated Rubisco molecules may have either enhanced carboxylase activity or higher carboxylase:oxygenase ratios. Tests of expression, however, must await the insertion of modified genes into the nucleus and chloroplasts. Methodology to accomplish chloroplast transformation is as yet unavailable. Recently, we have obtained the first transformation of cyanobacteria by a colE1 plasmid. We regard this transformation as an appropriate model for chloroplast transformation.     

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.     

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.     

Stability and Dissociation of the Large Subunit RuBisCO Binding Protein Complex in Vitro and in Organello

We are studying the stability of the binding protein which associates with newly synthesized large subunits of ribulose bisphosphate carboxylase. In chloroplast extracts, it has been shown that a dodecameric complex of the large subunit binding protein dissociates extensively into binding protein monomers and 7S (117 kilodaltons) large subunit-containing complexes in the presence of ATP. The concentrations of ATP which bring this about are quite low, prompting some investigators to suggest that the dodecameric complex might not exist in vivo. We have found, however, that in concentrated chloroplast extracts, at protein concentrations which are closer to those which occur in organello, the dissociation of the binding protein complex by ATP is much less extensive. For this reason, we have tested the stability of the binding protein in organello, by illuminating chloroplasts followed by lysis and polyacrylamide gel electrophoresis of the extracts. Radioactive large subunits associated with the dodecameric binding protein dissociated extensively in the light. The results are consistent with the idea that the high molecular weight form of the binding protein can function as a reservoir of large subunits which can be tapped in vivo, in a reaction dependent on light and ATP.     

Isolation of a Protein Containing Covalently Linked Large and Small Subunits of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Botryococcus braunii

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and a 66-kD protein were co-purified from solubilized microsomal preparations of the green alga Botryococcus braunii by Green A agarose, sucrose density gradient, MonoQ, and gel filtration. The 66-kD protein remained intact after 6 M urea treatment and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It could be detected in the soluble fraction of the cell-free extract but appeared to be more abundant in the microsomal preparations. It cross-reacted with antibodies raised against Rubisco holoenzyme, large and small subunits, indicating that the 66-kD protein contains both the large and the small subunits of Rubisco. The N-terminal amino acid sequence of this protein and that of a proteolytic fragment showed high homology with the mature Rubisco small subunits, and the sequence of another proteolytic fragment showed high homology with that of the Rubisco large subunit. It is concluded that the 66-kD protein is produced by cross-linking of large and small sub-units of Rubisco in the cell.     

Assembly of Newly Imported Oxygen-Evolving Complex Subunits in Isolated Chloroplasts: Sites of Assembly and Mechanism of Binding

We have examined the assembly of the nuclear-encoded subunits of the oxygen-evolving complex (OEC) after their import into isolated intact chloroplasts. We showed that all three subunits examined (OE33, OE23, and OE17) partition between the thylakoid lumen and a site on the inner surface of the thylakoid membrane after import in a homologous system (e.g., pea or spinach subunits into pea or spinach chloroplasts, respectively). Although some interspecies protein import experiments resulted in OEC subunit binding, maize OE17 did not bind thylakoid membranes in chloroplasts isolated from peas. Newly imported OE33 and OE23 were washed from the membranes at the same concentrations of urea and NaCl as the native, indigenous proteins; this observation suggests that the former subunits are bound productively within the OEC. Inhibition of neither chloroplast protein synthesis nor light- or ATP-dependent energization of the thylakoid membrane significantly affected these assembly reactions, and we present evidence suggesting that incoming subunits actively displace those already bound to the thylakoid membrane. Transport of OE33 took place primarily in the stromal-exposed membranes and proceeded through a protease-sensitive, mature intermediate. Initial binding of OE33 to the thylakoid membrane occurred primarily in the stromal-exposed membranes, from where it migrated with measurable kinetics to the granal region. In contrast, OE23 assembly occurred in the granal membrane regions. This information is incorporated into a model of the stepwise assembly of oxygen-evolving photosystem II.