Rubisco Assembly Research Articles

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.

Assessment of plant chaperonin-60 gene function in Escherichia coli

Brassica napus chaperonin-60 alpha and chaperonin-60 beta genes expressed separately and in combination produce three novel Escherichia coli strains: alpha, beta, and alpha beta. In beta and alpha beta cells, the plant gene products assemble efficiently into tetradecameric cpn60(14) species, including novel hybrids containing both bacterial and plant gene products. The levels of authentic groEL14 are reduced in these cells (Cloney, L. P., Wu, H. B., and Hemmingsen, S. M. (1992) J. Biol. Chem. 267, 23327-23332). The assembly of cyanobacterial ribulose-P2 carboxylase (rubisco) in E. coli requires the activities of the endogenous chaperonin proteins. Furthermore, the extent to which assembly occurs is limited by the normal levels of expression of the groE operon (Goloubinoff, P., Gatenby, A. A., and Lorimer, G. H. (1989) Nature 337, 44-47). We have now monitored the accumulation of cyanobacterial rubisco in E. coli alpha, beta, and alpha beta cells to assess the activity of the plant cpn60 gene products and effects on endogenous chaperonin functions. Expression of cpn-60 alpha alone did not enhance rubisco assembly, which is consistent with our previous observation that p60cpn-60 alpha required the presence of p60cpn-60 beta for assembly into cpn60(14) species. In contrast, expression of cpn-60 beta alone resulted in markedly enhanced rubisco assembly in cells that accumulated normal levels of both endogenous chaperonin polypeptides (groEL and groES). This demonstrates that assembled p60cpn-60 beta is functional as a chaperonin in E. coli. Co-expression of cpn-60 alpha and cpn-60 beta in cells with normal levels of expression of groES and groEL suppressed rubisco assembly. Increased expression of groES in cells in which cpn-60 alpha and cpn-60 beta were co-expressed relieved this suppression and resulted in enhanced rubisco assembly. Implications with respect to dependence of chloroplast cpn60 function on cpn10 are discussed.

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.

Theory of chaperonin action: inertial model for enhancement of prokaryotic Rubisco assembly.

We have performed a computational simulation of the aggregation and chaperonin-dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P. Goloubinoff et al. (1989, Nature 342, 884-889) and P. V. Viitanen et al. (1990, Biochemistry 29, 5665-5671). The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco-I, with itself and with aggregates of Rubisco-I, leading up to dodecamers. Four rate constants, applying to forward or reverse steps in the aggregation process, were included. Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G. & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp. 315-322). Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one. The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al. (1989). The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin. This is followed by self-association of the folded Rubisco monomer to yield the active dimeric Rubisco. A modification of the model that simulates temperature effects was also constructed. The most important results we obtained indicate that the chaperonin-dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin. In addition, the simulations predict values for the association rate constant of Rubisco-I with the chaperonin, and other variables, that are subject to experimental verification.

Biochemical and molecular studies of photosynthesis

Abstract Recent research on photosynthesis is reviewed, including electron transport and the complexities of the Rubisco enzyme, its biosynthesis, assembly and activity parameters. Aspects of gene expression are also considered, together with interactions between plant organelles, source—sink relationships in the allocation of photosynthate, and the influence of the environment on photosynthesis-related reactions.

Molecular chaperones and chloroplast biogenesis

It is now two years since it was first proposed that many cellular processes require the mediation of a family of proteins termed molecular chaperones (1). During these two years evidence supporting the concept that molecular chaperones mediate universal cellular functions has grown (2–4). More and more proteins are being identified which carry out chaperone roles in a growing variety of cellular processes. In this article we briefly discuss the concept of molecular chaperones, and speculate on their possible roles in chloroplast biogenesis in the hope of stimulating research in this area. It is particularly appropriate to discuss the chaperone concept at this Congress, since its origin springs from research on the assembly of the photosynthetic enzyme Rubisco carried out at the University of Warwick since 1979 (5).

Rubisco assembly: a model system for studying the mechanism of chaperonin action.

The chloroplast enzyme Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) catalyzes the carboxylation of ribulose bisphosphate in the Calvin cycle and the oxygenation of the same substrate in the photorespiratory pathway. Because these two competing reactions determine the net photosynthetic yield of many higher plants, it is important to understand their mechanisms. This requires not only a model of Rubisco atomic structure, but also experimental systems that allow the structure to be manipulated by in vitro mutagenesis. The requirement for a structural model has been fulfilled recently (Chapman et al., 1987, 1988; Andersson et al., 1989). Unfortunately, however, in vitro mutagenesis of higher plant Rubisco has not been possible because the subunits of higher plant Rubisco, either when dissociated from each other chemically in vitro (Voordouw, Van der Vies, and Boumeister, 1984) or when synthesized in Escherichia coli (Bradley, Van der Vies, and Gatenby, 1986), fail to assemble into active enzyme. Severa1 groups have attempted to overcome this problem by studying Rubisco assembly from either native subunits (Roy, Cannon, and Gilson, 1988a), prokaryotic subunits expressed in E. coli (Gatenby, 1988), or alga1 subunits (Newman and Cattolico, 1988). These studies indicate that the post-translational assembly of Rubisco holoenzyme in both prokaryotes and eukaryotes is a complex process that may require helper proteins called “chaperonins” (Gatenby, 1988; Hemmingsen et al., 1988; Roy and Cannon, 1988; Roy et al., 1988a; Ellis, Van der Vies, and Hemmingsen, 1989). Chaperonins are functionally analogous to other molecular chaperones such as nucleoplasmin, which is involved in nucleosome assembly, and the bovine microsomal binding protein, which participates in immunoglobulin transport (Pelham, 1986; Ellis et al., 1989). The molecular chaperones, including the chaperonins, are supposed to interact transiently with other proteins, promoting assembly of those proteins into functional complexes. The molecular chaperones do not form part of the final structure of the protein complexes whose assembly they promote. In this article I review recent experiments that persuasively support the idea that chaperonins are required for Rubisco assembly.

Rubisco Assembly: A Model System for Studying the Mechanism of Chaperonin Action

Rubisco Assembly: A Model System for Studying the Mechanism of Chaperonin Action

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

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.

GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli

Assembly of foreign prokaryotic ribulose bisphosphate carboxylases (Rubiscos) in Escherichia coli requires both heat-shock proteins groEL and groES. GroEL is related to a chloroplast protein implicated in Rubisco assembly. Bacteria and chloroplasts therefore have a conserved mechanism that uses auxiliary proteins to assist in the assembly of Rubisco.

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 Rubisco large subunit binding protein

Newly synthesized Rubisco large subunits made by isolated intact chloroplasts fromPisum sativumare bound non-covalently to another protein, termed the Rubisco large subunit binding protein. This protein is implicated in the assembly of Rubisco in higher plant chloroplasts. The binding protein has been purified fromPisum sativumin the form of an oligomer of relative molecular mass (Mr) about 720000. Analysis on polyacrylamide gels containing sodium dodecyl sulphate reveals equal amounts of two different types of subunit, termed alpha (Mrabout 61000) and beta (Mrabout 60000); thus the oligomer has the composition a α6β6. The alpha and beta subunits have been separated; their amino-terminal sequences are different, and antibodies raised against one subunit do not cross-react with the other subunit. Antibodies raised against the binding protein do not cross-react with the Rubisco large subunit, but do cross-react with polypeptides ofMrabout 60000 in extracts of chloroplasts from wheat, barley and tobacco, and in extracts of leucoplasts from castor-bean endosperm. The binding protein is made as a higher-molecular-mass precursor whan leaf polysomes are translated in a wheatgerm extract containing chloramphenicol, but is not synthesized by isolated intact chloroplasts. Thus the binding protein subunits are synthesized by cytoplasmic ribosomes and hence are likely to be encoded by nuclear genes. EtiolatedPisumplants contain binding protein, but exposure to light does not cause the same dramatic increase in amount that is seen in the case of Rubisco. Treatment of stromal extracts with Mg-ATP in the range 0.1-5.0 mM causes dissociation of the binding protein oligomer into monomeric subunits; CTP, GTP, UTP, AMP and cyclic AMP do not have this effect. Mg2+is required for dissociation but can be replaced by Ca2+. Newly synthesized large subunits are released when the binding protein oligomer is dissociated, but re-attach when the dissociation is reversed by removal of ATP.

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.