Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

Roy, Harry; Chaudhari, Panna; Cannon, Susan

doi:10.1104/pp.86.1.44

Cited by 13 publications

(12 citation statements)

References 14 publications

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“…Since these are the immediate source of large subunits for the in vitro assembly of RuBisCO, the increased supply of these could permit greater assembly in vitro. This behavior was predicted by Roy et al (22). The data presented here are in agreement with that prediction: there is an increase in the relative amount of large subunits in the low mol wt, assembly-competent complexes when the large subunits are synthesized under hypotonic conditions.…”

Section: Discussionsupporting

confidence: 81%

“…Large subunits and practically no other soluble radiolabeled polypeptides were found in all gradient fractions, as previously reported (19). The (22).…”

Section: Resultssupporting

confidence: 59%

“…Earlier data (4,16,22) indicated that the low mol wt complexes are the immediate donor for the in vitro assembly of RuBisCO. We therefore analyzed the low mol wt large subunit complexes synthesized in organello at 188 mm sorbitol and compared them with the complexes synthesized at 377 mM sorbitol.…”

Section: Resultsmentioning

confidence: 99%

“…RuBisCO small subunits were isolated as previously described (22) and were used to supplement the assembly reactions. Samples were analyzed by nondenaturing polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, sucrose velocity gradient centrifugation, and liquid scintillation counting as previously described (4,13,19,20).…”

Section: Analysis Of Productsmentioning

confidence: 99%

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Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Chaudhari¹,

Roy²

1989

Plant Physiol.

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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 coil. 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 vftro 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.

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Section: Discussionsupporting

confidence: 81%

“…Large subunits and practically no other soluble radiolabeled polypeptides were found in all gradient fractions, as previously reported (19). The (22).…”

Section: Resultssupporting

confidence: 59%

Section: Resultsmentioning

confidence: 99%

Section: Analysis Of Productsmentioning

confidence: 99%

See 2 more Smart Citations

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Chaudhari¹,

Roy²

1989

Plant Physiol.

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“…The components added to the translation reaction in this fashion were included to give final concentrations as follows: Bethesda Research Laboratories 1Ox biosynthesis reaction mix (50 1LM amino acids minus leucine or methionine, 10 mM phosphocreatine, 40 mi KCl, 0.4 mM ATP, 0. The assembly of large subunits sythesized in vitro was carried out essentially as described for the assembly of in organello-synthesized large subunits (4,22,23,25). However, DTT was not added to the assembly reaction because it was already present during synthesis; also, lower salt concentrations were used.…”

Section: In Vitro Protein Synthesis In Chloroplast Lysatesmentioning

confidence: 99%

Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast Extracts

Hubbs¹,

Roy²

1992

Plant Physiol.

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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.

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

et al. 1989

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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.

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Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

Cited by 13 publications

References 14 publications

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast Extracts

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

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