Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1992 Sep;100(1):272–281. doi: 10.1104/pp.100.1.272

Synthesis and Assembly of Large Subunits into Ribulose Bisphosphate Carboxylase/Oxygenase in Chloroplast Extracts 1

Alan Hubbs 1, Harry Roy 1
PMCID: PMC1075549  PMID: 16652958

Abstract

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.

Full text

PDF
272

Images in this article

277Figure 3
on p.277
277Figure 4
on p.277
279Figure 5
on p.279

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arnon D. I. COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS. Plant Physiol. 1949 Jan;24(1):1–15. doi: 10.1104/pp.24.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bard J. D., Bourque D. P., Zaitlin D. Coupled transcription-translation in chloroplast lysates. Methods Enzymol. 1986;118:270–295. doi: 10.1016/0076-6879(86)18078-5. [DOI] [PubMed] [Google Scholar]
  3. Bard J., Bourque D. P., Hildebrand M., Zaitlin D. In vitro expression of chloroplast genes in lysates of higher plant chloroplasts. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3983–3987. doi: 10.1073/pnas.82.12.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bloom M. V., Milos P., Roy H. Light-dependent assembly of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1013–1017. doi: 10.1073/pnas.80.4.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bottomley W., Spencer D., Whitfeld P. R. Protein synthesis in isolated spinach chloroplasts: comparison of light-driven and ATP-driven synthesis. Arch Biochem Biophys. 1974 Sep;164(1):106–117. doi: 10.1016/0003-9861(74)90012-5. [DOI] [PubMed] [Google Scholar]
  6. Bottomley W., Whitfeld P. R. Cell-free transcription and translation of total spinach chloroplast DNA. Eur J Biochem. 1979 Jan 2;93(1):31–39. doi: 10.1111/j.1432-1033.1979.tb12791.x. [DOI] [PubMed] [Google Scholar]
  7. Ellis R. J., Blair G. E., Hartley M. R. The nature and function of chloroplast protein synthesis. Biochem Soc Symp. 1973;(38):137–162. [PubMed] [Google Scholar]
  8. Gatenby A. A., van der Vies S. M., Rothstein S. J. Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli. Eur J Biochem. 1987 Oct 1;168(1):227–231. doi: 10.1111/j.1432-1033.1987.tb13409.x. [DOI] [PubMed] [Google Scholar]
  9. Goldstein E. S., Reichman M. E., Penman S. The regulation of protein synthesis in mammalian cells VI. Soluble and polyribosome associated components in controlling in vitro polypeptide initiation in HeLa cells. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4752–4756. doi: 10.1073/pnas.71.12.4752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. González R. G., Haxo R. S., Schleich T. Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions. Biochemistry. 1980 Sep 2;19(18):4299–4303. doi: 10.1021/bi00559a023. [DOI] [PubMed] [Google Scholar]
  11. Jackson R. J., Hunt T. Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 1983;96:50–74. doi: 10.1016/s0076-6879(83)96008-1. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lizardi P. M. Genetic polymorphism of silk fibroin studied by two-dimensional translation pause fingerprints. Cell. 1979 Oct;18(2):581–589. doi: 10.1016/0092-8674(79)90074-6. [DOI] [PubMed] [Google Scholar]
  14. Milos P., Roy H. ATP-released large subunits participate in the assembly of RuBP carboxylase. J Cell Biochem. 1984;24(2):153–162. doi: 10.1002/jcb.240240206. [DOI] [PubMed] [Google Scholar]
  15. Mullet J. E., Klein R. R., Grossman A. R. Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates. Eur J Biochem. 1986 Mar 3;155(2):331–338. doi: 10.1111/j.1432-1033.1986.tb09495.x. [DOI] [PubMed] [Google Scholar]
  16. Roy H., Chaudhari P., Cannon S. Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts : Influence of Added Small Subunits and of Conditions during Synthesis. Plant Physiol. 1988 Jan;86(1):44–49. doi: 10.1104/pp.86.1.44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schmidt G. W., Mishkind M. L. Rapid degradation of unassembled ribulose 1,5-bisphosphate carboxylase small subunits in chloroplasts. Proc Natl Acad Sci U S A. 1983 May;80(9):2632–2636. doi: 10.1073/pnas.80.9.2632. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Sheard B., Miall S. H., Peacocke A. R., Walker I. O., Richards R. E. Proton magnetic relaxation studies of the binding of manganese ions to Escherichia coli ribosomes. J Mol Biol. 1967 Sep 28;28(3):389–402. doi: 10.1016/s0022-2836(67)80088-3. [DOI] [PubMed] [Google Scholar]
  19. Stewart M. L., Grollman A. P., Huang M. T. Aurintricarboxylic acid: inhibitor of initiation of protein synthesis. Proc Natl Acad Sci U S A. 1971 Jan;68(1):97–101. doi: 10.1073/pnas.68.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tabita F. R., Small C. L. Expression and assembly of active cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase in Escherichia coli containing stoichiometric amounts of large and small subunits. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6100–6103. doi: 10.1073/pnas.82.18.6100. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES