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. 1988 May;7(5):1307–1314. doi: 10.1002/j.1460-2075.1988.tb02945.x

Imported large subunits of ribulose bisphosphate carboxylase/oxygenase, but not imported β-ATP synthase subunits, are assembled into holoenzyme in isolated chloroplasts

Anthony A Gatenby 1,3, Thomas H Lubben 2, Paul Ahlquist 1, Kenneth Keegstra 2
PMCID: PMC458409  PMID: 16453842

Abstract

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.

Keywords: chimeric proteins, coupling factor, import, plastid genes, RuBP carboxylase

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Selected References

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

  1. Andrews T. J., Lorimer G. H. Catalytic properties of a hybrid between cyanobacterial large subunits and higher plant small subunits of ribulose bisphosphate carboxylase-oxygenase. J Biol Chem. 1985 Apr 25;260(8):4632–4636. [PubMed] [Google Scholar]
  2. Barraclough R., Ellis R. J. Protein synthesis in chloroplasts. IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts. Biochim Biophys Acta. 1980 Jun 27;608(1):19–31. doi: 10.1016/0005-2787(80)90129-x. [DOI] [PubMed] [Google Scholar]
  3. Chua N. H., Schmidt G. W. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6110–6114. doi: 10.1073/pnas.75.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cline K., Andrews J., Mersey B., Newcomb E. H., Keegstra K. Separation and characterization of inner and outer envelope membranes of pea chloroplasts. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3595–3599. doi: 10.1073/pnas.78.6.3595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cline K., Werner-Washburne M., Lubben T. H., Keegstra K. Precursors to two nuclear-encoded chloroplast proteins bind to the outer envelope membrane before being imported into chloroplasts. J Biol Chem. 1985 Mar 25;260(6):3691–3696. [PubMed] [Google Scholar]
  6. Gatenby A. A. The properties of the large subunit of maize ribulose bisphosphate carboxylase/oxygenase synthesised in Escherichia coli. Eur J Biochem. 1984 Oct 15;144(2):361–366. doi: 10.1111/j.1432-1033.1984.tb08472.x. [DOI] [PubMed] [Google Scholar]
  7. Gray J. C., Bird C. R., Courtice G. R., Hird S. M., Howe C. J., Huttly A. K., Phillips A. L., Smith A. G., Willey D. L., Bowman C. M. Chloroplast genes for photosynthetic membrane components of higher plants. Biochem Soc Trans. 1986 Feb;14(1):25–27. [PubMed] [Google Scholar]
  8. Gurevitz M., Somerville C. R., McIntosh L. Pathway of assembly of ribulosebisphosphate carboxylase/oxygenase from Anabaena 7120 expressed in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6546–6550. doi: 10.1073/pnas.82.19.6546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hill H. A., Tew D. G., Walton N. J. An opsonised microelectrode. Observation of the respiratory burst of a single human neutrophil. FEBS Lett. 1985 Oct 28;191(2):257–263. doi: 10.1016/0014-5793(85)80020-x. [DOI] [PubMed] [Google Scholar]
  10. Hurt E. C., Goldschmidt-Clermont M., Pesold-Hurt B., Rochaix J. D., Schatz G. A mitochondrial presequence can transport a chloroplast-encoded protein into yeast mitochondria. J Biol Chem. 1986 Sep 5;261(25):11440–11443. [PubMed] [Google Scholar]
  11. Krebbers E. T., Larrinua I. M., McIntosh L., Bogorad L. The maize chloroplast genes for the beta and epsilon subunits of the photosynthetic coupling factor CF1 are fused. Nucleic Acids Res. 1982 Aug 25;10(16):4985–5002. doi: 10.1093/nar/10.16.4985. [DOI] [PMC free article] [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. Lubben T. H., Keegstra K. Efficient in vitro import of a cytosolic heat shock protein into pea chloroplasts. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5502–5506. doi: 10.1073/pnas.83.15.5502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nelson N. Functional assembly of the chloroplast H+-ATPase and photosynthetic reaction centres. Biochem Soc Trans. 1986 Feb;14(1):5–7. doi: 10.1042/bst0140005. [DOI] [PubMed] [Google Scholar]
  15. Nelson N., Nelson H., Schatz G. Biosynthesis and assembly of the proton-translocating adenosine triphosphatase complex from chloroplasts. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1361–1364. doi: 10.1073/pnas.77.3.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Roy H., Bloom M., Milos P., Monroe M. Studies on the assembly of large subunits of ribulose bisphosphate carboxylase in isolated pea chloroplasts. J Cell Biol. 1982 Jul;94(1):20–27. doi: 10.1083/jcb.94.1.20. [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. Schmidt G. W., Mishkind M. L. The transport of proteins into chloroplasts. Annu Rev Biochem. 1986;55:879–912. doi: 10.1146/annurev.bi.55.070186.004311. [DOI] [PubMed] [Google Scholar]
  19. Schreier P. H., Seftor E. A., Schell J., Bohnert H. J. The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J. 1985 Jan;4(1):25–32. doi: 10.1002/j.1460-2075.1985.tb02312.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shinozaki K., Yamada C., Takahata N., Sugiura M. Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4050–4054. doi: 10.1073/pnas.80.13.4050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Takeda M., Vassarotti A., Douglas M. G. Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor. J Biol Chem. 1985 Dec 15;260(29):15458–15465. [PubMed] [Google Scholar]
  23. Vaessen R. T., Kreike J., Groot G. S. Protein transfer to nitrocellulose filters. A simple method for quantitation of single proteins in complex mixtures. FEBS Lett. 1981 Feb 23;124(2):193–196. doi: 10.1016/0014-5793(81)80134-2. [DOI] [PubMed] [Google Scholar]
  24. Van den Broeck G., Timko M. P., Kausch A. P., Cashmore A. R., Van Montagu M., Herrera-Estrella L. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. 1985 Jan 31-Feb 6Nature. 313(6001):358–363. doi: 10.1038/313358a0. [DOI] [PubMed] [Google Scholar]
  25. van der Vies S. M., Bradley D., Gatenby A. A. Assembly of cyanobacterial and higher plant ribulose bisphosphate carboxylase subunits into functional homologous and heterologous enzyme molecules in Escherichia coli. EMBO J. 1986 Oct;5(10):2439–2444. doi: 10.1002/j.1460-2075.1986.tb04519.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. von Heijne G. Why mitochondria need a genome. FEBS Lett. 1986 Mar 17;198(1):1–4. doi: 10.1016/0014-5793(86)81172-3. [DOI] [PubMed] [Google Scholar]

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