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. 1973 Jun 1;57(3):798–814. doi: 10.1083/jcb.57.3.798

ISOLATION OF CYTOPLASMIC AND CHLOROPLAST RIBOSOMES AND THEIR DISSOCIATION INTO ACTIVE SUBUNITS FROM CHLAMYDOMONAS REINHARDTII

Nam-Hai Chua 1, G Blobel 1, P Siekevitz 1
PMCID: PMC2109006  PMID: 4698907

Abstract

A mixture of cytoplasmic (80S) and chloroplast (70S) ribosomes from Chlamydomonas reinhardtii was freed of contaminating membranes by sedimentation of the postmitochondrial supernatant through a layer of 1.87 M sucrose. The purified ribosomes were separated into 80S and 70S fractions by centrifugation at a relatively low speed on a 10–40% sucrose gradient containing 25 mM KCl and 5 mM MgCl2. Both the 80S and 70S ribosomes were dissociated into compact subunits by centrifugations in 5–20% high-salt sucrose gradients. The dissociations of both ribosomal species under these conditions were not affected by the addition of puromycin, indicating that the ribosomes as isolated were devoid of nascent chains. Subunits derived from the 80S ribosomes had apparent sedimentation coefficients of 57S and 37S whereas those from the 70S ribosomes had apparent sedimentation coefficients of 50S and 33S. In the presence of polyuridylic acid and cofactors, the 80S and 70S ribosomes incorporated [14C]phenylalanine into material insoluble in hot TCA. The requirements for incorporation were found to be similar to those described for eukaryotic and prokaryotic ribosomes. Experiments with antibiotics showed that the activity of the 80S ribosomes was sensitive to cycloheximide, whereas that of the 70S ribosomes was inhibited by streptomycin. The isolated subunits, when mixed together in an incorporation medium, were also active in the polymerization of phenylalanine in vitro.

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Selected References

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  1. App A. A., Bulis M. G., McCarthy W. J. Dissociation of ribosomes and seed germination. Plant Physiol. 1971 Jan;47(1):81–86. doi: 10.1104/pp.47.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BOARDMAN N. K., FRANCKI R. I., WILDMAN S. G. PROTEIN SYNTHESIS BY CELL-FREE EXTRACTS FROM TOBACCO LEAVES. II. ASSOCIATION OF ACTIVITY WITH CHLOROPLAST RIBOSOMES. Biochemistry. 1965 May;4:872–876. doi: 10.1021/bi00881a012. [DOI] [PubMed] [Google Scholar]
  3. BRAWERMAN G., EISENSTADT J. M. TEMPLATE AND RIBOSOMAL RIBONUCLEIC ACIDS ASSOCIATED WITH THE CHLOROPLASTS AND THE CYTOPLASM OF EUGLENA GRACILIS. J Mol Biol. 1964 Dec;10:403–411. doi: 10.1016/s0022-2836(64)80061-9. [DOI] [PubMed] [Google Scholar]
  4. Blobel G. Isolation of a 5S RNA-protein complex from mammalian ribosomes. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1881–1885. doi: 10.1073/pnas.68.8.1881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blobel G., Sabatini D. Dissociation of mammalian polyribosomes into subunits by puromycin. Proc Natl Acad Sci U S A. 1971 Feb;68(2):390–394. doi: 10.1073/pnas.68.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boardman N. K., Francki R. I., Wildman S. G. Protein synthesis by cell-free extracts of tobacco leaves. 3. Comparison of the physical properties and protein synthesizing activities of 70 s chloroplast and 80 s cytoplasmic ribosomes. J Mol Biol. 1966 Jun;17(2):470–487. doi: 10.1016/s0022-2836(66)80157-2. [DOI] [PubMed] [Google Scholar]
  7. Bourque D. P., Boynton J. E., Gillham N. W. Studies on the structure and cellular location of various ribosome and ribosomal RNA species in the green alga Chlamydomonas reinhardi. J Cell Sci. 1971 Jan;8(1):153–183. doi: 10.1242/jcs.8.1.153. [DOI] [PubMed] [Google Scholar]
  8. Boynton J. E., Gillham N. W., Burkholder B. Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, II. A New Mendelian Mutation. Proc Natl Acad Sci U S A. 1970 Nov;67(3):1505–1512. doi: 10.1073/pnas.67.3.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. EISENSTADT J. M., BRAWERMAN G. THE PROTEIN-SYNTHESIZING SYSTEMS FROM THE CYTOPLASM AND THE CHLOROPLASTS OF EUGLENA GRACILIS. J Mol Biol. 1964 Dec;10:392–402. doi: 10.1016/s0022-2836(64)80060-7. [DOI] [PubMed] [Google Scholar]
  10. Faber A. J., Tamaoki T. Isolation of active ribosomal subunits from L5178Y mouse lymphoma cells. Arch Biochem Biophys. 1972 Mar;149(1):289–294. doi: 10.1016/0003-9861(72)90324-4. [DOI] [PubMed] [Google Scholar]
  11. Faust C. H., Jr, Matthaei H. A systematic study of the isolation of murine plasma cell ribosomal subunits, their sedimentation properties and activity in polyphenylalanine synthesis. Biochemistry. 1972 Jul 4;11(14):2682–2691. doi: 10.1021/bi00764a021. [DOI] [PubMed] [Google Scholar]
  12. Friedman H., Lu P., Rich A. Temperature control of initiation of protein synthesis in Escherichia coli. J Mol Biol. 1971 Oct 14;61(1):105–121. doi: 10.1016/0022-2836(71)90209-9. [DOI] [PubMed] [Google Scholar]
  13. Gorman D. S., Levine R. P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665–1669. doi: 10.1073/pnas.54.6.1665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hamilton M. G., Pavlovec A., Petermann M. L. Molecular weight, buoyant density, and composition of active subunits of rat liver ribosomes. Biochemistry. 1971 Aug 31;10(18):3424–3427. doi: 10.1021/bi00794a017. [DOI] [PubMed] [Google Scholar]
  15. Hauge J. G. Pressure-induced dissociation of ribosomes during ultracentrifugation. FEBS Lett. 1971 Sep 15;17(1):168–172. doi: 10.1016/0014-5793(71)80589-6. [DOI] [PubMed] [Google Scholar]
  16. Hoober J. K., Blobel G. Characterization of the chloroplastic and cytoplasmic ribosomes of Chlamydomonas reinhardi. J Mol Biol. 1969 Apr 14;41(1):121–138. doi: 10.1016/0022-2836(69)90130-2. [DOI] [PubMed] [Google Scholar]
  17. Infante A. A., Baierlein R. Pressure-induced dissociation of sedimenting ribosomes: effect on sedimentation patterns. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1780–1785. doi: 10.1073/pnas.68.8.1780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Infante A. A., Krauss M. Dissociation of ribosomes induced by centrifugation: evidence for doubting conformational changes in ribosomes. Biochim Biophys Acta. 1971 Aug 12;246(1):81–99. [PubMed] [Google Scholar]
  19. KUCAN Z., LIPMANN F. DIFFERENCES IN CHLORAMPHENICOL SENSITIVITY OF CELL-FREE AMINO ACID POLYMERIZATION SYSTEMS. J Biol Chem. 1964 Feb;239:516–520. [PubMed] [Google Scholar]
  20. LYTTLETON J. W. Isolation of ribosomes from spinach chloroplasts. Exp Cell Res. 1962 Mar;26:312–317. doi: 10.1016/0014-4827(62)90183-0. [DOI] [PubMed] [Google Scholar]
  21. Lawford G. R. The effect of incubation with puromycin on the dissociation of rat liver ribosomes into active subunits. Biochem Biophys Res Commun. 1969 Sep 24;37(1):143–150. doi: 10.1016/0006-291x(69)90892-4. [DOI] [PubMed] [Google Scholar]
  22. Lee S. G., Evans W. R. Hybrid ribosome formation from Escherichia coli and chloroplast ribosome subunits. Science. 1971 Jul 16;173(3993):241–242. doi: 10.1126/science.173.3993.241. [DOI] [PubMed] [Google Scholar]
  23. Lin C. Y., Key J. L. Dissociation of N(2) Gas-induced Monomeric Ribosomes and Functioning of the Derived Subunits in Protein Synthesis in Pea. Plant Physiol. 1971 Nov;48(5):547–552. doi: 10.1104/pp.48.5.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loening U. E. Molecular weights of ribosomal RNA in relation to evolution. J Mol Biol. 1968 Dec;38(3):355–365. doi: 10.1016/0022-2836(68)90391-4. [DOI] [PubMed] [Google Scholar]
  25. Martin T. E., Hartwell L. H. Resistance of active yeast ribosomes to dissociation by KCl. J Biol Chem. 1970 Mar 25;245(6):1504–1506. [PubMed] [Google Scholar]
  26. Martin T. E., Rolleston F. S., Low R. B., Wool I. G. Dissociation and reassociation of skeletal muscle ribosomes. J Mol Biol. 1969 Jul 14;43(1):135–149. doi: 10.1016/0022-2836(69)90084-9. [DOI] [PubMed] [Google Scholar]
  27. Martin T. E., Wool I. G. Active hybrid 80 s particles formed from subunits of rat, rabbit and protozoan (Tetrahymena pyriformis) ribosomes. J Mol Biol. 1969 Jul 14;43(1):151–161. doi: 10.1016/0022-2836(69)90085-0. [DOI] [PubMed] [Google Scholar]
  28. Mechler B., Mach B. Preparation and properties of ribosomal subunits from mouse plasmocytoma tumors. Eur J Biochem. 1971 Aug 25;21(4):552–564. doi: 10.1111/j.1432-1033.1971.tb01501.x. [DOI] [PubMed] [Google Scholar]
  29. Miskin R., Zamir A., Elson D. Inactivation and reactivation of ribosomal subunits: the peptidyl transferase activity of the 50 s subunit of Escherihia coli. J Mol Biol. 1970 Dec 14;54(2):355–378. doi: 10.1016/0022-2836(70)90435-3. [DOI] [PubMed] [Google Scholar]
  30. Morimoto T., Blobel G., Sabatini D. D. Ribosome crystallization in chicken embryos. I. Isolation, characterization, and in vitro activity of ribosome tetramers. J Cell Biol. 1972 Feb;52(2):338–354. doi: 10.1083/jcb.52.2.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ohad I., Siekevitz P., Palade G. E. Biogenesis of chloroplast membranes. I. Plastid dedifferentiation in a dark-grown algal mutant (Chlamydomonas reinhardi). J Cell Biol. 1967 Dec;35(3):521–552. doi: 10.1083/jcb.35.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Parisi B., Ciferri O. Protein synthesis by cell-free extracts from castor bean seedlings. I. Preparation and characteristics of the amino acid incorporating system. Biochemistry. 1966 May;5(5):1638–1645. doi: 10.1021/bi00869a027. [DOI] [PubMed] [Google Scholar]
  33. Pestka S. Inhibitors of ribosome functions. Annu Rev Microbiol. 1971;25:487–562. doi: 10.1146/annurev.mi.25.100171.002415. [DOI] [PubMed] [Google Scholar]
  34. Rawson J. R., Stutz E. Isolation and characterization of Euglena gracilis cytoplasmic and chloroplast ribosomes and their ribosomal RNA components. Biochim Biophys Acta. 1969 Oct 22;190(2):368–380. doi: 10.1016/0005-2787(69)90087-2. [DOI] [PubMed] [Google Scholar]
  35. SAGER R., WEINSTEIN I. B., ASHKENAZI Y. Coding ambiguity in cell-free extracts of chlamydomonas. Science. 1963 Apr 19;140(3564):304–306. doi: 10.1126/science.140.3564.304. [DOI] [PubMed] [Google Scholar]
  36. SCHWARTZ J. H., EISENSTADT J. M., BRAWERMAN G., ZINDER N. D. BIOSYNTHESIS OF THE COAT PROTEIN OF COLIPHAGE F2 BY EXTRACTS OF EUGLENA GRACILIS. Proc Natl Acad Sci U S A. 1965 Jan;53:195–200. doi: 10.1073/pnas.53.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. SPENCER D., WILDMAN S. G. THE INCORPORATION OF AMINO ACIDS INTO PROTEIN BY CELL-FREE EXTRACTS FROM TOBACCO LEAVES. Biochemistry. 1964 Jul;3:954–959. doi: 10.1021/bi00895a019. [DOI] [PubMed] [Google Scholar]
  38. Sager R., Hamilton M. G. Cytoplasmic and chloroplast ribosomes of Chlamydomonas: ultracentrifugal characterization. Science. 1967 Aug 11;157(3789):709–711. doi: 10.1126/science.157.3789.709. [DOI] [PubMed] [Google Scholar]
  39. Schlessinger D., Mangiarotti G., Apirion D. The formation and stabilization of 30S and 50S ribosome couples in Escherichia coli. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1782–1789. doi: 10.1073/pnas.58.4.1782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Spirin A. S. On the equilibrium of the association-dissociation reaction of ribosomal subparticles and on the existance of the so-called '60 S intermediate' ('swollen 70 S') during centrifugation of the equilibrium mixture. FEBS Lett. 1971 May 20;14(5):349–353. doi: 10.1016/0014-5793(71)80298-3. [DOI] [PubMed] [Google Scholar]
  41. Stutz E., Noll H. Characterization of cytoplasmic and chloroplast polysomes in plants: evidence for three classes of ribosomal RNA in nature. Proc Natl Acad Sci U S A. 1967 Mar;57(3):774–781. doi: 10.1073/pnas.57.3.774. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. TAKANAMI M., OKAMOTO T. INTERACTION OF RIBOSOMES AND SYNTHETIC POLYRIBONUCLEOTIDES. J Mol Biol. 1963 Oct;7:323–333. doi: 10.1016/s0022-2836(63)80027-3. [DOI] [PubMed] [Google Scholar]
  43. TS'O P. O., BONNER J., VINOGRAD J. Structure and properties of microsomal nucleoprotein particles from pea seedlings. Biochim Biophys Acta. 1958 Dec;30(3):570–582. doi: 10.1016/0006-3002(58)90104-5. [DOI] [PubMed] [Google Scholar]
  44. Vasconcelos A. C., Bogorad L. Proteins of cytoplasmic, chloroplast, and mitochondrial ribosomes of some plants. Biochim Biophys Acta. 1971 Jan 28;228(2):492–502. doi: 10.1016/0005-2787(71)90054-2. [DOI] [PubMed] [Google Scholar]
  45. Zamir A., Miskin R., Elson D. Inactivation and reactivation of ribosomal subunits: amino acyl-transfer RNA binding activity of the 30 s subunit of Escherichia coli. J Mol Biol. 1971 Sep 14;60(2):347–364. doi: 10.1016/0022-2836(71)90299-3. [DOI] [PubMed] [Google Scholar]
  46. van Diggelen O. P., Oostrom H., Bosch L. Association products of native and derived ribosomal subunits of E. coli and their stability during centrifugation. FEBS Lett. 1971 Dec 1;19(2):115–120. doi: 10.1016/0014-5793(71)80492-1. [DOI] [PubMed] [Google Scholar]
  47. van Kammen A. Characteristics of in vitro amino acid incorporation by the ribosomal fraction from tobacco leaves. Arch Biochem Biophys. 1967 Mar 20;118(3):517–524. doi: 10.1016/0003-9861(67)90384-0. [DOI] [PubMed] [Google Scholar]

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