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Isolation of Mitochondrial Ribosomes

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Isolation of Plant Organelles and Structures

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1511))

Abstract

Translation of mitochondrial encoded mRNAs by mitochondrial ribosomes is thought to play a major role in regulating the expression of mitochondrial proteins. However, the structure and function of plant mitochondrial ribosomes remains poorly understood. To study mitochondrial ribosomes, it is necessary to separate them from plastidic and cytosolic ribosomes that are generally present at much higher concentrations. Here, a straight forward protocol for the preparation of fractions highly enriched in mitochondrial ribosomes from plant cells is described. The method begins with purification of mitochondria followed by mitochondrial lysis and ultracentrifugation of released ribosomes through sucrose cushions and gradients. Dark-grown Arabidopsis cells were used in this example because of the ease with which good yields of pure mitochondria can be obtained from them. However, the steps for isolation of ribosomes from mitochondria could be applied to mitochondria obtained from other sources. Proteomic analyses of resulting fractions have confirmed strong enrichment of mitochondrial ribosomal proteins.

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References

  1. Adams KL, Palmer JD (2003) Evolution of mitochondrial gene content: gene loss and transfer to the nucleus. Mol Phylogenet Evol 29:380–395

    Article  CAS  PubMed  Google Scholar 

  2. Kwasniak M, Majewski P, Skibior R et al (2013) Silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein alters translation in Arabidopsis mitochondria. Plant Cell 25:1855–1867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Uyttewaal M, Mireau H, Rurek M et al (2008) PPR336 is associated with polysomes in plant mitochondria. J Mol Biol 375:626–636

    Article  CAS  PubMed  Google Scholar 

  4. Chang IF, Szick-Miranda K, Pan S et al (2005) Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes. Plant Physiol 137:848–862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Pinel C, Douce R, Mache R (1986) A study of mitochondrial ribosomes from the higher plant Solanum tuberosum L. Mol Biol Rep 11:93–97

    Article  CAS  PubMed  Google Scholar 

  6. Vasconce AC (1971) Proteins of cytoplasmic, chloroplast, and mitochondrial ribosomes of some plants. Biochim Biophys Acta 228:492–502

    Article  Google Scholar 

  7. Leaver CJ, Harmey MA (1972) Isolation and characterization of mitochondrial ribosomes from higher plants. Biochem J 129:37P–38P

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Pring DR (1974) Maize mitochondria: purification and characterization of ribosomes and ribosomal ribonucleic acid. Plant Physiol 53:677–683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Leaver CJ, Harmey MA (1976) Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component. Biochem J 157:275–277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Carroll AJ (2013) The Arabidopsis cytosolic ribosomal proteome: from form to function. Front Plant Sci 4:32

    PubMed  PubMed Central  Google Scholar 

  11. Carroll AJ, Heazlewood JL, Ito J et al (2008) Analysis of the Arabidopsis cytosolic ribosome proteome provides detailed insights into its components and their post-translational modification. Mol Cell Proteomics 7:347–369

    Article  CAS  PubMed  Google Scholar 

  12. Giavalisco P, Wilson D, Kreitler T et al (2005) High heterogeneity within the ribosomal proteins of the Arabidopsis thaliana 80S ribosome. Plant Mol Biol 57:577–591

    Article  CAS  PubMed  Google Scholar 

  13. Yamaguchi K, Subramanian AR (2003) Proteomic identification of all plastid-specific ribosomal proteins in higher plant chloroplast 30S ribosomal subunit - PSRP-2 (U1A-type domains), PSRP-3 alpha/beta (ycf65 homologue) and PSRP-4 (Thx homologue). Eur J Biochem 270:190–205

    Article  CAS  PubMed  Google Scholar 

  14. Yamaguchi K, Subramanian AR (2000) The plastid ribosomal proteins - identification of all the proteins in the 50 S subunit of an organelle ribosome (chloroplast). J Biol Chem 275:28466–28482

    Article  CAS  PubMed  Google Scholar 

  15. Yamaguchi K, Beligni MV, Prieto S et al (2003) Proteomic characterization of the Chlamydomonas reinhardtii chloroplast ribosome - identification of proteins unique to the 70 S ribosome. J Biol Chem 278:33774–33785

    Article  CAS  PubMed  Google Scholar 

  16. TPGSC (2011) Genome sequence and analysis of the tuber crop potato. Nature 475:189–195

    Article  Google Scholar 

  17. Leaver CJ, Harmey MA (1973) Plant mitochondrial nucleic acids. Biochem Soc Symp 38:175–193

    PubMed  Google Scholar 

  18. Sweetlove LJ, Taylor NL, Leaver CJ (2007) Isolation of intact, functional mitochondria from the model plant Arabidopsis thaliana. Methods Mol Biol 372:125–136

    Article  CAS  PubMed  Google Scholar 

  19. Havelund JF, Salvato F, Chen M et al (2014) Isolation of mitochondria from potato tubers. Bio-protocol 4(17):e1226

    Article  Google Scholar 

  20. Coleman JOD, Harley JL (1976) Mitochondria of mycorrhizal roots of fagus-sylvatica. New Phytol 76:317–330

    Article  CAS  Google Scholar 

  21. Valero E, Varon R, Garcia-Carmona F (1991) A kinetic study of irreversible enzyme inhibition by an inhibitor that is rendered unstable by enzymic catalysis. The inhibition of polyphenol oxidase by L-cysteine. Biochem J 277(Pt 3):869–874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Lamfrom H, Glowacki ER (1962) Controlled dissociation of rabbit reticulocyte ribosomes and its effect on hemoglobin synthesis. J Mol Biol 5:97–108

    Article  CAS  PubMed  Google Scholar 

  23. Vignais PV, Stevens BJ, Huet J et al (1972) Mitoribosomes from Candida utilis. Morphological, physical, and chemical characterization of the monomer form and of its subunits. J Cell Biol 54:468–492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Freienstein C, Blobel G (1975) Nonribosomal proteins associated with eukaryotic native small ribosomal subunits. Proc Natl Acad Sci U S A 72:3392–3396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hørder M (1974) Complex formation of inorganic pyrophosphate with magnesium. Biochim Biophys Acta 358:319–328

    Article  Google Scholar 

  26. Hardy SJ, Turnock G (1971) Stabilization of 70S ribosomes by spermidine. Nat New Biol 229:17–19

    Article  CAS  PubMed  Google Scholar 

  27. Caldwell P, Luk DC, Weissbach H et al (1978) Oxidation of the methionine residues of Escherichia coli ribosomal protein L12 decreases the protein’s biological activity. Proc Natl Acad Sci U S A 75:5349–5352

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Stevens L, Morrison MR (1968) Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus. Biochem J 108:633–640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Datta RK, Sen S, Ghosh JJ (1969) Effect of polyamines on the stability of brain-cortex ribosomes. Biochem J 114:847–854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the Australian Research Council through the Australian Research Council Centre of Excellence for Plant Energy Biology (grant number CE0561495), Australian Research Council Centre of Excellence for Translational Photosynthesis (grant number CE140100015), and an Australian Research Council Future Fellowship to Guillaume Tcherkez (grant number FT140100645). Special thanks to Professor A. Harvey Millar and Dr Nicolas Taylor of The University of Western Australia and Dr Joshua Heazlewood of The University of Melbourne for invaluable training, supervision, and support with proteomic analysis.

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Authors and Affiliations

  1. The ARC Centre of Excellence for Translational Photosynthesis, The Australian National University, 134 Linnaeus Way, Acton, Canberra, ACT, 2601, Australia

    Adam J. Carroll

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  1. Adam J. Carroll
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Correspondence to Adam J. Carroll .

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Editors and Affiliations

  1. The University of Western Australia, Perth, West Australia, Australia

    Nicolas L. Taylor

  2. The University of Western Australia, Perth, West Australia, Australia

    A. Harvey Millar

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Carroll, A.J. (2017). Isolation of Mitochondrial Ribosomes. In: Taylor, N., Millar, A. (eds) Isolation of Plant Organelles and Structures. Methods in Molecular Biology, vol 1511. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6533-5_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6533-5_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6531-1

  • Online ISBN: 978-1-4939-6533-5

  • eBook Packages: Springer Protocols

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