Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Small is big in Arabidopsis mitochondrial ribosome

Abstract

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Strategy for the identification of Arabidopsis mitochondrial ribosome protein composition.
Fig. 2: rPPR1 immuno-precipitates the Arabidopsis mitochondrial ribosome.
Fig. 3: Macroscopic phenotyping of rPPR mutants.
Fig. 4: Arabidopsis rPPR1-deficient plants have lower ribosome density along mitochondrial mRNAs.
Fig. 5: Arabidopsis mitoribosome cryo-EM map compared to animal mitoribosome and Arabidopsis cytoribosome.
Fig. 6: Architecture of Arabidopsis mitoribosome fitted with E. coli and yeast mitochondria ribosomes.

Data availability

Mass spectrometric data were deposited with the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD010324. Ribo-Seq sequencing data were deposited in the NCBI Gene Expression Omnibus under accession number GSE119655. Cryo-EM data were deposited in EMDataBank under accession number EMDB-4408 for Arabidopsis mitoribosome SSU, and EMDB-4409 for Arabidopsis mitoribosome LSU.

References

  1. 1.

    Roger, A. J., Muñoz-Gómez, S. A. & Kamikawa, R. The origin and diversification of mitochondria. Curr. Biol. 27, R1177–R1192 (2017).

    CAS  Article  Google Scholar 

  2. 2.

    Nunnari, J. & Suomalainen, A. Mitochondria: in sickness and in health. Cell 148, 1145–1159 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    De Silva, D., Tu, Y. T., Amunts, A., Fontanesi, F. & Barrientos, A. Mitochondrial ribosome assembly in health and disease. Cell Cycle 14, 226–2250 (2015).

    Google Scholar 

  4. 4.

    Chen, L. & Liu, Y.-G. Male sterility and fertility restoration in crops. Annu. Rev. Plant. Biol. 65, 579–606 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Horn, R., Gupta, K. J. & Colombo, N. Mitochondrion role in molecular basis of cytoplasmic male sterility. Mitochondrion 19, 198–205 (2014).

    CAS  Article  Google Scholar 

  6. 6.

    Giegé, P., Sweetlove, L. J., Cognat, V. & Leaver, C. J. Coordination of nuclear and mitochondrial genome expression during mitochondrial biogenesis in Arabidopsis. Plant Cell 17, 1497–1512 (2005).

    Article  Google Scholar 

  7. 7.

    Lightowlers, R. N., Rozanska, A. & Chrzanowska-Lightowlers, Z. M. Mitochondrial protein synthesis: figuring the fundamentals, complexities and complications of mammalian mitochondrial translation. FEBS Lett. 588, 2496–2503 (2014).

    CAS  Article  Google Scholar 

  8. 8.

    Hammani, K. & Giegé, P. RNA metabolism in plant mitochondria. Trends Plant Sci. 19, 380–389 (2014).

    CAS  Article  Google Scholar 

  9. 9.

    Lopez Sanchez, M. I. G. et al. RNA processing in human mitochondria. Cell Cycle 10, 2904–2916 (2011).

    Article  Google Scholar 

  10. 10.

    Barkan, A. & Small, I. Pentatricopeptide repeat proteins in plants. Annu. Rev. Plant Biol. 65, 415–442 (2014).

    CAS  Article  Google Scholar 

  11. 11.

    Chinnery, P. F. et al. The challenges of mitochondrial replacement. PLoS Genet. 10, e1004315 (2014).

  12. 12.

    Giegé, P. Pentatricopeptide repeat proteins. RNA Biol. 10, 1417–1418 (2013).

    Article  Google Scholar 

  13. 13.

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

    CAS  Article  Google Scholar 

  14. 14.

    Hammani, K. et al. An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation. Plant Cell 23, 730–740 (2011).

    CAS  Article  Google Scholar 

  15. 15.

    Haïli, N. et al. The MTL1 pentatricopeptide repeat protein is required for both translation and splicing of the mitochondrial NADH dehydrogenase subunit 7 mRNA in Arabidopsis. Plant Physiol. 170, 354–366 (2016).

    Article  Google Scholar 

  16. 16.

    Mai, N., Chrzanowska-Lightowlers, Z. M. A. & Lightowlers, R. N. The process of mammalian mitochondrial protein synthesis. Cell Tissue Res. 367, 5–20 (2017).

    CAS  Article  Google Scholar 

  17. 17.

    Greber, B. J. & Ban, N. Structure and function of the mitochondrial ribosome. Annu. Rev. Biochem. 85, 103–132 . (2016).

  18. 18.

    Planchard, N. et al. The translational landscape of Arabidopsis mitochondria. Nucleic Acids Res. 46, 6218–6228 (2018).

    CAS  Article  Google Scholar 

  19. 19.

    Barrell, B. G., Bankier, A. T. & Drouin, J. A different genetic code in human mitochondria. Nature 282, 189–194 (1979).

    CAS  Article  Google Scholar 

  20. 20.

    Helm, M. et al. Search for characteristic structural features of mammalian mitochondrial tRNAs. RNA 6, 1356–1379 (2000).

    CAS  Article  Google Scholar 

  21. 21.

    Pfeffer, S., Woellhaf, M. W., Herrmann, J. M. & Förster, F. Organization of the mitochondrial translation machinery studied in situ by cryoelectron tomography. Nat. Commun. 6, 6019 (2015).

    CAS  Article  Google Scholar 

  22. 22.

    Ott, M., Amunts, A. & Brown, A. Organization and regulation of mitochondrial protein synthesis. Annu. Rev. Biochem. 85, 77–101 (2016).

    CAS  Article  Google Scholar 

  23. 23.

    Salinas-Giegé, T. et al. Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii Thalia Salinas-Gieg e. Nucleic Acids Res. 45, 12963–12973 (2017).

    Article  Google Scholar 

  24. 24.

    Kitakawa, M. et al. Identification and characterization of the genes for mitochondrial ribosomal proteins of Saccharomyces cerevisiae. Eur. J. Biochem. 245, 449–456 (1997).

    CAS  Article  Google Scholar 

  25. 25.

    Goldschmidt-Reisin, S. et al. Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences. J. Biol. Chem. 273, 34828–34836 (1998).

    CAS  Article  Google Scholar 

  26. 26.

    Koc, E. C. et al. A proteomics approach to the identification of mammalian mitochondrial small subunit ribosomal proteins. J. Biol. Chem. 275, 32585–32591 (2000).

    CAS  Article  Google Scholar 

  27. 27.

    Koc, E. C. et al. The large subunit of the mammalian mitochondrial ribosome: analysis of the complement of ribosomal proteins present. J. Biol. Chem. 276, 43958–43969 (2001).

    CAS  Article  Google Scholar 

  28. 28.

    Bonen, L. & Calixte, S. Comparative analysis of bacterial-origin genes for plant mitochondrial ribosomal proteins. Mol. Biol. Evol. 23, 701–712 (2006).

    CAS  Article  Google Scholar 

  29. 29.

    Amunts, A., Brown, A., Toots, J., Scheres, S. H. W. & Ramakrishnan, V. The structure of the human mitochondrial ribosome. Science 348, 95–98 (2015).

    CAS  Article  Google Scholar 

  30. 30.

    Greber, B. J. et al. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348, 303–308 (2015).

    CAS  Article  Google Scholar 

  31. 31.

    Desai, N., Brown, A., Amunts, A. & Ramakrishnan, V. The structure of the yeast mitochondrial ribosome. Science 355, 528–531 (2017).

    CAS  Article  Google Scholar 

  32. 32.

    Chrzanowska-Lightowlers, Z., Rorbach, J. & Minczuk, M. Human mitochondrial ribosomes can switch structural tRNAs – but when and why? RNA Biol. 14, 1668–1671 (2017).

    Article  Google Scholar 

  33. 33.

    Brown, A. et al. Structure of the large ribosomal subunit from human mitochondria. Science 346, 718–722 (2014).

    CAS  Article  Google Scholar 

  34. 34.

    Leaver, C. J. & Harmey, M. A. Higher-plant mitochondrial ribosomes contain a 5S ribosomal ribonucleic acid component. Biochem. J. 157, 275–277 (1976).

    CAS  Article  Google Scholar 

  35. 35.

    Gold, V. A., Chroscicki, P., Bragoszewski, P. & Chacinska, A. Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo‐tomography. EMBO Rep. 18, 1786–1800 (2017).

    CAS  Article  Google Scholar 

  36. 36.

    Lurin, C. et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103 (2004).

    CAS  Article  Google Scholar 

  37. 37.

    Portereiko, M. F. Nuclear fusion defective 1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization. Plant Physiol. 141, 957–965 (2006).

    CAS  Article  Google Scholar 

  38. 38.

    Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).

    CAS  Article  Google Scholar 

  39. 39.

    Ramesh, M. & Woolford, J. L. Eukaryote-specific rRNA expansion segments function in ribosome biogenesis. RNA 22, 1153–1162 (2016).

    CAS  Article  Google Scholar 

  40. 40.

    Bieri, P., Leibundgut, M., Saurer, M., Boehringer, D. & Ban, N. The complete structure of the chloroplast 70S ribosome in complex with translation factor pY. EMBO J. 36, 475–486 (2017).

    CAS  Article  Google Scholar 

  41. 41.

    Boerema, A. P. et al. Structure of the chloroplast ribosome with chl-RRF and hibernation-promoting factor. Nat. Plants 4, 212–217 (2018).

    CAS  Article  Google Scholar 

  42. 42.

    Pusnik, M., Small, I., Read, L. K., Fabbro, T. & Schneider, A. Pentatricopeptide repeat proteins in Trypanosoma brucei function in mitochondrial ribosomes. Mol. Cell. Biol. 27, 6876–6888 (2007).

    CAS  Article  Google Scholar 

  43. 43.

    Aphasizheva, I., Maslov, D., Wang, X., Huang, L. & Aphasizhev, R. Pentatricopeptide repeat proteins stimulate mRNA adenylation/uridylation to activate mitochondrial translation in trypanosomes. Mol. Cell 42, 106–117 (2011).

    CAS  Article  Google Scholar 

  44. 44.

    Aphasizheva, I. et al. Ribosome-associated pentatricopeptide repeat proteins function as translational activators in mitochondria of trypanosomes. Mol. Microbiol. 99, 1043–1058 (2016).

    CAS  Article  Google Scholar 

  45. 45.

    Ramrath, D. et al. Evolutionary shift toward protein-based architecture in trypanosomal mitochondrial ribosomes. Science 7735, 422 (2018).

    Google Scholar 

  46. 46.

    Bieri, P., Greber, B. J. & Ban, N. High-resolution structures of mitochondrial ribosomes and their functional implications. Curr. Opin. Struct. Biol. 49, 44–53 (2018).

    CAS  Article  Google Scholar 

  47. 47.

    Kühl, I., Dujeancourt, L., Gaisne, M., Herbert, C. J. & Bonnefoy, N. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression. Nucleic Acids Res. 39, 8029–8041 (2011).

    Article  Google Scholar 

  48. 48.

    Tavares-Carreón, F. et al. The pentatricopeptide repeats present in Pet309 are necessary for translation but not for stability of the mitochondrial Cox1 mRNA in yeast. J. Biol. Chem. 283, 1472–1479 (2008).

    Article  Google Scholar 

  49. 49.

    Ruzzenente, B. et al. LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs. EMBO J. 31, 443–456 (2012).

    CAS  Article  Google Scholar 

  50. 50.

    Uyttewaal, M. et al. Characterization of Raphanus sativus pentatricopeptide repeat proteins encoded by the fertility restorer locus for Ogura cytoplasmic male sterility. Plant Cell 20, 3331–3345 (2008).

    CAS  Article  Google Scholar 

  51. 51.

    Herbert, C. J. et al. Yeast PPR proteins, watchdogs of mitochondrial gene expression. RNA Biol. 10, 1477–1494 (2013).

    CAS  Article  Google Scholar 

  52. 52.

    Wilson, D. N. & Doudna Cate, J. H. The structure and function of the eukaryotic ribosome. Cold Spring Harb. Perspect. Biol. 4, a011536–a011536 (2012).

    Article  Google Scholar 

  53. 53.

    Jobe, A., Liu, Z., Gutierrez-Vargas, C. & Frank, J. New insights into ribosome structure andfunction. Cold Spring Harb. Perspect. Biol. 7, a032615 (2018).

    Google Scholar 

  54. 54.

    Hazle, T. & Bonen, L. Comparative analysis of sequences preceding protein-coding mitochondrial genes in flowering plants. Mol. Biol. Evol. 24, 1101–1112 (2007).

    CAS  Article  Google Scholar 

  55. 55.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    CAS  Article  Google Scholar 

  56. 56.

    Gregori, J., Villareal, L., Sanchez, A., Baselga, J. & Villanueva, J. An effect size filter improves the reproducibility in spectral counting-based comparative proteomics. J. Proteom. 16, 55–65 (2013).

    Article  Google Scholar 

  57. 57.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    CAS  Article  Google Scholar 

  58. 58.

    Lamattina, L., Gonzalez, D., Gualberto, J. & Grienenberger, J. M. Higher plant mitochondria encode an homologue of the nuclear-encoded 30-kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217, 831–838 (1993).

    CAS  Article  Google Scholar 

  59. 59.

    Elthon, T. E., Nickels, R. L. & McIntosh, L. Monoclonal antibodies to the alternative oxidase of higher plant mitochondria. Plant Physiol. 89, 1311–1317 (1989).

    CAS  Article  Google Scholar 

  60. 60.

    Pineau, B., Layoune, O., Danon, A. & De Paepe, R. l-Galactono-1,4-lactone dehydrogenase isrequired for the accumulation of plant respiratory complex I. J. Biol. Chem. 283, 32500–32505 (2008).

    CAS  Article  Google Scholar 

  61. 61.

    Carrie, C. et al. Conserved and novel functions for Arabidopsis thaliana MIA40 in assembly of proteins in mitochondria and peroxisomes. J. Biol. Chem. 285, 36138–36148 (2010).

    CAS  Article  Google Scholar 

  62. 62.

    Hooper, C. M., Castleden, I. R., Tanz, S. K., Aryamanesh, N. & Millar, A. H. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations. Nucleic Acids Res. 45, D1064–D1074 (2017).

    CAS  Article  Google Scholar 

  63. 63.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  Article  Google Scholar 

  64. 64.

    Karpenahalli, M. R., Lupas, A. N. & Söding, J. TPRpred: a tool for prediction of TPR-, PPR- and SEL1-like repeats from protein sequences. BMC Bioinformatics 8, 2 (2007).

    Article  Google Scholar 

  65. 65.

    Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845 (2015).

    CAS  Article  Google Scholar 

  66. 66.

    Obayashi, T., Aoki, Y., Tadaka, S., Kagaya, Y. & Kinoshita, K. ATTED-II in 2018: a plant coexpression database based on investigation of the statistical property of the mutual rank index. Special Issue – Databases 59, 1–7 (2018).

    Google Scholar 

  67. 67.

    de la Rosa-Trevín, J. M. et al. Scipion: a software framework toward integration, reproducibility and validation in 3D electron microscopy. J. Struct. Biol. 195, 93–99 (2016).

    Article  Google Scholar 

  68. 68.

    de la Rosa-Trevín, J. M. et al. Xmipp 3.0: an improved software suite for image processing in electron microscopy. J. Struct. Biol. 184, 321–328 (2013).

    Article  Google Scholar 

  69. 69.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  Google Scholar 

  70. 70.

    Abrishami, V. et al. A pattern matching approach to the automatic selection of particles from low-contrast electron micrographs. Bioinformatics 29, 2460–2468 (2013).

    CAS  Article  Google Scholar 

  71. 71.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  72. 72.

    Ban, N. et al. A new system for naming ribosomal proteins. Curr. Opin. Struct. Biol. 24, 165–169 (2014).

    CAS  Article  Google Scholar 

  73. 73.

    Stupar, R. M. et al. Complex mtDNA constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: Implication of potential sequencing errors caused by large-unit repeats. Proc. Natl Acad. Sci. USA 98, 5099–5103 (2001).

    CAS  Article  Google Scholar 

  74. 74.

    Adams, K. L., Daley, D. O., Whelan, J. & Palmer, J. D. Genes for two mitochondrial ribosomal proteins in flowering plants are derived from their chloroplast or cytosolic counterparts. Plant Cell 14, 931–943 (2002).

    CAS  Article  Google Scholar 

  75. 75.

    Noeske, J. et al. High-resolution structure of the Escherichia coli ribosome. Nat. Struct. Mol. Biol. 22, 336–341 (2015).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by Centre National de la Recherche Scientifique, the University of Strasbourg, by Agence Nationale de la Recherche (ANR) grants (MITRA, no. ANR-16-CE11-0024-02) and (CytoRP, no. ANR-16-CE21-0001-01) to P.G., Y.H. and H.M., and by the LabEx consortium MitoCross within the framework of the French National Program Investissement d’Avenir (no. ANR-11-LABX-0057_MITOCROSS) and LabEx Saclay Plant Sciences-SPS (no. ANR-10-LABX-0040-SPS). Mass spectrometry instruments were funded by the University of Strasbourg, IdEx Equipement mi-lourd 2015 and the LabEx consortium NetRNA within the framework of the French National Program Investissement d’Avenir (no. ANR-10-LABEX-0036_NETRNA). Y.H. was supported by the LabEx consortium NetRNA.

Author information

Affiliations

Authors

Contributions

P.G., F.W., H.M. and Y.H. designed and coordinated the experiments. F.W., T.T.N., M.A., A.B., J.C., M.Q., P.H. and L.K. performed the experiments and analysed the results. P.G., F.W., H.M., T.T.N. and Y.H. wrote the manuscript.

Corresponding authors

Correspondence to Hakim Mireau or Yaser Hashem or Philippe Giegé.

Ethics declarations

Competing interests

The authors declare no competing interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–11 and Supplementary Table 8.

Reporting Summary

Supplementary Data

Supplementary Tables 1–7.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Waltz, F., Nguyen, TT., Arrivé, M. et al. Small is big in Arabidopsis mitochondrial ribosome. Nature Plants 5, 106–117 (2019). https://doi.org/10.1038/s41477-018-0339-y

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing