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Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast

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The Mitoribosome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2661))

Abstract

The biogenesis of mitoribosomes is an intricate process that relies on the coordinated synthesis of nuclear-encoded mitoribosomal proteins (MRPs) in the cytosol, their translocation across mitochondrial membranes, the transcription of rRNA molecules in the matrix as well as the assembly of the roughly 80 different constituents of the mitoribosome. Numerous chaperones, translocases, processing peptidases, and assembly factors of the cytosol and in mitochondria support this complex reaction. The budding yeast Saccharomyces cerevisiae served as a powerful model organism to unravel the different steps by which MRPs are imported into mitochondria, fold into their native structures, and assemble into functional ribosomes.

In this chapter, we provide established protocols to study these different processes experimentally. In particular, we describe methods to purify mitochondria from yeast cells, to import radiolabeled MRPs into isolated mitochondria, and to elucidate the assembly reaction of MRPs by immunoprecipitation. These protocols and the list of dos and don’ts will enable beginners and experienced scientists to study the import and assembly of MRPs.

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Acknowledgments

We thank Markus Räschle and Lavanya Deenadayalu for help in the context of the experimental data shown in Figs. 2 and 3. The authors were supported by grants of the Deutsche Forschungsgemeinschaft (HE2803/9-2) and the Forschungsinitiative Rheinland-Pfalz BioComp.

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  1. Lea Bertgen and Tamara Flohr contributed equally with all other contributors.

Authors and Affiliations

  1. Cell Biology, University of Kaiserslautern, Kaiserslautern, Germany

    Lea Bertgen, Tamara Flohr & Johannes M. Herrmann

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  1. Lea Bertgen
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  2. Tamara Flohr
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  3. Johannes M. Herrmann
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Correspondence to Johannes M. Herrmann .

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Editors and Affiliations

  1. Miller School of Medicine, University of Miami, Miami, FL, USA

    Antoni Barrientos

  2. Miller School of Medicine, University of Miami, Miami, FL, USA

    Flavia Fontanesi

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Bertgen, L., Flohr, T., Herrmann, J.M. (2023). Methods to Study the Biogenesis of Mitoribosomal Proteins in Yeast. In: Barrientos, A., Fontanesi, F. (eds) The Mitoribosome. Methods in Molecular Biology, vol 2661. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3171-3_10

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  • DOI: https://doi.org/10.1007/978-1-0716-3171-3_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3170-6

  • Online ISBN: 978-1-0716-3171-3

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