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Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes

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Mitochondrial Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2192))

Abstract

Mitochondria contain ribosomes (mitoribosomes) specialized in the synthesis of a handful of proteins essential for oxidative phosphorylation. Therefore, mitoribosome integrity and function are essential for the life of eukaryotic cells and lesions that affect them result in devastating human disorders. To broadly analyze the integrity and assembly state of mitoribosomes it is useful to start by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts. During centrifugation, mitoribosome subunits, monosomes and polysomes, and potentially accumulated assembly intermediates will sediment through the gradient at different rates. Sedimentation will depend on the centrifugal force applied and the density and viscosity of the gradient. Importantly, it will also depend on the size, shape, and density of the mitoribosome particles present in the samples under study. Variations of this technique, often coupled with additional downstream approaches, have been used to analyze the process of mitoribosome biogenesis, the composition of assembly intermediates, or to monitor the interaction of extraribosomal proteins with individual mitoribosome subunits or monosomes.

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Acknowledgements

We thank Dr. Priyanka Maiti for critical reading of the manuscript. This research was supported by NIH R35 Grant GM118141 (to A.B.) and MDA Grant MDA-381828 (to A.B.).

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Authors and Affiliations

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

    Austin Choi & Antoni Barrientos

  2. Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, USA

    Antoni Barrientos

Authors
  1. Austin Choi
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  2. Antoni Barrientos
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Corresponding author

Correspondence to Antoni Barrientos .

Editor information

Editors and Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Michal Minczuk

  2. Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solna, Sweden, Institutet Laboratory, Max Planck Institute Biology of Ageing - Karolinska, Karolinska Institutet, Stockholm, Sweden

    Joanna Rorbach

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Choi, A., Barrientos, A. (2021). Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_16

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  • DOI: https://doi.org/10.1007/978-1-0716-0834-0_16

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0833-3

  • Online ISBN: 978-1-0716-0834-0

  • eBook Packages: Springer Protocols

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