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Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway

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The Mitoribosome

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

Abstract

Studies of yeast mitoribosome assembly have been historically hampered by the difficulty of generating mitoribosome protein-coding gene deletion strains with a stable mitochondrial genome. The identification of mitochondrial DNA-stabilizing approaches allows for the generation of a complete set of yeast deletion strains covering all mitoribosome proteins and known assembly factors. These strains can be used to analyze the integrity and assembly state of mitoribosomes by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts, coupled to mass spectrometry analysis of mitoribosome composition. Subsequent hierarchical cluster analysis of mitoribosome subassemblies accumulated in mutant strains reveals details regarding the order of protein association during the mitoribosome biogenetic process. These strains also allow the expression of truncated protein variants to probe the role of mitochondrion-specific protein extensions, the relevance of protein cofactors, or the importance of RNA-protein interactions in functional sites of the mitoribosome. In this chapter, we will detail the methodology involved in these studies.

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Acknowledgment

This research was supported by the National Institutes of Health grant R35-GM118141 (AB).

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Authors and Affiliations

  1. Department of Molecular and Cellular Pharmacology, University of Miami, Miller School of Medicine, Miami, FL, USA

    Samuel Del’Olio

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

    Antoni Barrientos

Authors
  1. Samuel Del’Olio
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  2. Antoni Barrientos
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Corresponding author

Correspondence to Antoni Barrientos .

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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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Del’Olio, S., Barrientos, A. (2023). Systematic Analysis of Assembly Intermediates in Yeast to Decipher the Mitoribosome Assembly Pathway. 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_11

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  • DOI: https://doi.org/10.1007/978-1-0716-3171-3_11

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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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