Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes

Austin Choi, Antoni Barrientos

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages211-226
Number of pages16
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2192
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Gradient fractionation
  • Gradient maker
  • Gradient master
  • Immunoblotting
  • Mitochondrial ribosome
  • Mitoribosome profile
  • Sucrose gradient

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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