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Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria

  • Luke E. Formosa
  • Annette Hofer
  • Christin Tischner
  • Tina Wenz
  • Michael T. Ryan
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

Abstract

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Key words

Mitochondria Mitochondrial DNA (mtDNA) Oxidative phosphorylation (OXPHOS) Respiratory chain Complex assembly Pulse labeling Pulse-chase labeling Blue-native PAGE (BN-PAGE) 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Luke E. Formosa
    • 1
    • 3
  • Annette Hofer
    • 2
  • Christin Tischner
    • 2
  • Tina Wenz
    • 2
  • Michael T. Ryan
    • 3
  1. 1.Department of Biochemistry and Genetics, La Trobe Institute for Molecular ScienceLa Trobe UniversityMelbourneAustralia
  2. 2.Institute for Genetics and Cluster of Excellence: Cellular Stress Responses in Aging-Associated Diseases (CECAD)University of CologneCologneGermany
  3. 3.Department of Biochemistry and Molecular BiologyMonash UniversityMelbourneAustralia

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