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Methods Mol Biol. 2016;1351:115-29. doi: 10.1007/978-1-4939-3040-1_9.

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

Author information

1
Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.
2
Department of Biochemistry and Molecular Biology, Monash University, Building 77, Level 2, Clayton Campus, Melbourne, VIC, 3800, Australia.
3
Institute for Genetics and Cluster of Excellence: Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Z├╝lpicher Str. 47A, Cologne, 50674, Germany.
4
Department of Biochemistry and Molecular Biology, Monash University, Building 77, Level 2, Clayton Campus, Melbourne, VIC, 3800, Australia. Michael.Ryan@monash.edu.

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.

KEYWORDS:

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

PMID:
26530678
DOI:
10.1007/978-1-4939-3040-1_9
[Indexed for MEDLINE]

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