In Vivo and In Organello Analyses of Mitochondrial Translation

Publisher Summary This chapter describes in vivo and in organello methods currently used in the laboratory to analyze the translation capacity of mitochondria from mammalian cells, both for basic research and as a tool to determine the molecular effects of mitochondrial DNA (mtDNA) mutations. Different types of cell lines can be used for the in vivo analysis of mitochondrial translation. The protocol described in the chapter is for the more commonly used anchorage-dependent transformed cells and should be adapted for each particular case (e.g., cells growing in suspension do not need to be trypsinized but are concentrated by low speed centrifugation, to collect them and/or remove the growing medium and wash the cells prior to labeling). In organello analysis of protein synthesis, the isolated mitochondria must be fully functional and able to perform coupled respiration. This requires a quick and gentle purification procedure. Mitochondria possess their own translation system devoted to the synthesis of the mtDNA-encoded polypeptides. Analysis of the protein synthesis capacity of mitochondria can be a useful tool to study basic aspects of mtDNA expression, as well as to detect defects due to mutations in the mtDNA or in nuclear genes involved in this process. The combination of the different options for labeling and analysis of the translation products must be adapted for each particular application.