Analysis of mitochondrial translation products in Vivo and in organello in yeast

Abstract

Publisher Summary This chapter describes procedures for the in vivo and in organello labeling of mitochondrial translation products in the yeast Saccharomyces cerevisiae ( S. cerevisiae ). The proteins synthesized in yeast mitochondria are an excellent model system to study biogenesis of a functional protein because they can be labeled and identified easily without the need of antibody production or enzymatic assays. The yeast mitochondrial genome harbors intron-containing genes, and defects of mRNA maturation result in an altered expression of cytochrome b (Cyt b) and CoxI. All mitochondrially synthesized proteins have to assemble into higher molecular complexes. To investigate this process, mitochondria have the unique advantage that precomplexes consisting of nuclear-encoded proteins can be accumulated by the chloramphenicol treatment of yeast cultures prior to the isolation of mitochondria. Applications for the analysis of mitochondrial translation for the investigation of various aspects of organelle biogenesis, such as protein folding and assembly, protein degradation, and protein sorting, are discussed in the chapter.