Abstract
Publisher Summary This chapter describes a method for C- or N-terminal epitope tagging, detection, and initial characterization of nuclear-encoded proteins in mitochondria. The tagging of proteins with epitopes, fluorochromes, or affinity labels is an effective tool to study protein localization, interaction dynamics, and function. The chapter also addresses specialized methods employed for visualizing tagged and untagged mitochondrial proteins in yeast by indirect immunofluorescence. A technique has been developed that enables single-step tagging of chromosomal genes at their 5' or 3' ends by homologous recombination in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. This method is versatile as various tags, including multiple copies of HA, Myc, or glutathione S -transferase (GST) (detectable with commercially available antibodies), can be inserted into a target gene using a single set of primers. As cassette modules carrying the Aequorea victoria green fluorescent protein (GFP) and new GFP variants with altered emission spectra are available, this application can be adapted for single and double labeling in living cells. The chapter illustrates an example of a nuclear-encoded mitochondrial gene product that is tagged with 13-Myc and expressed under its normal promoter.
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