This chapter focuses on the analysis and purification of Adenosine triphosphate (ATP)-dependent mitochondrial lon protease of saccharomyces cerevisiae. ATP-dependent protein degradation in fractionated mitochondrial matrix can be monitored by measuring the degradation of radiolabeled casein to radioactive peptides soluble in trichloroacetic acid. Disruption of the yeast gene encoding the La protease homolog greatly lowers the ATP-dependent proteolytic activity of a mitochondrial matrix fraction and blocks the selective turnover of several mitochondrial matrix proteins in vivo. The disruption of the yeast gene results in an accumulation of electron dense bodies within the mitochondrial matrix as detected by electron microscopy, H and also converts the cells to p- mutants whose mitochondrial DNA (mtDNA) has suffered deletions or rearrangements. Thus, selective protein degradation mediated by Lon is required for the maintenance of mitochondrial homeostasis in saccharomyces cerevisiae. The combined application of an in vitro as well as an in vivo assay for the proteolytic activity of the mitochondrial Lon protease from Saccharomyces cerevisiae thus permits the study of the physiological role of Lon-mediated proteolysis in mitochondrial homeostasis.
ASJC Scopus subject areas
- Molecular Biology