The line of inquiry proposed in this research program is based on the hypothesis that the mitochondrial ATP-dependent Lon protease monitors both the integrity and expression of the mitochondrial genome as well as the folding and assembly of nascent polypeptides. The goals of this proposal are to analyze the structure and function of yeast Lon and to identify mammalian and yeast proteins that functionally overlap with Lon. A combination of molecular biology, genetics, biochemistry and structural biology will be used to address the following aims: To analyze the role of Lon in regulating the mtDNA-protein interactions. The DNA-binding function of Lon is conserved from bacteria to humans, however the physiological importance of this activity is not understood. The investigator will identify the endogenous mtDNA sequences bound by yeast Lon, analyze the role of Lon in mtDNA-protein complexes called nucleoids and identify the protein(s) responsible for increased double-stranded DNA-binding in mitochondria that lack Lon. To identify and characterize mammalian and yeast proteins that have a shared function with Lon. Genetic complementation of a temperature-sensitive Lon mutant using mammalian libraries designed for expression in yeast will be carried out. The mammalian proteins identified will be characterized in mammalian cell systems. In addition, a genome-wide survey of transcript level changes in yeast deleted for the Lon gene will be conducted using oligonucleotide probe microarrays. To elucidate the structural dynamics and function of the homo-heptameric ring-shaped Lon protease using circular dichroism and high-resolution electron microscopy. Mitochondrial dysfunction has been implicated in a wide variety of degenerative disorders, aging and cancer. The best-characterized genetic mutations that lead to a loss of mitochondrial function are deletions, point mutations and base substitutions in mitochondrial DNA. Cellular responses to stress caused by oxidative damage and aging are likely to involve the action of ATP-dependent proteases which selectively degrade denatured, aggregated and unassembled proteins. Understanding the molecular details of how ATP-dependent proteases ensure cellular homeostasis by monitoring protein biogenesis as well as the integrity and expression of the mitochondrial genome, will provide insight into mechanisms that help to prevent mitochondrial dysfunction.
