Mutations altering mitochondrial DNA have been linked to aging and degenerative diseases, as well as, to specific inherited disorders. Some patients with these disorders have mutations in the ATPase-6 subunit gene of F1F0 ATP synthase. The ATPase-6-leu156-arg mutation was detected in four unrelated families of patients with diseases variously diagnosed as Leigh Syndrome (NARP). A correlation was drawn between severity of symptoms, age of onset and the percentage of defective mitochondrial genomes. We contributed to the understanding of the disease mutation by modeling it in the Escherichia coli F1F0 ATP synthase, and determining that enzyme function was lost due to a defect in proton (H+) translocation. The effects of mitochondrial mutations on aging is more tenuous. However, a large number of missense mutations affecting the a subunit result in a loss of F1F0 ATP synthase in E. coli. Similar point mutations undoubtedly occur in the human ATPase-6 gene resulting in loss of oxidative phosphorylation, and most will remain undetected in the clinical setting. Our long-term goal is to gain an understanding of the mechanisms of proton (H+) translocation and the coupling of H+ translocation to catalysis in F1F0 adenosine triphosphate (ATP) synthase. The laboratory is committed to using a mutagenesis approach for investigating structure and function of the F0 subunits. The present proposal centers on using the molecular biology approach to study molecular interactions within F0. We propose studies involving the generation of second-site suppressor mutations, study of the effects of F0 mutations on F1, in depth investigation of a site in the b subunit likely to be involved in the coupling of H+ translocation to catalysis, and to take a novel approach for considering the molecular interactions between the subunits connecting the F1 sector to the F0 sector.
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