| ATP synthase (F1Fo) couples the movement of stored protons from cellular respiration through its mitochondrial membrane portion, Fo, with the phosphorylation of ADP to ATP by its F 1 portion. The central stalk of F1Fo and the alpha- and beta-subunit contact points during rotation are critical for coupled proton translocation by the ATP synthase. This thesis investigates mutations in genes from the alpha-subunit and the delta-subunit of the ATP synthase of yeast and analyzes the phenotypic and functional mitochondrial changes caused by these mutations.;Chapter 1 relates to an ATPase alpha-subunit gene variant that was identified in a patient with clinically suspicious mitochondrial disease who presented with oxidative phosphorylation (OXPHOS) deficiency and mtDNA depletion. This previously unreported gene variant, ATP synthase alpha-subunit p.Y321C, was modeled through an analogous mutation in the yeast Saccharomyces cerevisiae. The similar mutation in yeast, p.Y280C, is found on the alpha-subunit of ATP synthase, but is near the beta-subunit and near the surface of the gamma-subunit, as well as near the S. cerevisiae mgi mutation alphaA295V. This analogous yeast variant (p.Y280C) was transformed into an atp1Delta strain and resulted in a three fold increase in the percentage of petite colonies, reflecting an elevated rate of mtDNA loss. Isolated mitochondria from this mutant yeast also showed a decrease by 50% of the wild-type mitochondrial membrane potential measured upon addition of ATP and magnesium.[5] Test results show a significantly increased petite colony formation along with surprisingly small changes in respiratory functional testing. mgi mutations of this region also cause increased petite colony formation with concomitant increased loss of mitochondrial DNA [6]. Two additional replacements were made in the same residue (p.Y280A and p.Y280L) and again modeled in Saccharomyces cerevisiae in an attempt to prompt an enhanced effect. These subsequent mutants, however, showed a similar approximate three fold increase in petite formation, reflecting the same elevated rate of mtDNA loss, and demonstrating only a mild deficit in mitochondrial function testing in comparison to the wild type ATP1. This study suggests that p.Y280 of the alpha-subunit of ATP synthase is conserved because mutations of this residue lead to slight uncoupling and to significantly increased loss of mtDNA. This significant loss of mtDNA was also observed in the patient's liver and muscle cells. One hypothesis is that the disease mutation, p. Y321C, caused a loss of mtDNA over time in human cells and therefore caused the death of the patient.;The second chapter of this study relates to the role of the mitochondrial delta-subunit of the F1 portion of the mitochondrial Saccharomyces cerevisiae ATP synthase. Specifically, two truncation mutations of the carboxyl-terminal helices A and B of the delta-subunit were made to determine the role of this C-terminus in the function of ATP synthase. As part of the central stalk, the mitochondrial delta-subunit is at the base of the gamma-subunit, next to the epsilon-subunit, and just above the Fo portion and the proton pore. This work also demonstrates that the delta-subunit carboxyl-terminal helices, A and B together, are essential for coupling, with deletion of both helices resulting in a Deltadelta-subunit phenotype. Also, the carboxyl-terminal helix B is necessary for a functional ATP synthase, although not as critical for coupling of ATP synthase as both carboxyl helices A and B together. This study also demonstrates that the helices of the mitochondrial delta-subunit act differently from the helices of the bacterial homolog subunit epsilon, which are not important in coupling. |
