Characterization of Subunit e of the Yeast Mitochondrial F₁F₀-ATP Synthase

The F1 Fo-ATP synthase of mitochondria, bacteria and chloroplasts, is a multi-subunit complex responsible for the synthesis of adenosine 5\u27 triphosphate (ATP). Moreover, the F1 Fo-ATP synthase of yeast and mammalian mitochondria contain a small protein, termed subunit e. Although subunit e is not essential for the enzymatic activity of the F1 Fo-ATP synthase, subunit e plays several other roles within mitochondria. Prior to this dissertation, it was known that Saccharomyces cerevisiae (S. cerevisiae ) subunit e is required for the modulation of mitochondrial morphology, the prevention of rho0 /rho- cell conversion, and the stabilization of subunit g, and F1 Fo-ATP synthase dimers. However, it was not understood how subunit e carries out these particular functions. Thus, to gain insight into the mechanisms of S. cerevisiae subunit e, the functional significance of the conserved N-terminal hydrophobic segment and the putative C-terminal coiled-coil region was analyzed. C-terminal truncated derivatives of subunit e were created and utilized to determine if the truncated forms could compensate for the loss of subunit e. As the N-terminal hydrophobic segment was shown to be functionally significant, the roles of the conserved R8 and G15 (G 15 XXXG19 motif) of this hydrophobic segment was examined next. The amino acid residues were mutated by site-directed mutagenesis, and the mutated protein was subjected to functional analyses. Lastly, as establishing the neighbors of subunit e could aid in the understanding of how subunit e functions, the molecular environment was analyzed using a cross-linking approach. This dissertation highlights the importance of the hydrophobic segment for the stabilization of subunit g, maintenance of mitochondrial DNA and the formation of mitochondrial morphology. Additionally, the coiled-coil is necessary for rho- cell prevention, stabilization of F1 Fo-ATP synthase dimers and subunit e stabilization. The GXXXG motif was demonstrated to be necessary for all of the known functions of subunit e, while the R8 residue is necessary for subunit g stabilization. This dissertation also demonstrates that subunit e is in proximity to subunit k, phosphatidylethanolamine, and subunits 6 and 9 are proposed neighboring proteins