Date of Award
Spring 5-2009
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Stuart, Rosemary A.
Second Advisor
Blumenthal, Edward
Third Advisor
Noel, Dale
Abstract
Oxal is a member of the highly-conserved Oxal/YidC/Alb3 protein family. This family of proteins is found to support the membrane insertion of proteins throughout prokaryotes and eukaryotes. Oxal is a major protein translocases in the mitochondrion and is required for the biogenesis of all oxidative phosphorylation (OXPHOS) complexes located in the inner mitochondrial membrane which contain mitochondrially-encoded subunits. Namely, the stable assemblies of the F]F0-ATP synthase, cytochrome c oxidase (COX) and the cytochrome bc\ complexes occur in an Oxal-dependent fashion. Due to the combined defects on the OXPHOS system in the absence of Oxal function, in the budding yeast Saccharomyces cerevisiae, cells null for OXA1 (Aoxal) are viable, however, they fail to carry out OXPHOS and, thus, are unable to grow on non-fermentable carbon sources such as glycerol. Although the role of Oxal is understood, the molecular mechanism explaining how Oxal functions is not well defined.
Oxal is predicted to span the inner mitochondrial membrane five times with an Nout-Cjn topology. The first transmembrane region (TM1) of Oxal is interesting as it is highly conserved throughout Oxal homologues and spans the membrane in a unique fashion. The area encompassing the TM1 domain of Oxal spans a region of approximately 30 hydrophobic amino acid residues (residues 119-148 in yeast), 10 amino acids longer than traditional membrane-spanning segments, therefore, it is difficult to precisely place this region in the membrane. The high level of conservation and unusual length of the TM1 domain indicated that this region may play a pivotal role in Oxal protein function.
A site-directed mutational analysis was performed throughout this dissertation study to characterize the conserved amino acid residues within the TM1 region of Oxal. Specifically, we analyzed the importance of these residues and the role they play in Oxal's ability to facilitate the biogenesis of inner membrane OXPHOS complexes and supporting mitochondrial protein synthesis. Results from this study show that there are separate domains which contribute independently to its functions. Moreover, our data highlights the importance of the conserved residues within the TM1 region of Oxal, particularly for their role in COX complex biogenesis and mitochondrial protein synthesis.