Date of Award
Fall 2006
Document Type
Dissertation - Restricted
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Stuart, Rosemary
Second Advisor
Munroe, Stephen
Third Advisor
Noel, Dale
Abstract
Mitochondria are essential organelles, which are not only responsible of the synthesis of energy in the cells in form of ATP hut are also required for other cellular functions like lipid metabolism, calcium signaling, FeS cluster biogenesis and apoptosis. Mitochondria produces ATP by a process termed as oxidative phosphorylation (OXPHOS), where the electrons from NADH are passed along the respiratory chain complexes to molecular oxygen. This process of electron transport is coupled to the translocation of protons from the matrix to the intermembrane space and creates an electrochemical gradient which is utilized by the F1F0-ATP synthase to generate ATP in the matrix. The electrochemical gradient and ATP also support other essential mitochondrial functions like mitochondrial fusion, fission, segregation, protein insertion and assembly of protein complexes. These OX-PHOS complexes are embedded within the mitochondrial inner membrane, where together they form higher-ordered assembly states called 'supercomplexes'. Although the organization of these complexes into supercomplexes is accepted in literature and the formation is evolutionary conserved, the precise function(s) of these supercomplexes is currently unknown. This dissertation study centres on the molecular characterization of one of these supercomplexes, the dimeric ATP synthase complex. Throughout the course of this work a relationship between this supercomplex and another OX-PHOS complex, the cytochrome bc1-cytochrome oxidase supercomplex, was also established. The dimeric ATP synthase complex contains three dimer-specific subunits, subunit g (Sug), subunit e (Su e) and subunit k (Su k), which play an essential role in the physical association of the two neighboring ATP synthase complexes. Although both Su g and Sue are non-essential subunits of the ATP synthase complex, their presence is required to develop normal mitochondrial morphology, in particular the development of cristae structures of the inner membrane. Another interesting phenotype associated with the null mutants of Su g (also Sue) is that they display reduced levels of cytochrome oxidase (COX) enzymatic activity. Prior to this dissertation, these phenotypes of Su g and Su e were known; however, the role of Su g in these processes remained to be elucidated. Thus, to gain insight into the functions of these proteins, the S. cerevisiae Su g protein was further characterized in this dissertation. The molecular environment of Su g within the ATP synthase was determined. Using a chemical cross-linking approach, interacting patner protein(s) of Su g was identified. Alignment of subunit g amino acid sequences indicates a highly conserved GXXXG motif (G is Gly, and X represents any amino acid) located in its transmembrane segment, and a partially conserved carboxy-terminal domain present in the intermembrane space region. The GXXXG motif may function as a dimerization motif and supports helix-helix interaction between two neighboring transmembrane helices. These conserved regions of Su g were also analyzed to determine their importance for the function(s) of Su g. Lastly, the relationship between dimeric-ATP synthase subunit g and cytochrome oxidase (COX) complex was also explored to understand how a dimer-specific Su g affects the enzymatic activity and the assembly of the COX complex. In conclusion, the data obtained indicate that both Su g and Su e are important for the assembly of dimeric ATP synthase complex and the cytochrome bc,-COX supercomplex, and together these supercomplexes may form an organized platfonn of OX-PH OS complexes to ensure the maintenance of highly efficient OX-PHOS activity. The majority of ATP within a cell is produced in the mitochondria through the process of OX-PHOS and dysfunctions in this process are often associated with metabolic disorders, which affect variety of organs like heart, liver, skeletal muscles and brain. At present, the molecular nature of many of the mitochondrial disorders is unknown. Therefore, to cure these disorders, it is important to first fully understand these disorders, which can be achieved by studying the OXPHOS complexes at the molecular level of their subunit composition, organization and the assembly within the inner membrane. Thus, deciphering the function(s) of Su g protein could lead to better understanding of the respiratory chain complexes and may provide insight into the mitochondrial function(s) and disorders.