Characterization of Coq2 and Coq7 proteins, dual function polypeptides in Saccharomyces cerevisiae coenzyme Q biosynthesis

Coenzyme Q (ubiquinone or Q) is an isoprenylated benzoquinone lipid found in various cellular compartments of eukaryotic cells. Besides its function in the respiratory electron transport chain, Q serves as a redox active cofactor in different metabolic processes and as a lipophilic antioxidant. At least nine proteins, Coq1p through Coq9p, are required for de novo Q biosynthesis, and hence respiration, in the budding yeast Saccharomyces cerevisiae. Previous studies have provided genetic and physical evidence suggesting that in yeast the Coq polypeptides assemble into a protein complex. This dissertation describes my studies demonstrating dual-functionality of Coq7p, characterization of Coq2 and Coq7 proteins as components of the Q biosynthetic complex, and work investigating effects of growth in different carbon sources on Q content and steady state levels of Coq proteins.; The goal of the study described in Chapter 2 is to define the multiple functional roles of Coq7 protein. Eight uncharacterized coq7 point mutants were sequenced and five missense mutations were identified. Complementation of yeast coq7 mutants with E. coli UbiF provided genetic evidence for enzymatic hydroxylation activity and a structural role for the yeast Coq7 protein in Q biosynthesis. Moreover, gel filtration chromatography and blue native gel electrophoresis showed that Coq7p co-migrated with Coq3 and Coq4 polypeptides as a high molecular mass complex.; Chapter 3 details different biochemical analyses to further characterize the Coq2 and Coq7 polypeptides. Submitochondrial localization data indicated that Coq2p is an integral membrane protein while Coq7p is peripherally associated to the inner mitochondrial membrane protein, facing toward the matrix side. Gel filtration and blue native gel electrophoresis results provided the first evidence for interaction between Coq2 protein and the Q biosynthetic complex. A working model for the complex in which Coq2p serves as an anchor for the other Coq polypeptides to the inner mitochondrial membrane was proposed.; In chapter 4, potential effects of growth in media containing different carbon catabolites (e.g. dextrose, glycerol, ethanol, and galactose) on the content of Q6 and DMQ6, and steady state levels of Coq polypeptides in wild-type yeast were examined. Preliminary data suggest that quinone contents, DMQ 6/Q6 ratio, and level of each Coq protein vary in response to specific carbon sources provided in the growth media.