Coenzyme Q (ubiquinone or Qn) is a benzoquinone lipid of varying isoprenoid tail length ‘n’, with essential functions within the respiratory electron transport chain and in cellular antioxidant defense. Several Coq polypeptides coordinate to drive the biosynthesis of Qn at the inner mitochondrial membrane, and are organized into a high-molecular weight complex known as the ‘CoQ synthome’, Absence of individual Coq enzymes results in decreased content of Qn and other Coq proteins, in addition to destabilization of the CoQ synthome. Patients with partial defects in Q10 biosynthesis suffer from a variety of debilitating diseases. Therefore, the studies outlined here seek to produce a more comprehensive understanding of the Qn biosynthetic pathway, allowing for more targeted therapeutic strategies. This work employs Saccharomyces cerevisiae as a model organism due to the high level of COQ gene functional conservation between yeast and humans. In S. cerevisiae, Coq11 was recently identified to associate with members of the CoQ synthome, and was required for efficient de novo Q6 biosynthesis. The function of Coq11 remains uncharacterized.
Chapter 1 provides an overview of the coenzyme Q biosynthetic pathway in both humans and yeast, while highlighting the identification of Coq11 as a novel member of the CoQ synthome. Chapter 2 explores the functional relationship between Coq11 and Coq10, which have evolved as protein fusions in several fungal genomes. Data collected with help from Dr. Roland Stocker’s laboratory, Dr. Mario Barros, and Jenny Ngo from Dr. Orian Shirai’s laboratory, has demonstrated that the coq10Δ mutant respiratory deficiency, sensitivity to lipid peroxidation, and low de novo Q6 biosynthesis is rescued by deletion of COQ11. Further, yeast lacking COQ11 have increased expression of several Coq proteins and a stabilized CoQ synthome. These results indicate that Coq11 may serve as a modulator of Q6 biosynthesis. Chapter 3 outlines additional experimental characterization towards understanding the role of Coq11 within the Q6 biosynthetic pathway. With assistance from Dr. Lukas Susac from the laboratory of Professor Juli Feigon, various strategies for Coq11 purification have been documented, with some showing promise for future purification attempts. We have also revealed that Coq11 deletion in combination with deletion of two phenylacrylic acid decarboxylases, Pad1 and Fdc1, fails to effect Q6 biosynthesis. In collaboration with Dr. Mario Barros, several Coq11 overexpression vectors have been constructed and evaluated for their ability to restore Q6 biosynthesis in the coq11∆ mutant. The data reveal that yeast does not tolerate Coq11 overexpression from the majority of constructs, suggesting that Coq11 is a negative regulator of Q6 biosynthesis. In Chapter 4, two novel phosphatidylethanolamine methyltransferase deletion mutants, cho2 and opi3, were identified to have significantly higher Q6 compared to wild type. Dr. Anita Ayer from the Stocker laboratory conducted a preliminary large-scale screen of the S. cerevisiae diploid homozygous knockout library for mutants displaying altered Q6 content. With help from Dr. Lucía Fernández-del-Río, we confirmed that the cho2∆ and opi3∆ mutants have increased Q6 as well as a stabilized CoQ synthome, despite retaining wild-type amounts of Coq proteins. Finally, Appendices I-VIII contain previous publications that detail various aspects of coenzyme Q characterization in several organisms, in addition to a methods paper regarding the detection of protein-protein interaction networks. Together, this work provides novel insights regarding the biosynthesis, cellular functions, and regulation of coenzyme Q.
