Characterization of the CoQ-Synthome proteins and binding partners, their phosphorylation status, and alternative splicing and regulation in Saccharomyces cerevisiae

Coenzyme Q (also termed ubiquinone or Q) is an electron carrier in the mitochondrial respiratory chain that functions as an essential component in energy metabolism processes. Q transfers electrons from NADH and succinate to cytochrome c, a heme protein, and also serves as a vital lipid soluble antioxidant via its redox activity, whereby it is able to undergo a radical intermediate. Thus, sufficient de novo Q biosynthesis is crucial for proper health maintenance in humans. Patients with deficiencies in Q suffer from a wide spectrum of health disorders, including kidney disease, neurodegenerative diseases, ataxia, and cardiovascular complications. Additionally, decreased Q levels have been linked to aging. Saccharomyces cerevisiae (baker’s yeast) serves as an excellent model for studies on Q because of its powerful molecular genetics and its close homology to human Q biosynthesis and function. Q biosynthesis in S. cerevisiae (which makes Q6 with six isoprene units, versus humans whose Q10 has ten isoprene units) takes place in the mitochondria. Fourteen known mitochondrial proteins are responsible for facilitating this process—Coq1-Coq11 (Coq11 being recently discovered and, renamed from YLR290C, by Allan and Awad), Yah1 (ferredoxin), Arh1 (ferredoxin reductase), and Hfd1 (aldehyde dehydrogenase). Null coq1-coq9 mutants lack Q6, are respiratory impaired, and sensitive to lipid autoxidation stress by polyunsaturated fatty acids. Also, many of the proteins necessary for the biosynthesis of Q6 are associated in a high molecular weight complex that localizes in the inner mitochondrial membrane, as part of what has been termed the ‘CoQ-Synthome’.Chapter 2 investigates potential additional protein partners of the CoQ-Synthome. Via tandem affinity purification and proteomic and mass spectrometry analysis—facilitated by dual-tagged Coq polypeptides—YLR290C was identified as a CoQ-Synthome binding partner. This protein of unknown function co-purified with Coq4, Coq5, and Coq7 and was verified via Immunoblotting and proteomic analysis. Additionally, yeast null mutants for ylr290c showed impaired 12C-Q6 and 13C6-Q6 levels, indicating that this novel binding partner was needed for efficient Q biosynthesis. Altogether, the entirety of the data supported YLR290C as a novel Coq polypeptide, and hence it was renamed “Coq11”. Additionally, previous work elucidated that the stability of the CoQ-Synthome relied on the presence of the Coq8 protein, the putative kinase of the system, which was not previously shown to be a member of the complex. Moreover, several Coq polypeptides—namely Coq3, Coq5, and Coq7—are phosphorylated in a Coq8-dependent manner. Chapter 2 demonstrates the novel finding that Coq8 was captured in association with Coq6, a known member of the high molecular-weight complex, hence asserting a physical association between Coq8 and Coq6. Thus, it is now believed that Coq8 is a novel member of the CoQ-Synthome.Given the findings contained in Chapter 2 related to the association of Coq6 with the putative kinase of the system, Coq8, the question now remains whether Coq6 is a phosphorylated Coq polypeptide, similar to Coq3, Coq5, and Coq7. Chapter 3 further investigates this potential for phosphorylation as it related to Coq6. In fact, 2-Dimensional Isoelectric Focusing Assays conducted on dual-tagged Coq6 showed that indeed it is a phosphorylated polypeptide. With this novel information regarding Coq6, purified dual-tagged Coq6 was subjected to phosphoproteomic analysis, the results of which proved inconclusive. Hence, predictive software and algorithms were employed to assist in the elucidation of potential sites of phosphorylation on Coq6. Additionally, a putative model of Coq6 was generated using the predictive software PHYRE2. Using this information, site-directed mutagenesis of Coq6 was conducted in order to investigate the phenotypes of particular sites that were predicted to be “high potential for phosphorylation sites” based on the predictive algorithms and the Coq6 model. Chapter 4 details the extensive story of alternative splicing in yeast, and in particular, as it relates to the Coenzyme Q biosynthetic pathway. Previous findings from another collaborating laboratory showed that Coq7 contained several phosphorylated residues that were regulatory in terms of Q biosynthesis. Namely, when three particular residues were mutated to be constitutively nonphosphorylatable, Q levels were dramatically higher compared to the WT. In addition, the phosphatase responsible for the dephosphorylation of Coq7 was identified as Ptc7, a known mitochondrial phosphatase. Chapter 4 highlights the alternative splicing of PTC7, which results in two distinct and functional isoforms of this gene: Ptc7s (spliced) and Ptc7ns (nonspliced). Particularly, it is the spliced form which was previously cited as the mitochondrial phosphatase; the nonspliced form is, indeed, a novel protein of unknown function. Chapter 5 will further delve into the potential function of Ptc7ns. In addition to highlighting a rare case of alternative splicing in yeast, and particularly that of a gene (PTC7) involved in Q biosynthesis, Chapter 4 also uncovers the gene that is responsible for this alternative splicing event. In particular, SNF2, which stands for sucrose-nonfermentable, is a nutrient sensing gene that modulates the splicing of PTC7 and is, thereby, able to control Q-biosynthesis in yeast. Moreover, Chapter 4 highlights how SNF2 is necessary for the metabolic shift from fermentation to respiration in yeast, and how nonconsensus splicing is initiated when SNF2 becomes absent from the cell. Chapter 5 provides insight and perspectives on the current projects at hand and suggests future directions for each portion of the thesis projects. Additional experimental techniques and approaches are highlighted and ideas regarding how to advance each project further are proposed.Finally, the Appendix contains the recent publication that elucidates kaempherol as a novel coenzyme Q biosynthetic precursor. When tested in comparison to various polyphenolic compounds, kaempherol supplementation showed the greatest effect on increasing Q content in mammalian cell lines. The data, overall, suggests that kaempherol is able to be metabolized into a novel ring precursor for coenzyme Q biosynthesis