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Coenzyme Q biosynthesis in Saccharomyces cerevisiae: characterization of a Coq polypeptide biosynthetic complex and a coenzyme Q binding protein

Abstract

Coenzyme Q (Q or ubiquinone) is a redox-active lipid consisting of a fully substituted benzoquinone ring and a lipophilic polyisoprenoid moiety which serves to anchor Q in biological membranes. Q is essential to mitochondrial electron transport where it accepts electrons from NADH or succinate at complexes I and II respectively, and donates them to cytochrome c at complex III. In the yeast Saccharomyces cerevisiae Q is synthesized by the products of eleven nuclear-encoded genes: COQ1-COQ9, YAH1, and ARH1. The product of an additional gene, COQ10, is not required for the biosynthesis of Q but is necessary for its function in efficient electron transport and respiration.

Chapter 2 investigates the role of Coq10p in both mitochondrial respiration and de novo Q synthesis through analysis of coq10 null mutants and the Coq10p prokaryotic homolog CC1736. Expression of CC1736 harboring a mitochondrial leader sequence in yeast coq10 mutants restored respiration and growth on a non-fermentable carbon source, and binding assays demonstrated that CC1736 is capable of binding different isoforms of Q and a late-stage Q biosynthetic intermediate. Lipid analysis of the coq10 null mutant revealed significantly decreased de novo Q synthesis when compared to wild type, especially at early-log phase, indicating Coq10p is required for efficient de novo biosynthesis of Q.

Chapter 3 describes characterization of a mitochondrial multi-subunit Coq polypeptide complex required for Q biosynthesis. The complex was purified by tandem affinity purification of particular dual-tagged Coq proteins and Western blotting analysis of purified eluates showed that the biosynthetic complex includes Coq3p, Coq4p, Coq5p, Coq6p, Coq7p, and Coq9p. In addition, Coq8p was observed to co-purify with tagged Coq6p but not other tagged Coq proteins. The purified eluates were also subject to proteomic analysis to identify potentially novel binding partners, identifying two proteins, Ilv6p and YLR290C. Lipid analysis of the corresponding null mutants revealed that the ylr290c mutant has significantly reduced de novo Q synthesis, while the ilv6 mutant synthesizes wild-type levels of Q. Tandem affinity purification of tagged YLR290C demonstrated that it is associated with Coq4p, Coq5p, and Coq7p.

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