Associated Polypeptides Found within Yeast Mitochondria for the Biosynthesis of Coenzyme Q

Coenzyme Q, CoQ, or ubiquinone, is a water-insoluble antioxidant found in the lipid bilayer of the inner membrane of mitochondria where it serves as an essential component of the electron transport chain. Although biosynthesis of CoQ is not completely characterized several multi-subunit complexes and their proteins have been identified and are required for synthesis of CoQ. However the functions of several polypeptides for CoQ synthesis remain unknown. Biotinylated Coq3p has shown to interact with the polypeptide, Coq4p as well as several other Coq polypeptides. In this study, a dual tag, CNAP, containing ten consecutive histidine residues and a protein C epitope will aid in the immunoprecipitation of the other proteins associated with Coq3p. This tag will permit two tandem-affinity purification steps to enhance protein purification. Preliminary data confirm expression of Coq3p-CNAP rescues the coq3 knockout and therefore succeeds in preserving the mitochondrial complex and function. Upcoming experiments include preparation of yeast lipid extracts and assays of CoQ content via HPLC and tandem mass spectrometry. Digitonin solubilization of mitochondrial proteins will aid in the preparation of the final pull-downs of the Coq polypeptide complex. With more insight as to how Coq proteins associate, this project's data will hopefully elucidate many enigmas that exist in CoQ biosynthesis.