Peter T. Lee

A defect in coenzyme Q biosynthesis is responsible for the respiratory deficiency in Saccharomyces cerevisiae abc1 mutants.

Ubiquinone (coenzyme Q or Q) is an essential component of the mitochondrial respiratory chain in eukaryotic cells. There are eight complementation groups of Q-deficientSaccharomyces cerevisiae mutants designatedcoq1-coq8. Here we report that COQ8 isABC1 (for Activity of bc 1 complex), which was originally isolated as a multicopy suppressor of a cytochrome bmRNA translation defect (Bousquet, I., Dujardin, G., and Slonimski, P. P. (1991) EMBO J. 10, 2023–2031). Previous studies of abc1 mutants suggested that the mitochondrial respiratory complexes were thermosensitive and function inefficiently. Although initial characterization of the abc1 mutants revealed characteristics of Q-deficient mutants, levels of Q were reported to be similar to wild type. The suggested function of Abc1p was that it acts as a chaperone-like protein essential for the proper conformation and functioning of the bc 1 and its neighboring complexes (Brasseur, G., Tron, P., Dujardin, G., Slonimski, P. P. (1997) Eur. J. Biochem. 246, 103–111). Studies presented here indicate that abc1/coq8 null mutants are defective in Q biosynthesis and accumulate 3-hexaprenyl-4-hydroxybenzoic acid as the predominant intermediate. As observed in other yeast coq mutants, supplementation of growth media with Q6 rescues theabc1/coq8 null mutants for growth on nonfermentable carbon sources. Such supplementation also partially restores succinate-cytochrome c reductase activity in theabc1/coq8 null mutants. Abc1/Coq8p localizes to the mitochondria, and is proteolytically processed upon import. The findings presented here indicate that the previously reported thermosensitivity of the respiratory complexes of abc1/coq8mutants results from the lack of Q and a general deficiency in respiration, rather than a specific phenotype due to dysfunction of the Abc1 polypeptide. These results indicate that ABC1/COQ8 is essential for Q-biosynthesis and that the critical defect ofabc1/coq8 mutants is a lack of Q.

Yeast and Rat Coq3 and Escherichia coli UbiG Polypeptides Catalyze Both O-Methyltransferase Steps in Coenzyme Q Biosynthesis*

Ubiquinone (coenzyme Q or Q) is a lipid that functions in the electron transport chain in the inner mitochondrial membrane of eukaryotes and the plasma membrane of prokaryotes. Q-deficient mutants of Saccharomyces cerevisiae harbor defects in one of eight COQ genes (coq1–coq8) and are unable to grow on nonfermentable carbon sources. The biosynthesis of Q involves two separate O-methylation steps. In yeast, the first O-methylation utilizes 3,4-dihydroxy-5-hexaprenylbenzoic acid as a substrate and is thought to be catalyzed by Coq3p, a 32.7-kDa protein that is 40% identical to theEscherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the secondO-methylation step, demethyl-Q3 and Q3, have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q3 precursor as a substrate. In addition, E. coli UbiGp was purified and found to catalyze both O-methylation steps. Futhermore, antibodies to yeast Coq3p were used to determine that the Coq3 polypeptide is peripherally associated with the matrix-side of the inner membrane of yeast mitochondria. The results indicate that oneO-methyltransferase catalyzes both steps in Q biosynthesis in eukaryotes and prokaryotes and that Q biosynthesis is carried out within the matrix compartment of yeast mitochondria.

Genetic evidence for a multi-subunit complex in the O-methyltransferase steps of coenzyme Q biosynthesis

Coq3 O-methyltransferase carries out both O-methylation steps in coenzyme Q (ubiquinone) biosynthesis. The degree to which Coq3 O-methyltransferase activity and expression are dependent on the other seven COQ gene products has been investigated. A panel of yeast mutant strains harboring null mutations in each of the genes required for coenzyme Q biosynthesis (COQ1-COQ8) have been prepared. Mitochondria have been isolated from each member of the yeast coq mutant collection, from the wild-type parental strains and from respiratory deficient mutants harboring deletions in ATP2 or COR1 genes. These latter strains constitute Q-replete, respiratory deficient controls. Each of these mitochondrial preparations has been analyzed for COQ3-encoded O-methyltransferase activity and steady state levels of Coq3 polypeptide. The findings indicate that the presence of the other COQ gene products is required to observe normal levels of O-methyltransferase activity and the Coq3 polypeptide. However, COQ3 steady state RNA levels are not decreased in any of the coq mutants, relative to either wild-type or respiratory deficient control strains, suggesting either a decreased rate of translation or a decreased stability of the Coq3 polypeptide. These data are consistent with the involvement of the Coq polypeptides (or the Q-intermediates formed by the Coq polypeptides) in a multi-subunit complex. It is our hypothesis that a deficiency in any one of the COQ gene products results in a defective complex in which the Coq3 polypeptide is rendered unstable.