Tanya Jonassen

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.

Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: A gene required for ubiquinone biosynthesis with potential implications for aging

Abstract. Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span. The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis. In this study, RT-PCR and 5′ RACE were used to isolate both human and mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr 16(p12–13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods. The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C. elegans. Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression. The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source. The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants. The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging.

Demethoxy-Q, an intermediate of coenzyme Q biosynthesis, fails to support respiration in Saccharomyces cerevisiae and lacks antioxidant activity

Caenorhabditis elegans clk-1 mutants cannot produce coenzyme Q9 and instead accumulate demethoxy-Q9 (DMQ9). DMQ9 has been proposed to be responsible for the extended lifespan of clk-1 mutants, theoretically through its enhanced antioxidant properties and its decreased function in respiratory chain electron transport. In the present study, we assess the functional roles of DMQ6 in the yeast Saccharomyces cerevisiae. Three mutations designed to mirror the clk-1 mutations of C. elegans were introduced into COQ7, the yeast homologue of clk-1: E233K, predicted to disrupt the di-iron carboxylate site considered essential for hydroxylase activity; L237Stop, a deletion of 36 amino acid residues from the carboxyl terminus; and P175Stop, a deletion of the carboxyl-terminal half of Coq7p. Growth on glycerol, quinone content, respiratory function, and response to oxidative stress were analyzed in each of the coq7 mutant strains. Yeast strains lacking Q6 and producing solely DMQ were respiratory deficient and unable to support 6either NADH-cytochrome c reductase or succinate-cytochrome c reductase activities. DMQ6 failed to protect cells against oxidative stress generated by H2O2 or linolenic acid. Thus, in the yeast model system, DMQ does not support respiratory activity and fails to act as an effective antioxidant. These results suggest that the life span extension observed in the C. elegans clk-1 mutants cannot be attributed to the presence of DMQ per se.

Complementation of Saccharomyces cerevisiae coq7 mutants by mitochondrial targeting of the escherichia coli UbiF polypeptide : Two functions of yeast Coq7 polypeptide in coenzyme q biosynthesis

Coenzyme Q (ubiquinone or Q) functions in the respiratory electron transport chain and serves as a lipophilic antioxidant. In the budding yeast Saccharomyces cerevisiae, Q biosynthesis requires nine Coq proteins (Coq1–Coq9). Previous work suggests both an enzymatic activity and a structural role for the yeast Coq7 protein. To define the functional roles of yeast Coq7p we test whether Escherichia coli ubiF can functionally substitute for yeast COQ7. The ubiF gene encodes a flavin-dependent monooxygenase that shares no homology to the Coq7 protein and is required for the final monooxygenase step of Q biosynthesis in E. coli. The ubiF gene expressed at low copy restores growth of a coq7 point mutant (E194K) on medium containing a non-fermentable carbon source, but fails to rescue a coq7 null mutant. However, expression of ubiF from a multicopy vector restores growth and Q synthesis for both mutants, although with a higher efficiency in the point mutant. We attribute the more efficient rescue of the coq7 point mutant to higher steady state levels of the Coq3, Coq4, and Coq6 proteins and to the presence of demethoxyubiquinone, the substrate of UbiF. Coq7p co-migrates with the Coq3 and Coq4 polypeptides as a high molecular mass complex. Here we show that addition of Q to the growth media also stabilizes the Coq3 and Coq4 polypeptides in the coq7 null mutant. The data suggest that Coq7p, and the lipid quinones (demethoxyubiquinone and Q) function to stabilize other Coq polypeptides.

Ubiquinone is not required for proton conductance by uncoupling protein 1 in yeast mitochondria.

Q (coenzyme Q or ubiquinone) is reported to be a cofactor obligatory for proton transport by UCPs (uncoupling proteins) in liposomes [Echtay, Winkler and Klingenberg (2000) Nature (London) 408, 609-613] and for increasing the binding of the activator retinoic acid to UCP1 [Tomás, Ledesma and Rial (2002) FEBS Lett. 526, 63-65]. In the present study, yeast ( Saccharomyces cerevisiae ) mutant strains lacking Q and expressing UCP1 were used to determine whether Q was required for UCP function in mitochondria. Wild-type yeast strain and two mutant strains (CENDeltaCOQ3 and CENDeltaCOQ2), both not capable of synthesizing Q, were transformed with the mouse UCP1 gene. UCP1 activity was measured as fatty acid-dependent, GDP-sensitive proton conductance in mitochondria isolated from the cells. The activity of UCP1 was similar in both Q-containing and -deficient yeast mitochondria. We conclude that Q is neither an obligatory cofactor nor an activator of proton transport by UCP1 when it is expressed in yeast mitochondria.

Reproductive Fitness and Quinone Content of Caenorhabditis elegans clk-1 Mutants Fed Coenzyme Q Isoforms of Varying Length

Caenorhabditis elegans clk-1 mutants lack coenzyme Q9 and accumulate the biosynthetic intermediate demethoxy-Q9. A dietary source of ubiquinone (Q) is required for larval growth and development of the gonad and germ cells. We considered that uptake of the shorter Q8 isoform present in the Escherichia coli food may contribute to the Clk phenotypes of slowed development and reduced brood size observed when the animals are fed Q-replete E. coli. To test the effect of isoprene tail length, N2 and clk-1 animals were fed E. coli engineered to produce Q7, Q8, Q9, or Q10. Wild-type nematodes showed no change in reproductive fitness regardless of the Qn isoform fed. clk-1(e2519) fed the Q9 diet showed increased egg production; however, this diet did not improve reproductive fitness of the clk-1(qm30) animals. Furthermore, animals with the more severe clk-1(qm30) allele become sterile and their progeny inviable when fed Q7-containing bacteria. The content of Q7 in the mitochondria of clk-1 animals was decreased relative to Q8, suggesting less effective transport of Q7 to the mitochondria, impaired retention, or decreased stability. Additionally, regardless of E. coli diet, clk-1(qm30) animals contain a dysfunctional dense form of mitochondria. The gonads of clk-1(qm30) worms fed Q7-containing food were severely shrunken and disordered. The differential fertility of clk-1 mutant nematodes fed Q isoforms may result from changes in Q localization, altered recognition by Q-binding proteins, and/or potential defects in mitochondrial function resulting from the mutant CLK-1 polypeptide itself.