3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae

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Currently, eight genes are known to be involved in coenzyme Q 6 biosynthesis in Saccharomyces cerevisiae. Here, we report a new gene designated COQ9 that is also required for the biosynthesis of this lipoid quinone. The respiratory-deficient pet mutant C92 was found to be deficient in coenzyme Q and to have low mitochondrial NADH-cytochrome c reductase activity, which could be restored by addition of coenzyme Q 2 . The mutant was used to clone COQ9, corresponding to reading frame YLR201c on chromosome XII. The respiratory defect of C92 is complemented by COQ9 and suppressed by COQ8/ ABC1. The latter gene has been shown to be required for coenzyme Q biosynthesis in yeast and bacteria. Suppression by COQ8/ABC1 of C92, but not other coq9 mutants tested, has been related to an increase in the mitochondrial concentration of several enzymes of the pathway. Coq9p may either catalyze a reaction in the coenzyme Q biosynthetic pathway or have a regulatory role similar to that proposed for Coq8p.Biosynthesis of coenzyme Q (ubiquinone) in eukaryotes occurs in mitochondria. Eight genes designated COQ1-8 have been shown to be involved in the biosynthesis of this lipoid component of the electron transport chain of Saccharomyces cerevisiae (1). The products of these genes have been localized to the inner membrane (2-8) and, in some cases, were inferred to be present in a complex (4, 9, 10). The chemical intermediates detected in some mutants have revealed that the pathways in bacteria and in this yeast are identical up to the formation of 3-hexaprenyl-4-hydroxybenzoic acid (HHB) 1 (11,12), at which point they diverge for the next three steps, but then converge again in the last stages of biosynthesis (11,12).A hallmark of most yeast coq mutants is the accumulation of HHB when the biochemical block occurs at any step subsequent to the formation of this early intermediate (5-8, 13, 14). This may indicate that 1) most intermediates of the pathway are unstable and degraded; 2) the pathway is highly regulated; or 3) the enzymes are organized in a complex, which is highly sensitive to mutations in any one of its components. The latter possibility is supported by recent evidence indicating an interdependence of some Coq proteins for their stability (4). Most of the COQ gene products have been related to specific reactions of the eukaryotic pathway based on their homology to the bacterial counterparts (6, 7). The reactions catalyzed by the products of COQ4 and COQ8/ABC1, however, still need to be clarified (5,8).In the course of analyzing the biochemical defects of respiratory-deficient pet mutants of S. cerevisiae, we have identified a new gene that, when mutated, produces a phenotype similar to that of coenzyme Q mutants. This gene is defined by complementation group G61 of our pet mutant collection (1) and has been named COQ9. Homologs of COQ9 are present in a wide range of different eukaryotes, but not in bacteria, indicating that its function is specific to coenzyme Q biosynthesis in mitochondria. Suppression of a coq9 mutant by C...

Section: Coq9 Is Required For Biosynthesis Of Coenzyme Qmentioning

confidence: 98%

Section: Construction Of Hybrid Genes Expressing Coq9p Tagged With Hementioning

confidence: 99%

Section: Construction Of Hybrid Genes Expressing Coq9p Tagged With Hementioning

confidence: 99%

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COQ9, a New Gene Required for the Biosynthesis of Coenzyme Q in Saccharomyces cerevisiae

Johnson

Gin

Marbois

et al. 2005

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“…Cells the were harvested after 3 days of incubation at 30°C with shaking (200 rpm, A 600 nm ϭ 10), and the lipids were extracted as described (29). The lipid extracts were concentrated, the volume was adjusted to 1 ml with hexane, and 0.10-ml aliquots (5.0 -12.0 ϫ 10 3 cpm) were analyzed by normal phase HPLC employing a cyanopropyl column (Zorbax, 5 m, 4.6 ϫ 250 mm; MacMod Analytical, Chadds Ford, PA) as described (30). The column was equilibrated with a mobile phase composed of 98% solvent A (hexane) and 2% solvent B (isopropanol:hexane:water:methylene chloride, 52:41: 5:2) at a flow rate of 1 ml/min.…”

Section: Figmentioning

confidence: 99%

The Saccharomyces cerevisiae COQ6 Gene Encodes a Mitochondrial Flavin-dependent Monooxygenase Required for Coenzyme Q Biosynthesis

Gin

Hsu

Rothman

et al. 2003

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Coenzyme Q (Q) is a lipid that functions as an electron carrier in the mitochondrial respiratory chain in eukaryotes. There are eight complementation groups of Q-deficient Saccharomyces cerevisiae mutants, designated coq1-coq8. Here we have isolated the COQ6 gene by functional complementation and, in contrast to a previous report, find it is not an essential gene. coq6 mutants are unable to grow on nonfermentable carbon sources and do not synthesize Q but instead accumulate the Q biosynthetic intermediate 3-hexaprenyl-4-hydroxybenzoic acid. The Coq6 polypeptide is imported into the mitochondria in a membrane potential-dependent manner. Coq6p is a peripheral membrane protein that localizes to the matrix side of the inner mitochondrial membrane. Based on sequence homology to known proteins, we suggest that COQ6 encodes a flavindependent monooxygenase required for one or more steps in Q biosynthesis.

“…Yeast strains harboring null mutations in coq3, coq4, coq5, coq6, coq7, or coq8 accumulate the first lipid-associated intermediate 3-hexaprenyl-4-hydroxybenzoic acid (HHB) ( Fig. 1) (20,21). This lipid intermediate is detected after metabolic labeling with the dedicated precursor 4-[U- 14 C]hydroxybenzoic acid.…”

mentioning

confidence: 99%

Coq3 and Coq4 Define a Polypeptide Complex in Yeast Mitochondria for the Biosynthesis of Coenzyme Q

Marbois¹,

Gin²,

Faull

et al. 2005

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120

Coenzyme Q (Q) is a redox active lipid essential for aerobic respiration in eukaryotes. In Saccharomyces cerevisiae at least eight mitochondrial polypeptides, designated Coq1-Coq8, are required for Q biosynthesis. Here we present physical evidence for a coenzyme Qbiosynthetic polypeptide complex in isolated mitochondria. Separation of digitonin-solubilized mitochondrial extracts in one-and two-dimensional Blue Native PAGE analyses shows that Coq3 and Coq4 polypeptides comigrate as high molecular mass complexes. Similarly, gel filtration chromatography shows that Coq1p, Coq3p, Coq4p, Coq5p, and Coq6p elute in fractions higher than expected for their respective subunit molecular masses. Coq3p, Coq4p, and Coq6p coelute with an apparent molecular mass exceeding 700 kDa. Coq3 O-methyltransferase activity, a surrogate for Q biosynthesis and complex activity, also elutes at this high molecular mass. We have determined the quinone content in lipid extracts of gel filtration fractions by liquid chromatography-tandem mass spectrometry and find that demethoxy-Q 6 is enriched in fractions with Coq3p. Co-precipitation of biotinylated-Coq3 and Coq4 polypeptide from digitoninsolubilized mitochondrial extracts shows their physical association. This study identifies Coq3p and Coq4p as defining members of a Q-biosynthetic Coq polypeptide complex. Coenzyme Q (ubiquinone or Q) 1 is a redox active lipid containing a long polyprenyl tail attached to a fully substituted benzoquinone ring. The number (n) of isoprene units in the polyprenyl tail (Q n ) is distinct in different organisms; humans produce Q 10 , Caenorhabditis elegans Q 9 , Escherichia coli Q 8 , and Saccharomyces cerevisiae Q 6 . Within the inner mitochondrial membrane Q functions in respiratory electron transport, where it transfers two electrons from either complex I or complex II to complex III (1). Q is present in almost all organisms, with the quantity roughly matching the respiratory capability of the tissue from which it is isolated (2). In addition to this role, Q has been found to act as a chain-breaking antioxidant (3) and to be involved in the electron transport chains of plasma and lysosomal membranes (4, 5). Q supplementation in humans slows the functional decline in patients with Parkinson disease (6), is useful for treating patients with respiratory chain defects (7), and has promise as a treatment in other neurodegenerative diseases (8).As observed with other redox active compounds, Q has the potential to act as a source of oxidative stress. In certain species, the amount of reactive oxygen species generated by mitochondria has been related to Q content (9, 10). These described dual roles as antioxidant and prooxidant associate Q with both extended and shortened life spans. C. elegans fed E. coli diets lacking Q 8 , and C. elegans clk-1 mutants with defects in Q biosynthesis show increased life spans, and we have proposed that the decreased levels of Q are associated with a decreased level of the Q semiquinone radical (Q . ) and a decreased propensity to...