Molecular characterization of the human COQ5 C-methyltransferase in coenzyme Q10 biosynthesis

Coq5 catalyzes the only C-methylation involved in the biosynthesis of coenzyme Q (Q or ubiquinone) in humans and yeast Saccharomyces cerevisiae. As one of eleven polypeptides required for Q production in yeast, Coq5 has also been shown to assemble with the multi-subunit complex termed the CoQ-synthome. In humans, mutations in several COQ genes cause primary Q deficiency, and a decrease in Q biosynthesis is associated with mitochondrial, cardiovascular, kidney and neurodegenerative diseases. In this study, we characterize the human COQ5 polypeptide and examine its complementation of yeast coq5 point and null mutants. We show that human COQ5 RNA is expressed in all tissues and that the COQ5 polypeptide is associated with the mitochondrial inner membrane on the matrix side. Previous work in yeast has shown that point mutations within or adjacent to conserved COQ5 methyltransferase motifs result in a loss of Coq5 function but not Coq5 steady state levels. Here, we show that stabilization of the CoQ-synthome within coq5 point mutants or by over-expression of COQ8 in coq5 null mutants permits the human COQ5 homolog to partially restore coq5 mutant growth on respiratory media and Q₆ content. Immunoblotting against the human COQ5 polypeptide in isolated yeast mitochondria shows that the human Coq5 polypeptide migrates in two-dimensional blue-native/SDS-PAGE at the same high molecular mass as other yeast Coq proteins. The results presented suggest that human and Escherichia coli Coq5 homologs expressed in yeast retain C-methyltransferase activity but are capable of rescuing the coq5 yeast mutants only when the CoQ-synthome is assembled.     

CABC1 gene mutations cause ubiquinone deficiency with cerebellar ataxia and seizures

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS) in that it distributes electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. Primary coenzyme Q(10) deficiency represents a clinically heterogeneous condition suggestive of genetic heterogeneity, and several disease genes have been previously identified. The CABC1 gene, also called COQ8 or ADCK3, is the human homolog of the yeast ABC1/COQ8 gene, one of the numerous genes involved in the ubiquinone biosynthesis pathway. The exact function of the Abc1/Coq8 protein is as yet unknown, but this protein is classified as a putative protein kinase. We report here CABC1 gene mutations in four ubiquinone-deficient patients in three distinct families. These patients presented a similar progressive neurological disorder with cerebellar atrophy and seizures. In all cases, enzymological studies pointed to ubiquinone deficiency. CoQ(10) deficiency was confirmed by decreased content of ubiquinone in muscle. Various missense mutations (R213W, G272V, G272D, and E551K) modifying highly conserved amino acids of the protein and a 1 bp frameshift insertion c.[1812_1813insG] were identified. The missense mutations were introduced into the yeast ABC1/COQ8 gene and expressed in a Saccharomyces cerevisiae strain in which the ABC1/COQ8 gene was deleted. All the missense mutations resulted in a respiratory phenotype with no or decreased growth on glycerol medium and a severe reduction in ubiquinone synthesis, demonstrating that these mutations alter the protein function.     

Site-directed mutagenesis and structural modeling of Coq10p indicate the presence of a tunnel for coenzyme Q6 binding

Coq10p is a protein required for coenzyme Q function, but its specific role is still unknown. It is a member of the START domain superfamily that contains a hydrophobic tunnel implicated in the binding of lipophilic molecules. We used site-directed mutagenesis, statistical coupling analysis and molecular modeling to probe structural determinants in the Coq10p putative tunnel. Four point mutations were generated (coq10-K50E, coq10-L96S, coq10-E105K and coq10-K162D) and their biochemical properties analysed, as well as structural consequences. Our results show that all mutations impaired Coq10p function and together with molecular modeling indicate an important role for the Coq10p putative tunnel.     

Identification of Coq11, a New Coenzyme Q Biosynthetic Protein in the CoQ-Synthome in Saccharomyces cerevisiae

Coenzyme Q (Q or ubiquinone) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail and is required for mitochondrial electron transport. In the yeast Saccharomyces cerevisiae, Q is synthesized by the products of 11 known genes, COQ1–COQ9, YAH1, and ARH1. The function of some of the Coq proteins remains unknown, and several steps in the Q biosynthetic pathway are not fully characterized. Several of the Coq proteins are associated in a macromolecular complex on the matrix face of the inner mitochondrial membrane, and this complex is required for efficient Q synthesis. Here, we further characterize this complex via immunoblotting and proteomic analysis of tandem affinity-purified tagged Coq proteins. We show that Coq8, a putative kinase required for the stability of the Q biosynthetic complex, is associated with a Coq6-containing complex. Additionally Q₆ and late stage Q biosynthetic intermediates were also found to co-purify with the complex. A mitochondrial protein of unknown function, encoded by the YLR290C open reading frame, is also identified as a constituent of the complex and is shown to be required for efficient de novo Q biosynthesis. Given its effect on Q synthesis and its association with the biosynthetic complex, we propose that the open reading frame YLR290C be designated COQ11.     

Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis

The COQ3 gene in Saccharomyces cerevisiae encodes an O-methyltransferase required for two steps in the biosynthetic pathway of ubiquinone (coenzyme Q, or Q). This enzyme methylates an early Q intermediate, 3,4-dihydroxy-5-polyprenylbenzoic acid, as well as the final intermediate in the pathway, converting demethyl-Q to Q. This enzyme is also capable of methylating the distinct prokaryotic early intermediate 2-hydroxy-6-polyprenyl phenol. A full-length cDNA encoding the human homologue of COQ3 was isolated from a human heart cDNA library by sequence homology to rat Coq3. The clone contained a 933-base pair open reading frame that encoded a polypeptide with a great deal of sequence identity to a variety of eukaryotic and prokaryotic Coq3 homologues. In the region between amino acids 89 and 255 in the human sequence, the rat and human homologues are 87% identical, whereas human and yeast are 35% identical. When expressed in multicopy, the human construct rescued the growth of a yeast coq3 null mutant on a nonfermentable carbon source and restored coenzyme Q biosynthesis, although at lower levels than that of wild type yeast. In vitro methyltransferase assays using farnesylated analogues of intermediates in the coenzyme Q biosynthetic pathway as substrates showed that the human enzyme is active with all three substrates tested.     

Overexpression of the Coq8 kinase in Saccharomyces cerevisiae coq null mutants allows for accumulation of diagnostic intermediates of the coenzyme Q6 biosynthetic pathway

Most of the Coq proteins involved in coenzyme Q (ubiquinone or Q) biosynthesis are interdependent within a multiprotein complex in the yeast Saccharomyces cerevisiae. Lack of only one Coq polypeptide, as in Δcoq strains, results in the degradation of several Coq proteins. Consequently, Δcoq strains accumulate the same early intermediate of the Q(6) biosynthetic pathway; this intermediate is therefore not informative about the deficient biosynthetic step in a particular Δcoq strain. In this work, we report that the overexpression of the protein Coq8 in Δcoq strains restores steady state levels of the unstable Coq proteins. Coq8 has been proposed to be a kinase, and we provide evidence that the kinase activity is essential for the stabilizing effect of Coq8 in the Δcoq strains. This stabilization results in the accumulation of several novel Q(6) biosynthetic intermediates. These Q intermediates identify chemical steps impaired in cells lacking Coq4 and Coq9 polypeptides, for which no function has been established to date. Several of the new intermediates contain a C4-amine and provide information on the deamination reaction that takes place when para-aminobenzoic acid is used as a ring precursor of Q(6). Finally, we used synthetic analogues of 4-hydroxybenzoic acid to bypass deficient biosynthetic steps, and we show here that 2,4-dihydroxybenzoic acid is able to restore Q(6) biosynthesis and respiratory growth in a Δcoq7 strain overexpressing Coq8. The overexpression of Coq8 and the use of 4-hydroxybenzoic acid analogues represent innovative tools to elucidate the Q biosynthetic pathway.     

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.     

The yeast Coq4 polypeptide organizes a mitochondrial protein complex essential for coenzyme Q biosynthesis

Coenzyme Q is a redox active lipid essential for aerobic respiration. The Coq4 polypeptide is required for Q biosynthesis and growth on non-fermentable carbon sources, however its exact function in this pathway is not known. Here we probe the functional roles of Coq4p in a yeast Q biosynthetic polypeptide complex. A yeast coq4-1 mutant harboring an E226K substitution is unable to grow on nonfermentable carbon sources. The coq4-1 yeast mutant retains significant Coq3p O-methyltransferase activity, and mitochondria isolated from coq4-1 and coq4-2 (E₁₂₁K) yeast point mutants contain normal steady state levels of Coq polypeptides, unlike the decreased levels of Coq polypeptides generally found in strains harboring coq gene deletions. Digitonin-solubilized mitochondrial extracts prepared from yeast coq4 point mutants show that Coq3p and Coq4 polypeptides no longer co-migrate as high molecular mass complexes by one- and two-dimensional Blue Native-PAGE. Similarly, gel filtration chromatography confirms that O-methyltransferase activity, Coq3p, Coq4p, and Coq7p migration are disorganized in the coq4-1 mutant mitochondria. The data suggest that Coq4p plays an essential role in organizing a Coq enzyme complex required for Q biosynthesis.     

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.     

The submitochondrial distribution of ubiquinone affects respiration in long-lived Mclk1+/? mice

Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1^+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1^+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ₁₀ was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3^+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.     

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 the Escherichia coli O-methyltransferase, UbiG. In this study, farnesylated analogs corresponding to the second O-methylation step, demethyl-Q(3) and Q(3), have been chemically synthesized and used to study Q biosynthesis in yeast mitochondria in vitro. Both yeast and rat Coq3p recognize the demethyl-Q(3) 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 one O-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.     

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q₆ increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q₆ or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q₆, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.     

Pathogenicity of two COQ7 mutations and responses to 2,4‐dihydroxybenzoate bypass treatment

Primary ubiquinone (co‐enzyme Q) deficiency results in a wide range of clinical features due to mitochondrial dysfunction. Here, we analyse and characterize two mutations in the ubiquinone biosynthetic gene COQ7. One mutation from the only previously identified patient (V141E), and one (L111P) from a 6‐year‐old girl who presents with spasticity and bilateral sensorineural hearing loss. We used patient fibroblast cell lines and a heterologous expression system to show that both mutations lead to loss of protein stability and decreased levels of ubiquinone that correlate with the severity of mitochondrial dysfunction. The severity of L111P is enhanced by the particular COQ7 polymorphism (T103M) that the patient carries, but not by a mitochondrial DNA mutation (A1555G) that is also present in the patient and that has been linked to aminoglycoside‐dependent hearing loss. We analysed treatment with the unnatural biosynthesis precursor 2,4‐dihydroxybenzoate (DHB), which can restore ubiquinone synthesis in cells completely lacking the enzymatic activity of COQ7. We find that the treatment is not beneficial for every COQ7 mutation and its outcome depends on the extent of enzyme activity loss.     

Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q₂. Rescue of respiration by Q₂ is a characteristic of mutants blocked in coenzyme Q₆ synthesis. Unlike Q₆ deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q₆. The physiological significance of earlier observations that purified Coq10p contains bound Q₆ was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q₂. This suggests that in vivo binding of Q₆ by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.     

The role of UbiX in Escherichia coli coenzyme Q biosynthesis

The reversible redox chemistry of coenzyme Q serves a crucial function in respiratory electron transport. Biosynthesis of Q in Escherichia coli depends on the ubi genes. However, very little is known about UbiX, an enzyme thought to be involved in the decarboxylation step in Q biosynthesis in E. coli and Salmonella enterica. Here we characterize an E. coli ubiX gene deletion strain, LL1, to further elucidate E. coli ubiX function in Q biosynthesis. LLI produces very low levels of Q, grows slowly on succinate as the sole carbon source, accumulates 4-hydroxy-3-octaprenyl-benzoate, and has reduced UbiG O-methyltransferase activity. Expression of either E. coli ubiX or the Saccharomyces cerevisiae ortholog PAD1, rescues the deficient phenotypes of LL1, identifying PAD1 as an ortholog of ubiX. Our results suggest that both UbiX and UbiD are required for the decarboxylation of 4-hydroxy-3-octaprenyl-benzoate in E. coli coenzyme Q biosynthesis, especially during logarithmic growth.     

Coq3 and Coq4 define a polypeptide complex in yeast mitochondria for the biosynthesis of coenzyme Q

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 Q-biosynthetic 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 co-migrate 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 digitonin-solubilized mitochondrial extracts shows their physical association. This study identifies Coq3p and Coq4p as defining members of a Q-biosynthetic Coq polypeptide complex.     

COQ9, a new gene required for the biosynthesis of coenzyme Q in Saccharomyces cerevisiae

Currently, eight genes are known to be involved in coenzyme Q6 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 Q2. 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.     

Ubiquinone biosynthesis in Saccharomyces cerevisiae. Isolation and sequence of COQ3, the 3,4-dihydroxy-5-hexaprenylbenzoate methyltransferase gene

Ubiquinone (or coenzyme Q) is a lipid component of the respiratory chain in the inner mitochondrial membrane, in which it functions in electron transport. Recent reports show that ubiquinone and ubiquinone biosynthetic enzymes are present in both mitochondrial and nonmitochondrial membranes of cells (Kalen, A., Appelkvist, E.-L., Chojnacki, T., and Dallner, G. (1990) J. Biol. Chem. 265, 1158-1164) although the functions that ubiquinone may play outside of the mitochondrion are not understood. To study coenzyme Q synthesis and function we cloned the 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase gene by functional complementation of a yeast coenzyme Q mutant strain, defective in the COQ3 gene (Tzagoloff, A., and Dieckmann, C. L. (1990) Microbiol. Rev. 54, 211-225). This gene restores both coenzyme Q synthesis in the mutant strain and the ability to grow on media containing glycerol, a nonfermentable substrate. A one-step in situ gene replacement with the cloned DHHB methyltransferase DNA directs integration to the yeast COQ3 locus on chromosome XV of Saccharomyces cerevisiae, establishing that the COQ3 locus encodes the DHHB methyltransferase structural gene. The predicted amino acid sequence of the yeast DHHB methyltransferase contains a methyltransferase consensus sequence and shows a 40% identity with an open reading frame of Escherichia coli, the gyrA5' hypothetical protein. This open reading frame is adjacent to the gyrA gene and close to the mapped location of the ubiG gene at 48 min on the E. coli chromosome. These results suggest that the E. coli gyrA5' open reading frame encodes a methyltransferase and may correspond to the ubiG gene, which is required for ubiquinone biosynthesis.     

New advances in coenzyme Q biosynthesis

Summary Coenzyme Q (or ubiquinone) is the product of two distinct biosynthetic pathways: the lipid tail of coenzyme Q is formed via the isoprene biosynthetic pathway, and the quinone ring derives from the metabolism of either shikimic acid or tyrosine. In general, eukaryotic organisms use the classical mevalonate pathway to form isopentenyl- and dimethylallyl-diphosphate, the five carbon building blocks of the polyisoprenoid tail, and prokaryotes use 1-deoxy-D-xylulose-5-phosphate, formed via the Rohmer pathway. The quinone ring precursor is 4-hydroxybenzoic acid, which is formed directly from chorismate inSaccharomyces cerevisiae andEscherichia coli, or from tyrosine in animal cells. Ring modification steps including prenylation, decarboxylation, and successive hydroxylation and methylation steps form the fully substituted benzoquinone ring of coenzyme Q. Many of the genes and polypeptides involved in coenzyme Q biosynthesis have been isolated and characterized by utilizing strains ofE. coli andS. cerevisiae with mutations in theubi andCOQ genes, respectively. This article reviews recent progress in characterizing the biosynthesis of coenzyme Q inE. coli, S. cerevisiae, and other eukaryotic organisms.     

Expression of the human atypical kinase ADCK3 rescues coenzyme Q biosynthesis and phosphorylation of Coq polypeptides in yeast coq8 mutants

Coenzyme Q (ubiquinone or Q) is a lipid electron and proton carrier in the electron transport chain. In yeast Saccharomyces cerevisiae eleven genes, designated COQ1 through COQ9, YAH1 and ARH1, have been identified as being required for Q biosynthesis. One of these genes, COQ8 (ABC1), encodes an atypical protein kinase, containing six (I, II, III, VIB, VII, and VIII) of the twelve motifs characteristically present in canonical protein kinases. Here we characterize seven distinct Q-less coq8 yeast mutants and show that unlike the coq8 null mutant, each maintained normal steady-state levels of the Coq8 polypeptide. The phosphorylation states of Coq polypeptides were determined with two-dimensional gel analyses. Coq3p, Coq5p, and Coq7p were phosphorylated in a Coq8p-dependent manner. Expression of a human homolog of Coq8p, ADCK3(CABC1) bearing an amino-terminal yeast mitochondrial leader sequence, rescued growth of yeast coq8 mutants on medium containing a nonfermentable carbon source and partially restored biosynthesis of Q(6). The phosphorylation state of several of the yeast Coq polypeptides was also rescued, indicating a profound conservation of yeast Coq8p and human ADCK3 protein kinase function in Q biosynthesis.