Coenzyme Q (Q) is a redox active lipid that is an essential component of the electron transport chain. Here, we show that steady state levels of Coq3, Coq4, Coq6, Coq7 and Coq9 polypeptides in yeast mitochondria are dependent on the expression of each of the other COQ genes. Submitochondrial localization studies indicate Coq9p is a peripheral membrane protein on the matrix side of the mitochondrial inner membrane. To investigate whether Coq9p is a component of a complex of Qbiosynthetic proteins, the native molecular mass of Coq9p was determined by Blue Native-PAGE. Coq9p was found to co-migrate with Coq3p and Coq4p at a molecular mass of approximately 1 MDa. A direct physical interaction was shown by the immunoprecipitation of HA-tagged Coq9 polypeptide with Coq4p, Coq5p, Coq6p and Coq7p. These findings, together with other work identifying Coq3p and Coq4p interactions, identify at least six Coq polypeptides in a multi-subunit Q biosynthetic complex.

Coenzyme Q (CoQ) is an essential electron carrier in the respiratory chain whose deficiency has been implicated in a wide variety of human mitochondrial disease manifestations. Its multi-step biosynthesis involves production of polyisoprenoid diphosphate in a reaction that requires the enzymes be encoded by PDSS1 and PDSS2. Homozygous mutations in either of these genes, in humans, lead to severe neuromuscular disease, with nephrotic syndrome seen in PDSS2 deficiency. We now show that a presumed autoimmune kidney disease in mice with the missense Pdss2kd/kd genotype can be attributed to a mitochondrial CoQ biosynthetic defect. Levels of CoQ9 and CoQ10 in kidney homogenates from B6.Pdss2kd/kd mutants were significantly lower than those in B6 control mice. Disease manifestations originate specifically in glomerular podocytes, as renal disease is seen in Podocin/cre,Pdss2loxP/loxP knockout mice but not in conditional knockouts targeted to renal tubular epithelium, monocytes, or hepatocytes. Liver-conditional B6.Alb/cre,Pdss2loxP/loxP knockout mice have no overt disease despite demonstration that their livers have undetectable CoQ9 levels, impaired respiratory capacity, and significantly altered intermediary metabolism as evidenced by transcriptional profiling and amino acid quantitation. These data suggest that disease manifestations of CoQ deficiency relate to tissue-specific respiratory capacity thresholds, with glomerular podocytes displaying the greatest sensitivity to Pdss2 impairment.

Ubiquinone (coenzyme Q) functions as an electron transporter in aerobic respiration and oxidative phosphorylation in the respiratory chain [1]. In addition, many reports suggest that ubiquinone also functions as a lipid-soluble antioxidant in cellular biomembranes, scavenging reactive oxygen species [2][3][4][5]. Indeed, several studies using yeast strains that do not produce ubiquinone suggest that an in vitro function of ubiquinone is to protect against oxidants [6,7]. Another phenotype of such ubiquinone-deficient fission yeast is that they generate high levels of hydrogen sulfide [8][9][10]. As Schizosaccharomyces pombe and other eukaryotes are known to carry sulfide-ubiquinone reductase, an enzyme that oxidizes sulfide via ubiquinone [11], it has been suggested that ubiquinone is linked to sulfide metabolism in many organisms. In addition, it was The isoprenoid chain of ubiquinone (Q) is determined by trans-polyprenyl diphosphate synthase in micro-organisms and presumably in mammals. Because mice and humans produce Q 9 and Q 10 , they are expected to possess solanesyl and decaprenyl diphosphate synthases as the determining enzyme for a type of ubiquinone. Here we show that murine and human solanesyl and decaprenyl diphosphate synthases are heterotetramers composed of newly characterized hDPS1 (mSPS1) and hDLP1 (mDLP1), which have been identified as orthologs of Schizosaccharomyces pombe Dps1 and Dlp1, respectively. Whereas hDPS1 or mSPS1 can complement the S. pombe dps1 disruptant, neither hDLP1 nor mDLP1 could complement the S. pombe dLp1 disruptant. Thus, only hDPS1 and mSPS1 are functional orthologs of SpDps1. Escherichia coli was engineered to express murine and human SpDps1 and ⁄ or SpDlp1 homologs and their ubiquinone types were determined. Whereas transformants expressing a single component produced only Q 8 of E. coli origin, double transformants expressing mSPS1 and mDLP1 or hDPS1 and hDLP1 produced Q 9 or Q 10 , respectively, and an in vitro activity of solanesyl or decaprenyl diphosphate synthase was verified. The complex size of the human and murine long-chain transprenyl diphosphate synthases, as estimated by gel-filtration chromatography, indicates that they consist of heterotetramers. Expression in E. coli of heterologous combinations, namely, mSPS1 and hDLP1 or hDPS1 and mDLP1, generated both Q 9 and Q 10 , indicating both components are involved in determining the ubiquinone side chain. Thus, we identified the components of the enzymes that determine the side chain of ubiquinone in mammals and they resembles the S. pombe, but not plant or Saccharomyces cerevisiae, type of enzyme.Abbreviations

Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain.

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and ␣-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H 2 O 2 and Cu 2؉. Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H 2 S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system. Ubiquinone is known to be an electron transporter in the respiratory chain in prokaryotes and eukaryotes. It varies among organisms in the length of its isoprenoid side chain. For example, Saccharomyces cerevisiae uses ubiquinone-6 (UQ-6), Escherichia coli uses UQ-8, and Schizosaccharomyces pombe uses UQ-10 (9, 16, 37). It has been shown that the type of ubiquinone in organisms is determined by the polyprenyl diphosphate synthase enzyme, which catalyzes the condensation reaction of isopentenyl diphosphate with allylic diphosphate to give a defined length of the isoprenoid (22, 26). When polyprenyl diphosphate synthase genes from other sources were expressed in S. cerevisiae and E. coli, the ubiquinone generated was of the same type as that expressed in the donor organism (22)(23)(24)(25)(26). By this method, we successfully produced various ubiquinone species (UQ-5 to UQ-10) in the S. cerevisiae COQ1 mutant (22), which in turn indicates that p-hydroxybenzoate (PHB) polyprenyl diphosphate transferase, which catalyzes the condensation reaction between the isoprenoid side chain and PHB, has a broad substrate specificity. This is supported by consistent observations showing that purified PHB polyprenyl diphosphate transferases from Pseudomonas putida (12, 40) and E. coli (17) have fairly wide substrate specificities in terms of polyprenols. In contrast, PHB g...