Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation.

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5' and 3', respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q(10). Here we show that the Q(9) content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q(9) content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.

Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in prenyl diphosphate synthase subunit 2.

Homozygous mice carrying kd (kidney disease) mutations in the gene encoding prenyl diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal disease. The PDSS2 polypeptide in concert with PDSS1 synthesizes the polyisoprenyl tail of coenzyme Q (Q or ubiquinone), a lipid quinone required for mitochondrial respiratory electron transport. We have shown that a deficiency in Q content is evident in Pdss2kd/kd mouse kidney lipid extracts by 40 days of age and thus precedes the onset of proteinuria and kidney disease by several weeks. The presence of the kd (V117M) mutation in PDSS2 does not prevent its association with PDSS1. However, heterologous expression of the kd mutant form of PDSS2 together with PDSS1 in Escherichia coli recapitulates the Q deficiency observed in the Pdss2kd/kd mouse. Dietary supplementation with Q10 provides a dramatic rescue of both proteinuria and interstitial nephritis in the Pdss2kd/kd mutant mice. The results presented suggest that Q may be acting as a potent lipid-soluble antioxidant, rather than by boosting kidney mitochondrial respiration. Such Q10 supplementation may have profound and beneficial effects in treatment of certain forms of focal segmental glomerulosclerosis that mirror the renal disease of the Pdss2kd/kd mouse.

Heteromer formation of a long-chain prenyl diphosphate synthase from fission yeast Dps1 and budding yeast Coq1.

Ubiquinone is an essential factor for the electron transfer system and is also a known lipid antioxidant. The length of the ubiquinone isoprenoid side-chain differs amongst living organisms, with six isoprene units in the budding yeast Saccharomyces cerevisiae, eight units in Escherichia coli and 10 units in the fission yeast Schizosaccharomyces pombe and in humans. The length of the ubiquinone isoprenoid is determined by the product generated by polyprenyl diphosphate synthases (poly-PDSs), which are classified into homodimer (i.e. octa-PDS IspB in E. coli) and heterotetramer [i.e. deca-PDSs Dps1 and D-less polyprenyl diphosphate synthase (Dlp1) in Sc. pombe and in humans] types. In this study, we characterized the hexa-PDS (Coq1) of S. cerevisiae to identify whether this enzyme was a homodimer (as in bacteria) or a heteromer (as in fission yeast). When COQ1 was expressed in an E. coli ispB disruptant, only hexa-PDS activity and ubiquinone-6 were detected, indicating that the expression of Coq1 alone results in bacterial enzyme-like functionality. However, when expressed in fission yeast Deltadps1 and Deltadlp1 strains, COQ1 restored growth on minimal medium in the Deltadlp1 but not Deltadps1 strain. Intriguingly, ubiquinone-9 and ubiquinone-10, but not ubiquinone-6, were identified and deca-PDS activity was detected in the COQ1-expressing Deltadlp1 strain. No enzymatic activity or ubiquinone was detected in the COQ1-expressing Deltadps1 strain. These results indicate that Coq1 partners with Dps1, but not with Dlp1, to be functional in fission yeast. Binding of Coq1 and Dps1 was demonstrated by coimmunoprecipitation, and the formation of a tetramer consisting of Coq1 and Dps1 was detected in Sc. pombe. Thus, Coq1 is functional when expressed alone in E. coli and in budding yeast, but is only functional as a partner with Dps1 in fission yeast. This unusual observation indicates that different folding processes or protein modifications in budding yeast/E. coli versus those in fission yeast might affect the formation of an active enzyme. These results provide important insights into the process of how PDSs have evolved from homo- to hetero-types.

Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.

Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production. A study from the abc1Sp (coq8Sp) mutant.

We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.