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.

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.

Endogenous synthesis of coenzyme Q in eukaryotes.

Coenzyme Q (Q) functions in the mitochondrial respiratory chain and serves as a lipophilic antioxidant. There is increasing interest in the use of Q as a nutritional supplement. Although, the physiological significance of Q is extensively investigated in eukaryotes, ranging from yeast to human, the eukaryotic Q biosynthesis pathway is best characterized in the budding yeast Saccharomyces cerevisiae. At least ten genes (COQ1-COQ10) have been shown to be required for Q biosynthesis and function in respiration. This review highlights recent knowledge about the endogenous synthesis of Q in eukaryotes, with emphasis on S. cerevisiae as a model system.

Saccharomyces cerevisiae Coq9 polypeptide is a subunit of the mitochondrial coenzyme Q biosynthetic complex.

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 Q-biosynthetic 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.

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.