Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome.

Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA) in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB). To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model.

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

Resveratrol and para-coumarate serve as ring precursors for coenzyme Q biosynthesis.

Coenzyme Q (Q or ubiquinone) is a redox-active polyisoprenylated benzoquinone lipid essential for electron and proton transport in the mitochondrial respiratory chain. The aromatic ring 4-hydroxybenzoic acid (4HB) is commonly depicted as the sole aromatic ring precursor in Q biosynthesis despite the recent finding that para-aminobenzoic acid (pABA) also serves as a ring precursor in Saccharomyces cerevisiae Q biosynthesis. In this study, we employed aromatic (13)C6-ring-labeled compounds including (13)C6-4HB, (13)C6-pABA, (13)C6-resveratrol, and (13)C6-coumarate to investigate the role of these small molecules as aromatic ring precursors in Q biosynthesis in Escherichia coli, S. cerevisiae, and human and mouse cells. In contrast to S. cerevisiae, neither E. coli nor the mammalian cells tested were able to form (13)C6-Q when cultured in the presence of (13)C6-pABA. However, E. coli cells treated with (13)C6-pABA generated (13)C6-ring-labeled forms of 3-octaprenyl-4-aminobenzoic acid, 2-octaprenyl-aniline, and 3-octaprenyl-2-aminophenol, suggesting UbiA, UbiD, UbiX, and UbiI are capable of using pABA or pABA-derived intermediates as substrates. E. coli, S. cerevisiae, and human and mouse cells cultured in the presence of (13)C6-resveratrol or (13)C6-coumarate were able to synthesize (13)C6-Q. Future evaluation of the physiological and pharmacological responses to dietary polyphenols should consider their metabolism to Q.

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.