Coq3 and Coq4 Define a Polypeptide Complex in Yeast Mitochondria for the Biosynthesis of Coenzyme Q

Abstract

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 Qbiosynthetic 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 comigrate 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 digitoninsolubilized mitochondrial extracts shows their physical association. This study identifies Coq3p and Coq4p as defining members of a Q-biosynthetic Coq polypeptide complex. Coenzyme Q (ubiquinone or Q) 1 is a redox active lipid containing a long polyprenyl tail attached to a fully substituted benzoquinone ring. The number (n) of isoprene units in the polyprenyl tail (Q n ) is distinct in different organisms; humans produce Q 10 , Caenorhabditis elegans Q 9 , Escherichia coli Q 8 , and Saccharomyces cerevisiae Q 6 . Within the inner mitochondrial membrane Q functions in respiratory electron transport, where it transfers two electrons from either complex I or complex II to complex III (1). Q is present in almost all organisms, with the quantity roughly matching the respiratory capability of the tissue from which it is isolated (2). In addition to this role, Q has been found to act as a chain-breaking antioxidant (3) and to be involved in the electron transport chains of plasma and lysosomal membranes (4, 5). Q supplementation in humans slows the functional decline in patients with Parkinson disease (6), is useful for treating patients with respiratory chain defects (7), and has promise as a treatment in other neurodegenerative diseases (8).As observed with other redox active compounds, Q has the potential to act as a source of oxidative stress. In certain species, the amount of reactive oxygen species generated by mitochondria has been related to Q content (9, 10). These described dual roles as antioxidant and prooxidant associate Q with both extended and shortened life spans. C. elegans fed E. coli diets lacking Q 8 , and C. elegans clk-1 mutants with defects in Q biosynthesis show increased life spans, and we have proposed that the decreased levels of Q are associated with a decreased level of the Q semiquinone radical (Q . ) and a decreased propensity to...

Coenzyme Q supplementation or Over-Expression of the Yeast Coq8 Putative Kinase Stabilizes Multi-Subunit Coq Polypeptide Complexes in Yeast Coq Null Mutants

“…It was shown that DMQ 6 co-elutes with Coq3 O-methyltransferase activity and high molecular mass Coq polypeptide complexes during size exclusion chromatography of digitonin extracts of mitochondria [18]. Yeast coq7 null mutants cultured in the presence of exogenous Q 6 were able to synthesize DMQ 6 , and steady-state levels of Coq4 polypeptides were restored, indicating that the presence of Q 6 itself may stabilize the Coq polypeptide complexes [23,34].…”

“…The yeast Coq4 and Coq9 polypeptides co-purify with other Coq polypeptides and both migrate at high molecular mass in separation of digitonin extracts of mitochondria [18][19][20][21]. Thus, we used the Coq4 and Coq9 polypeptides as sensitive indicators of the state of the high molecular mass Coq complexes.…”

“…For example, certain coq7 point mutants retain steady-state levels of the Coq7 polypeptide and accumulate demethoxy-Q 6 (DMQ 6 ), a late-stage Q-intermediate missing just one methoxy group [22,23]. Some of the Coq polypeptides physically interact -biotinylated Coq3 co-purifies with Coq4 [18], and Coq9 tagged with the hemagglutinin epitope co-purifies with Coq4, Coq5, Coq6, and Coq7 polypeptides [20]. These studies were performed with digitonin-extracts of purified mitochondria.…”

Genetic Evidence for an Interaction of the UbiG O -Methyltransferase with UbiX in Escherichia coli Coenzyme Q Biosynthesis

“…Gel filtration chromatography shows that Coq3p, Coq4p, Coq6p, and Coq7p coelute as a high-molecular-weight complex (22,28). Coq3p, Coq4p, and Coq7 comigrate as highmolecular-mass complexes as assessed by two-dimensional blue native analysis (22,28).…”

“…E. coli strains harbored plasmids expressing the designated genes as follows: pAHG, UbiG K-12 ; pUbiG THU , UbiG THU ; pHZ1, UbiX THU ; and pPZ2, UbiX K-12 . L-methionine as previously described (22,24). O-methyltransferase activity assays were linearly dependent on time of incubation, substrate concentration, and amount of permeabilized cells.…”