Complementation of coenzyme Q‐deficient yeast by coenzyme Q analogues requires the isoprenoid side chain

Abstract

The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial respiration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ (ΔCoQ) are viable, but cannot grow on nonfermentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a ΔCoQ strain on the nonfermentable substrate glycerol [James AM et al. (2005) J Biol Chem280, 21295–21312]. In the present study we investigated the structural requirements of exogenously supplied CoQ2 for growth on glycerol and found that the first double bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ2 analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ6) is extremely hydrophobic and transported to mitochondria via the endocytic pathway when supplied exogenously. We found that CoQ2 does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of ΔCoQ yeast in the presence of exogenous CoQ2. Using a ΔCoQ double‐knockout library we identified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a signal for growth during the shift to respiration.

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

“…This effect of supplementation with exogenous Q 6 is known to require uptake; Q 6 binds to soluble proteins derived from peptone in the growth medium and is taken up by cells and transported to mitochondria via an endocytic pathway [84]. James et al, [85] identified 16 yeast ORFs required for utilization of exogenous Q 4 in a yeast double knockout library ( ΔORFΔcoq2 ). We determined the steady-state levels of the Coq4, Coq7, and Coq9 polypeptides as indicators of the CoQ-synthome, and scanned for Q 6 -intermediates by HPLC tandem mass spectrometry.…”

Genes and lipids that impact uptake and assimilation of exogenous coenzyme Q in Saccharomyces cerevisiae

“…The methodology used was a combination of previously described methods [39][40][41]. Briefly, LUVETs (large unilamellar vesicles produced by extrusion) were created from 25 mg/mL L-α-phosphatidylcholine (PC) from soybean (Type III-S, Millipore-Sigma) in chloroform, supplemented with 1-pyrene dodecanoic acid (Pyr12, Santa Cruz Biotechnology) to achieve a final concentration of 4 µM.…”

“…After 90 s, 0.5 mL of a CoQcontaining vesicle population was added and the fluorescence was monitored for a total of 10 min. CoQ collisionally quenches Pyr12 fluorescence if both are present in the same vesicle and are capable of physical interaction [39,42]. On mixing the two populations of vesicles, a further decrease in the fluorescence signal can only occur if the CoQn isoform is able to move from vesicle to vesicle.…”

“…On mixing the two populations of vesicles, a further decrease in the fluorescence signal can only occur if the CoQn isoform is able to move from vesicle to vesicle. This is because the gain in fluorescence in the CoQn vesicle population from CoQ loss is less than the loss in fluorescence in the previously CoQn-less population from the gain of CoQ (for a discussion see [39]). Data are expressed as the ratio of fluorescence at a given point in time (I) to initial fluorescence just before the addition of the second population of vesicles (I0).…”

“…To select for mutants that contain a kanMX cassette, YPD + 200 mg/L G418 (Santa Cruz Biotechnology, Inc) plates were used. To select for ORFΔcoq2Δ mutants obtained from a previously created library [39], synthetic dextrose media without arginine and histidine (SD -His -Arg) (20 g/L glucose, 1.7 g/L yeast nitrogen base without amino acids and ammonium sulfate, 1 g/L monosodium glutamic acid, and 2 g/L of amino acid supplement lacking histidine and arginine) plates were used. These SD -His -Arg plates were supplemented with 200 mg/L G418, 70 mg/L canavanine (Millipore-Sigma) and 100 mg/L nourseothricin sulfate (ClonNat) (Gold Biotechnology).…”