Ubiquinone accumulates in the mitochondria of yeast mutated in the ubiquinone binding protein, Qcr8p.

In Saccharomyces cerevisiae, the trans-membrane helix of Qcr8p, the ubiquinone binding protein of complex III, contributes to the Q binding site. In wild-type cells, residue 62 of the helix is non-polar (proline). Substitution of proline 62 with a polar, uncharged residue does not impair the ability of the cells to respire, complex III assembly is unaffected, ubiquinone occupancy of the Q binding site is unchanged, and mitochondrial ubiquinone levels are in the wild-type range. Substitution with a +1 charged residue is associated with partial respiratory competence, impaired complex III assembly, and loss of cytochrome b. Although ubiquinone occupancy of the Q binding site is similar to wild-type, total mitochondrial ubiquinone doubled in these mutants. Mutants with a +2 charged substitution at position 62 are unable to respire. These results suggest that the accumulation of ubiquinone in the mitochondria may be a compensatory mechanism for impaired electron transport at cytochrome b.

Ubiquinone binding protein used for determination of coenzyme Q.

The conventional method of assaying for the ubiquinone (CoQ) content of biological samples is to partition CoQ into an organic phase and separate it from contaminants by high-performance liquid chromatography (HPLC). HPLC is an accurate method of quantifying CoQ content but is not ideal for routine clinical analyses. This paper describes the development of a rapid method for assaying the CoQ content of biological samples based on the binding of CoQ to a CoQ binding peptide. The 14-amino acid binding peptide was chemically synthesized, and conditions for immobilizing the peptide on microfuge tubes were established. CoQ could be selectively bound to the immobilized peptide, eluted, and determined spectrophotometrically. Limits of detection for the method were 0.25 to 5 nmol CoQ. To test biological samples, CoQ was isolated from cultures of Saccharomyces cerevisiae grown in oleic acid medium. The recovery of CoQ samples using the binding assay ranged from 99 to 102% of the values obtained with HPLC. The assay described here provides an inexpensive, rapid method for determining the CoQ content of large numbers of biological samples in a variety of laboratory settings.

The yeast gene COQ5 is differentially regulated by Mig1p, Rtg3p and Hap2p.

Ubiquinone (CoQ) is an important component of the electron transport chain and serves to regenerate cellular anti-oxidants. In Saccharomyces cerevisiae, expression of the COQ5 gene, encoding for a C-methyltransferase involved in CoQ synthesis, is transcriptionally regulated by carbon source. We have identified three transcription factors involved in this regulation. Mig1p repressed COQ5 expression on dextrose, while Rtg1p/Rtg3p heterodimers up-regulated COQ5 expression on oleic acid. Hap2p modulated the response to oleic acid but did not have an effect in other nonfermentable carbon sources such as glycerol. These results suggest that the regulation of COQ5 gene expression by carbon source is multifactorial and involves the interaction of various transcription factors.

The regulation of COQ5 gene expression by energy source.

The degree of severity of cardiomyopathy is inversely correlated with tissue levels of coenzyme Q (Q), suggesting that Q synthesis may impact the progression of the disease. It has been suggested that Q functions as an endogenously synthesized anti-oxidant, in addition to regenerating the potent anti-oxidants, vitamins E and C. However, very little is known about the mechanisms that regulate Q synthesis. Using the simple eukaryote Saccharomyces cerevisiae as a model, experiments have been designed to investigate the regulation of Q synthesis at the genetic level. To investigate the regulation of COQ5 gene expression by energy source, mRNA content was evaluated in yeast cells treated with dextrose, glycerol or oleic acid. After 1.5 h, more COQ5 mRNA is produced by oleic acid treated cells than by glycerol treated. Experiments performed using COQ5 promoter deletion/reporter constructs demonstrate a specific response to oleic acid. Additional promoter deletion analysis demonstrates that a non-fermentable carbon source element is also present, responding to both glycerol and oleic acid. The specific oleic acid response appears to be regulated by the Rtg family of transcription factors. This family of proteins is required for oleic acid-induced expression of genes of beta-oxidation and peroxisomal proliferation, and plays an important role in co-ordinating mitochondrial/peroxisomal/nuclear communication in response to oleic acid, as well as defects in cellular respiration.