3-HEXAPRENYL-4-HYDROXYBENZOIC ACID FORMS A PREDOMINANT INTERMEDIATE POOL IN UBIQUINONE BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE

The biosynthesis of ubiquinone (coenzyme Q) was studied in Saccharomyc es cerevisiae, Lipid extracts were prepared from wild-type yeast grown in the presence of p-[U-C-14]- and p-[carboxy-C-14] hydroxybenzoic ac id. Ergosterol was removed by adsorption to digitonin-celite, and radi olabeled lipids were purified by sequential reverse-phase and normal-p hase HPLC steps. Radiolabeled peaks were identified by comparison with synthetic standards using retention time and electron ionization mass spectrometric criteria. The recovery and identification of the unstab le 5-hexaprenyl-4-hydroxybenzoic acid molecule were facilitated by tre atment of the lipid extract with diazomethane under conditions that re sulted in the formation of the stable derivatives methyl 3-hexaprenyl- 4-hydroxybenzoate or methyl 3-hexaprenyl-4-methoxybenzoate. In station ary-phase yeast cultures, the major radioactive lipid products are coe nzyme Q and 3-hexaprenyl-4-hydroxybenzoic acid, constituting 62 and 38 % of the radioactive lipids, respectively. However, under log-phase gr owth conditions the biosynthetic intermediate 3-hexaprenyl-4-hydroxybe nzoic acid predominates (accounting for 81% of the radioactive lipids) , The data indicate that in wild-type yeast, 3-hexaprenyl-4-hydroxyben zoic acid forms a predominant intermediate pool in ubiquinone biosynth esis and that in log-phase growth this ubiquinone intermediate is pres ent at fourfold higher abundance than the end product, The physiologic al rationale for this high concentration of a membrane-bound intermedi ate is unclear. (C) 1995 Academic Press, Inc.