A sensitive method was developed for measuring farnesyl diphosphate (FPP) accumulation in a mutant strain of Saccharomyces cerevisiae. The strain was blocked at squalene synthase (ERG9 gene) in the isoprenoid pathway and had the catalytic domain of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene integrated into the chromosome. It required ergosterol for growth and produced E,E-farnesol. The method was based on the isolation of FPP using the anion exchanger Macro Prep High Q and conversion of FPP to E,E-farnesol with alkaline phosphatase. Farnesol was measured using gas chromatography-mass spectrometry. Background farnesol in the cell-free extract was also retained by the anion exchanger, but was removed with repeated washing with methanol. Both 1M NaCl and 40% (v/v) methanol were required in the elution buffer to effectively elute FPP. The preparation of cell-free extract in Bis-Tris propane/HCl, pH 7, buffer containing 0.025% (w/v) Triton X-100 and 15mM MgCl2 provided optimum conditions for the stabilization of FPP. © 2003 Elsevier Science (USA). All rights reserved.
CITATION STYLE
Song, L. (2003). Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants. Analytical Biochemistry, 317(2), 180–185. https://doi.org/10.1016/S0003-2697(03)00138-6
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