Detection of farnesyl diphosphate accumulation in yeast ERG9 mutants.
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 15 mM MgCl(2) provided optimum conditions for the stabilization of FPP.
Song
Linsheng
L
Bio-Technical Resources, 1035 South 7th Street, Manitowoc, WI 54220, USA. song@biotechresources.com
D000469:Alkaline Phosphatase / Q000378:metabolism; D005204:Farnesol / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006863:Hydrogen-Ion Concentration; D009154:Mutation; D011106:Polyisoprenyl Phosphates / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes; D013045:Species Specificity
D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D005204:Farnesol; C004808:farnesyl pyrophosphate; D013186:Farnesyl-Diphosphate Farnesyltransferase; D000469:Alkaline Phosphatase