Fig. 1. Measurement of PA levels in yeast subcellular fractions by the fluorometric coupled enzyme assay.

Wild type (WT) or pah1Δ mutant (5) cells were grown to the stationary phase in 250 ml YEPD (1% yeast extract, 2% peptone, and 2% glucose) medium. The cells (~ 4 g wet weight) of each culture were harvested by centrifugation, treated with lyticase, and the resulting spheroplasts were lysed using a Dounce homogenizer. 90% of the cell lysate was fractionated by differential centrifugation (16), and the lipids were extracted (19) from the lysate and subcellular fractions. The lipid extracts were solubilized in 0.5 ml of 1% Triton X-100 (Surfact-Amps, < 1.0 μeq/ml peroxides, Thermo Scientific), and 20 μl of the samples were treated with 2,400 units (μmol/min) Pseudomonas sp. lipoprotein lipase (Wako). The lipase was inactivated by boiling for 10 min and the denatured protein was removed by centrifugation. Glycerol-3-phosphate derived from PA was oxidized by 0.5 unit (μmol/min) Aerococcus viridans glycerol-3-phosphate oxidase (Sigma-Aldrich) to produce hydrogen peroxide, which was then used for the conversion of Amplex Red (10-Acetyl-3,7-dihydroxyphenoxazine, Thermo Scientific) to resorufin by 0.5 unit (μmol/min) horseradish peroxidase (Sigma-Aldrich) (7). The last two steps in the coupled enzyme reaction were carried out for 30 min at room temperature in a black 96-well plate, and the resulting fluorescence was immediately measured by Agilent Technologies Cary Eclipse Fluorescence Spectrometer. The Amplex Red stop solution (7), which is ineffective in stopping the peroxidase reaction under the conditions of the assay, was not used in this work. A standard curve with dioleoyl PA (Avanti Polar Lipids) (200 to 1,000 pmol, linear range) was used to quantify the phospholipid in the extracted lipids. The data are averages ± S.D. (error bars) from triplicate determinations. *, p < 0.01.