Characterization of the DDL1 gene. A, phospholipase assay and measurement of the released FFA. The assays were conducted using the purified recombinant Ddl1 protein. The released FFA, estimated using a fluorescent FFA estimation kit, are represented as μmol of FFA released/min/mg of protein. B, phospholipase assay using PE and PG with different fatty acid compositions. The assay was conducted for 30 min at 30 °C in the presence of 2 μg of the purified recombinant Ddl1 and 100 μm substrates. The released FFA were scraped from the TLC plates, and the samples were prepared and subjected to gas chromatography analysis: DOPE, 18:1–18:1 PE; SOPE, 18:0–18:1 PE; POPE, 16:0–18:1 PE; DOPG, 18:1–18:1 PG; SOPG, 18:0–18:1 PG; POPE, 16:0–18:1 PG. C, site-directed mutagenesis of the lipase motif and phospholipase activity. Upper insert, the recombinant Ddl1 mutant proteins were purified from yeast similar to the wild-type Ddl1 protein, and an immunoblot analysis was performed. Lower insert, 1 μg of the purified wild-type and mutant Ddl1 proteins was used for the phospholipase assay at 30 °C for 30 min. A portion of the representative TLC plate is shown. D, effect of the DDHD domain on the phospholipase activity of Ddl1. The purified recombinant truncated Ddl1(tDdl1) protein (left panel) and an immunoblot of the tDdl1 (middle panel) are shown. Right panel, 1 μg of the purified wild-type and tDdl1 proteins was used for the phospholipase assay at 30 °C for 30 min. The reaction was stopped, and the lipids were resolved on a TLC plate and quantified with GeneTools software. M, protein marker; −E, without enzyme; WT, pYES2/NT B-DDL1. The values are presented as the mean ± S.E. (n = 3), and significance was determined at **, p < 0.01.