Antibodies were raised against the two membrane-bound lysophosphatidic acid acyltransferase (LPAAT) enzymes from Limnanthes douglasii (meadowfoam), LAT1 and LAT2, using the predicted soluble portion of each protein as recombinant protein antigens. The antibodies can distinguish between the two acyltransferase proteins and demonstrate that both migrate in an anomalous fashion on SDS/PAGE gels. The antibodies were used to determine that LAT1 is present in both leaf and developing seeds, whereas LAT2 is only detectable in developing seeds later than 22daf (days after flowering). Both proteins were found exclusively in microsomal fractions and their amount was determined using the recombinant antigens as quantification standards. LAT1 is present at a level of 27pg/μg of membrane protein in leaf tissue and ≤ 12.5pg/μg of membrane protein in developing embryos. The amount of LAT2 reaches a peak at 305pg/μg of membrane protein 25daf and is not expressed 20daf or before. This is the first study to quantify these membrane-bound proteins in a plant tissue. The maximal level of LAT2 protein coincides with the maximal level of erucic acid synthesis in the seeds. Both full-length proteins were expressed in the Escherichia coli LPAAT mutant JC201, and membranes from these strains were used to investigate the substrate selectivity of these two enzymes, demonstrating that they are different. Finally, we report that LAT2 and a maize LPAAT enzyme (MAT1) can functionally replace the E. coli plsC gene after its deletion in the chromosome, whereas LAT1 and a coconut LPAAT (Coco1) cannot. This is probably due to differences in substrate utilization.
Abbreviations used: daf, days after flowering; DTT, dithiothreitol; GPAT, glycerol 3-phosphate acyltransferase; IPTG, isopropyl β-d-thiogalactoside; ORF, open reading frame; LPAAT, lysophosphatidic acid acyltransferase; MALDI—TOF, matrix-assisted laser-desorption ionization—time-of-flight; PA, phosphatidic acid; Pefabloc, 4-(2-aminoethyl)benzenesulphonylfluoride hydrochloride; TAG, triacylglycerol; TBS, Tris-buffered saline.