Locations and contributions of the phosphotransferases EPT1 and CEPT1 to the biosynthesis of ethanolamine phospholipids

Abstract

The final step of the CDP-ethanolamme pathway is catalyzed by ethanolamme phosphotransferase 1 (EPT1) and cholme/ethanolamme phosphotransferase 1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamme phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for phosphatidylethanolamine (PE) production, and l-alkyl-2-acylglycerol (AAG) for the generation of l-alkyl-2-acyl-glycerophosphoethanolamine (plasmanyl-PE). Here, we investigated the intracellular location and contribution to ethanolamme phospholipid biosynthesis of EPT1 and CEPT1 in HEK293 cells. Immunohistochemical analyses revealed that EPT1 localizes to the Golgi apparatus and CEPT1 to the endoplasmic reticulum. We created EPT1-, CEPT1-, and EPTI-CEPT1-deficient cells, and labeling of these cells with radio- or deuterium-labeled ethanolamme disclosed that EPT1 is more important for the de novo biosynthesis of plasmenyl-PE than is CEPT1. EPT1 also contributed to the synthesis of PE species containing the fatty acids 36:1, 36:4, 38:5, 38:4, 38:3, 40:6, 40:5, and 40:4. In contrast, CEPT1 was important for PE formation from shorter fatty acids such as 32:2, 32:1, 34:2, and 34:1. Brefeldin A treatment did no significantly affect the levels of the different PE species, indicating that the subcellular localization of the two enzymes is not responsible for their substrate preferences. In vitro enzymatic analysis revealed that EPT1 prefers AAG 16-20:4 > DAG 18:0-20:4 > DAG 16:0-18:1 = AAG 16-18:1 as lipid acceptors and that CEPT1 greatly prefers DAG 16:0-18:1 to other acceptors. These results suggest that EPT1 and CEPT1 differ in organelle location and are responsible for the biosynthesis of distinct EP species.