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

Abstract

The final step of the CDP-ethanolamine pathway is catalyzed by ethanolamine phosphotransferase 1 (EPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). These enzymes are likely involved in the transfer of ethanolamine phosphate from CDP-ethanolamine to lipid acceptors such as 1,2-diacylglycerol (DAG) for phosphatidylethanolamine (PE) production, and 1-alkyl-2-acylglycerol (AAG) for the generation of 1-alkyl-2-acyl-glycerophosphoethanolamine (plasmanyl-PE). Here, we investigated the intracellular location and contribution to ethanolamine 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 ethanolamine 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.

Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation

“…PE synthesis is dependent to a lesser extent, mainly during T activation. T cells appear to be able to compensate for the loss of SELENOI to maintain levels of total PE (unlabeled and labeled), similar to recent studies using HeLa and HEK293 cell lines [ 34 , 35 ].…”

“…SELENOI is a versatile enzyme participating in two phospholipid synthesis pathways: it may transfer the ethanolamine headgroup from CDP-ethanolamine to either DAG or AAG to generate PE or plasmenyl PE, respectively. Although SELENOI participates in the biosynthesis of both, data from HeLa and HEK293 cell lines suggested that, in the absence of SELENOI, mainly plasmenyl PE levels are reduced since cells can utilize choline/ethanolamine phosphotransferase 1 (CEPT1) to maintain PE synthesis [ 34 , 35 ]. Our results suggest that the same is true for T cells as only plasmenyl PE in the labeled and unlabeled forms were lower in the SELENOI KO T cells.…”

Genetic diseases of the Kennedy pathways for membrane synthesis

“…Loss of function of EPT1 may be able to be compensated for, in part, by the presence of CEPT1, although this remains to be established. To this end, knockdown of EPT1 or CEPT1 in HEK293 cells resulted in viable cells [49], with metabolic labeling analyses revealing that EPT1 preferentially synthesized plasmenyl-PE and PE species with long chain unsaturated fatty acids, whereas CEPT1 preferentially synthesized diacyl-PE with shorter and more saturated fatty acids [49]. Similarly, analysis of cells derived from EPT1 HSP patients determined that the total level of PE was similar to controls, however, PE species with polyunsaturated fatty acids were reduced [47,48].…”

A highly efficient gene disruption strategy reveals lipid co-regulatory networks

“…Although we do not know the precise mechanism of this additional layer of co-regulation, the increase in C16 SLs or the decrease in C24 SLs might affect the activity of enzymes adding head groups on ether DAG, either by reducing the transfer of phosphocholine or increasing the transfer of phosphoethanolamine ( Figure 6E). Interestingly, an enzyme catalyzing the transfer of the phosphoethanolamine head group, ethanolamine phosphotransferase 1 (EPT1, encoded by SELENOI gene), is localized to the Golgi apparatus [33], where the biosynthesis of complex SLs takes place. This enzyme is more important for ether PE synthesis than for PE synthesis, and has a preference for substrates containing PUFAs [34].…”