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Differential contributions of choline phosphotransferases CPT1 and CEPT1 to the biosynthesis of choline phospholipids.

ABSTRACT: Choline phospholipids (PLs) such as phosphatidylcholine (PC) and 1-alkyl-2-acyl-sn-glycerophosphocholine are important components for cell membranes and also serve as a source of several lipid mediators. These lipids are biosynthesized in mammals in the final step of the CDP-choline pathway by the choline phosphotransferases choline phosphotransferase 1 (CPT1) and choline/ethanolamine phosphotransferase 1 (CEPT1). However, the contributions of these enzymes to the de novo biosynthesis of lipids remain unknown. Here, we established and characterized CPT1- and CEPT1-deficient human embryonic kidney 293 cells. Immunohistochemical analyses revealed that CPT1 localizes to the trans-Golgi network and CEPT1 to the endoplasmic reticulum. Enzyme assays and metabolic labeling with radiolabeled choline demonstrated that loss of CEPT1 dramatically decreases choline PL biosynthesis. Quantitative PCR and reintroduction of CPT1 and CEPT1 revealed that the specific activity of CEPT1 was much higher than that of CPT1. LC-MS/MS analysis of newly synthesized lipid molecular species from deuterium-labeled choline also showed that these enzymes have similar preference for the synthesis of PC molecular species, but that CPT1 had higher preference for 1-alkyl-2-acyl-sn-glycerophosphocholine with PUFA than did CEPT1. The endogenous level of PC was not reduced by the loss of these enzymes. However, several 1-alkyl-2-acyl-sn-glycerophosphocholine molecular species were reduced in CPT1-deficient cells and increased in CEPT1-deficient cells when cultured in 0.1% FBS medium. These results suggest that CEPT1 accounts for most choline PL biosynthesis activity, and that both enzymes are responsible for the production of different lipid molecular species in distinct organelles.

SUBMITTER: Horibata Y

PROVIDER: S-EPMC8387743 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The Saccharomyces cerevisiae CPT1 and EPT1 genes encode for a cholinephosphotransferase (CPT) and choline/ethanolaminephosphotransferase, respectively. Both Cpt1p and Ept1p activities display an absolute requirement for cations and phospholipids. A mixed-micelle assay was employed to determine cation and lipid activators of parental and chimaeric Cpt1p/Ept1p enzymes to gain insight into their mechanism(s) of activation. Mg2+, Mn2+ and Co2+ were the only cations capable of activating Cpt1p and Ept1p in vitro. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. Kinetic data revealed that only Mg2+ is present in appropriate amounts to activate CPT activity in vivo. The two enzymes displayed distinct activation profiles on the basis of their relative affinities for Mg2+, and Mn2+ and Co2+. This allowed the use of chimaeric enzymes to determine the mechanism of cation activation. Cations do not activate Cpt1p or Ept1p by complexing with the substrate, CDP-choline, but instead bind to disparate regions within the enzymes themselves. Cpt1p and Ept1p also displayed distinct phospholipid activation profiles. Phospholipid activation required a phosphate and/or glycero-phosphoester linkage, with the phospho-head group moiety positioned at the surface of the micelle. Assays with parental and chimaeric Cpt1p/Ept1p constructs revealed that the phospholipid binding/activation domains are not located within linear segments of the protein, but instead are contained within distinct and separate regions of the proteins that require an intact tertiary structure for formation. Phosphatidylcholine (and its structural analogue sphingomyelin) were the best lipid activators of Cpt1p, the main biologically relevant CPT activity in S. cerevisiae. Hence CPT displays product activation. Because phosphatidylcholine is an efficient activator of CPT activity (and hence Cpt1p is not subject to feedback inhibition by its product), Cpt1p is incapable of functioning as a direct monitor of membrane phosphatidylcholine composition.

Project description:Background:Phospholipids are important structural and signaling molecules in plant membranes. Some fluorescent dyes can stain general lipids of membranes, but labeling and visualization of specific lipid classes have yet to be developed for most components of the membrane. New techniques for visualizing membrane lipids are needed to further delineate their dynamic structural and signaling roles in plant cells. In this study we examined whether propargylcholine, a bioortholog of choline, can be used to label the major membrane lipid, phosphatidylcholine, and other choline phospholipids in plants. We established that propargylcholine is readily taken up by roots, and that its incorporation is not detrimental to plant growth. After plant tissue is harvested and fixed, a click-chemistry reaction covalently links the alkyne group of propargylcholine to a fluorescently-tagged azide, resulting in specific labeling of choline phospholipids. Results:Uptake of propargylcholine, followed by click chemistry with fluorescein or Alexa Fluor 594 azide was used to visualize choline phospholipids in cells of root, leaf, stem, silique and seed tissues from Arabidopsis thaliana. Co-localization with various subcellular markers indicated coinciding fluorescent signals in cell membranes, such as the tonoplast and the ER. Among different cell types in the leaf epidermis, guard cells displayed strong labeling. Mass spectrometry-based lipidomic analysis of the various plant tissues revealed that incorporation of propargylcholine was strongest in roots with approximately 50% of total choline phospholipids being labeled, but it was also incorporated in the other tissues including seeds. Phospholipid profiling confirmed that, in each tissue analyzed, incorporation of the bioortholog had little impact on the pool of choline plus choline-like phospholipids or other lipid species. Conclusion:We developed and validated a click-chemistry based method for fluorescence imaging of choline phospholipids using a bioortholog of choline, propargylcholine, in various cell-types and tissues from Arabidopsis. This click-chemistry method provides a direct way to metabolically tag and visualize specific lipid molecules in plant cells. This work paves the way for future studies addressing in situ localization of specific lipids in plants.

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Differential contributions of choline phosphotransferases CPT1 and CEPT1 to the biosynthesis of choline phospholipids.

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