Phospholipid and cation activation of chimaeric choline/ethanolamine phosphotransferases

Abstract

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.

Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

“…This same cation preference was also observed for a partially purified choline\ethanolamine-phosphotransferase activity from rat liver [33]. Two mechanisms have been proposed by which cations activate cholinephosphotransferases and ethanolaminephosphotransferases : either (1) cations bind to the CDP-aminoalcohol substrate or (2) there exists a defined cation-binding site within the enzyme [4,17]. Resolution of the mechanism of cation activation and also of the cation used in i o await protein purification.…”

“…The inhibitor was purified and identified as argininosuccinate. In addition, residues 252-277 of hCEPT1p align with residues 89-114 of the platelet-activating factor receptor [11][12][13] ; the S. cere isiae Cpt1p and Ept1p enzymes require activation by their products, PtdCho and PtdCho\PtdEtn respectively, in amounts indicative of a precise phospholipid-binding site within the enzymes [16,17]. Therefore this region might be capable of binding phospholipids.…”

“…The yeast CPT1 gene product encodes a cholinephosphotransferase (Cpt1p) [6,14,15] specific for the synthesis of PtdCho in itro and in i o, whereas the S. cere isiae gene EPT1 product codes for a dual-specificity choline\ethanolaminephosphotransferase (Ept1p) capable of synthesizing PtdCho and PtdEtn in itro but that synthesizes primarily PtdEtn in i o [6,16]. Analysis of chimaeric CPT1\EPT1 enzymes [6,16,17] mapped the active-site Abbreviations used : AAPT1p, plant CDP-aminoalcoholphosphotransferase protein ; CPT1, Saccharomyces cerevisiae cholinephosphotransferase gene ; Cpt1p, S. cerevisiae cholinephosphotransferase protein ; EPT1, S. cerevisiae choline/ethanolaminephosphotransferase gene ; Ept1p, S. cerevisiae choline/ethanolaminephosphotransferase protein ; EST, expressed sequence tag ; hCEPT1, human choline/ethanolaminephosphotransferase cDNA ; hCEPT1p, human choline/ethanolaminephosphotransferase protein ; PtdCho, phosphatidylcholine ; PtdEtn, phosphatidylethanolamine. 1 To whom correspondence should be addressed (e-mail cmcmaste!is.dal.ca).…”

Biochemistry, cell biology and molecular biology of lipids ofSaccharomyces cerevisiae

“…Finally, PtdEtn is formed in the endoplasmic reticulum by CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase (Ept1p). 165 Although Ept1p can utilize CDPethanolamine, CDP-monomethylethanolamine, CDP-dimethylethanolamine, and CDP-choline as substrates in vitro 97,167 the almost exclusive substrate in vivo is CDP-ethanolamine. 161,163 Similar to the utilization of free ethanolamine, yeast cells can incorporate choline into PtdCho through the Kennedy pathway (see Figure 1).…”

Regulation of Activities of Cytidine 5'-Diphospho-Choline: 1-O-Alkyl-2-Acetyl-sn-Glycerol Cholinephosphotransferase, an Enzyme Responsible for de novo Synthesis of Platelet-Activating Factor, by Membrane Phospholipids.

“…Although the effect of PA on mammalian DG-CPT has not been described, yeast DG-CPT, which shares a homologous structure with that of mammalian DG-CPT, 27) is reported to be activated by PA with other phospholipids including lysophospholipids. 28) These different effects of phospholipids on both enzymes might provide additional evidence that AAG-CPT is a separate enzyme from DG-CPT, although we must await the isolation of the gene for AAG-CPT to understand the relationship of the two enzymes.…”