Emerging roles of the oxysterol-binding protein family in metabolism, transport, and signaling.

OSBP (oxysterol-binding protein) and ORPs (OSBP-related proteins) constitute an enigmatic eukaryotic protein family that is united by a signature domain that binds oxysterols, sterols, and possibly other hydrophobic ligands. The human genome contains 12 OSBP/ORP family members genes, while that of the budding yeast Saccharomyces cerevisiae encodes seven OSBP homologues (Osh). Of these, Osh4 (also referred to as Kes1) has been the most widely studied to date. Recently, three-dimensional crystal structures of Osh4 with and without sterols bound within the core of the protein were determined. The core consists of 19 anti-parallel beta-sheets that form a near-complete beta-barrel. Recent work has suggested that Osh proteins facilitate the non-vesicular transport of sterols in vivo and in vitro, while other evidence supports a role for Osh proteins in the regulation of vesicular transport and lipid metabolism. This article will review recent advances in the study of ORP/Osh proteins and will discuss future research issues regarding the ORP/Osh family.

TSAd interacts with Smad2 and Smad3.

Smad-dependent signalling initiated by TGFbeta superfamily members can be modulated by a variety of interacting proteins. Using yeast two-hybrid, co-immunoprecipitation, and GST pull-down assays we identified T-cell SH2 adapter (TSAd) as a protein that interacts with Smad2 and Smad3. TSAd is an adapter protein thought to participate in many different signalling pathways. The objective of this study was to elucidate the domains important for interaction between TSAd and Smad proteins. Our results suggest a model for TSAd-Smad interaction that is facilitated by multiple TSAd domains, but primarily through the TSAd type I SH2 domain. Interestingly, we also found that both Smad2 and Smad3 interact with the Lck type I SH2 domain, but not the PI3K type III SH2 domain. This research raises the possibility that interaction between SH2-containing proteins and Smad proteins may represent another method to modulate Smad-dependent signalling.

Lipidomic analysis of the molecular specificity of a cholinephosphotransferase in situ.

Dynamic lipidomics using ESI-MS (tandem electrospray ionization mass spectrometry) of 9-deuterated choline (choline-d(9)) incorporation into mammalian cell PtdCho (phosphatidylcholine) permits assessment of the molecular specificity of synthesis. Bulk cell PtdCho synthesis occurs in spatially distinct locations, using separate CPTs (1,2 diacylglycerol CDP:choline cholinephosphotransferases). We assessed whether in vitro molecular selectivity of DAG (diacylglycerol) incorporation between CPTs is manifest in situ, by monitoring choline-d(9) incorporation into PtdCho and lyso-PtdCho molecular species over 3 h in control cells and in CHO-K1 cells overexpressing hCEPT1. Compared with controls, the basal molecular species composition of hCEPT1 overexpressors was significantly enriched in arachidonate. This was not due to net accretion of cellular PtdCho arguing against effects of inadequate unsaturated PtdCho degradation or remodelling. Rather, time-course analyses of PtdCho and lyso-PtdCho pools showed that both arachidonate-containing DAG incorporation and turnover of PtdCho is increased in hCEPT1 overexpressors. Increased choline-d(9) incorporation into arachidonyl lyso-PtdCho shows that both phospholipase A(1)- and A(2)-mediated turnover is involved. Spatially distinct molecular specificity of DAG incorporation into cellular PtdCho at the level of hCEPT1 exists in situ.

Uncoupling farnesol-induced apoptosis from its inhibition of phosphatidylcholine synthesis.

Genetic inactivation of the synthesis of phosphatidylcholine, the most abundant membrane lipid in eukaryotic cells, induces apoptosis. Administration of farnesol, a catabolite within the isoprenoid/cholesterol pathway, also induces apoptosis. The mechanism by which farnesol induces apoptosis is currently believed to be by direct competitive inhibition with diacylglycerol for cholinephosphotransferase, the final step in the phosphatidylcholine biosynthetic pathway. Our recent isolation of the first mammalian cholinephosphotransferase cDNA has enabled us to more precisely assess how farnesol affects phosphatidylcholine synthesis and the induction of apoptosis. Induced over-expression of cholinephosphotransferase in Chinese hamster ovary cells prevented the block in phosphatidylcholine biosynthesis associated with exposure to farnesol. However, induced over-expression of cholinephosphotransferase was not sufficient for the prevention of farnesol-induced apoptosis. In addition, exogenous administration of diacylglycerol prevented farnesol-induced apoptosis but did not relieve the farnesol-induced block in phosphatidylcholine synthesis. We also developed an in vitro lipid mixed micelle cholinephosphotransferase enzyme assay, as opposed to the delivery of the diacylglycerol substrate in a detergent emulsion, and demonstrated that there was no direct inhibition of cholinephosphotransferase by farnesol or its phosphorylated metabolites. The execution of apoptosis by farnesol appears to be a separate and distinct event from farnesol-induced inhibition of phosphatidylcholine biosynthesis and instead likely occurs through a diacylglycerol-mediated process that is downstream of phosphatidylcholine synthesis.

Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth.

Phosphatidylcholine and phosphatidylethanolamine are the most abundant phospholipids in eukaryotic cells and thus have major roles in the formation and maintenance of vesicular membranes. In yeast, diacylglycerol accepts a phosphocholine moiety through a CPT1-derived cholinephosphotransferase activity to directly synthesize phosphatidylcholine. EPT1-derived activity can transfer either phosphocholine or phosphoethanolamine to diacylglcyerol in vitro, but is currently believed to primarily synthesize phosphatidylethanolamine in vivo. In this study we report that CPT1- and EPT1-derived cholinephosphotransferase activities can significantly overlap in vivo such that EPT1 can contribute to 60% of net phosphatidylcholine synthesis via the Kennedy pathway. Alterations in the level of diacylglycerol consumption through alterations in phosphatidylcholine synthesis directly correlated with the level of SEC14-dependent invertase secretion and affected cell viability. Administration of synthetic di8:0 diacylglycerol resulted in a partial rescue of cells from SEC14-mediated cell death. The addition of di8:0 diacylglycerol increased di8:0 diacylglycerol levels 20-40-fold over endogenous long-chain diacylglycerol levels. Di8:0 diacylglcyerol did not alter endogenous phospholipid metabolic pathways, nor was it converted to di8:0 phosphatidic acid.

Novel members of the human oxysterol-binding protein family bind phospholipids and regulate vesicle transport.

Oxysterol-binding proteins (OSBPs) are a family of eukaryotic intracellular lipid receptors. Mammalian OSBP1 binds oxygenated derivatives of cholesterol and mediates sterol and phospholipid synthesis through as yet poorly undefined mechanisms. The precise cellular roles for the remaining members of the oxysterol-binding protein family remain to be elucidated. In yeast, a family of OSBPs has been identified based on primary sequence similarity to the ligand binding domain of mammalian OSBP1. Yeast Kes1p, an oxysterol-binding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p pathway for Golgi-derived vesicle transport. Specifically, inactivation of the KES1 gene resulted in the ability of yeast to survive in the absence of Sec14p, a phosphatidylinositol/phosphatidylcholine transfer protein that is normally required for cell viability due to its essential requirement in transporting vesicles from the Golgi. We cloned the two human members of the OSBP family, ORP1 and ORP2, with the highest degree of similarity to yeast Kes1p. We expressed ORP1 and ORP2 in yeast lacking Sec14p and Kes1p function and found that ORP1 complemented Kes1p function with respect to cell growth and Golgi vesicle transport, whereas ORP2 was unable to do so. Phenotypes associated with overexpression of ORP2 in yeast were a dramatic decrease in cell growth and a block in Golgi-derived vesicle transport distinct from that of ORP1. Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but instead bound phospholipids with both proteins exhibiting strong binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate. In Chinese hamster ovary cells, ORP1 localized to a cytosolic location, whereas ORP2 was associated with the Golgi apparatus, consistent with our vesicle transport studies that indicated ORP1 and ORP2 function at different steps in the regulation of vesicle transport.

Lipid metabolism and vesicle trafficking: more than just greasing the transport machinery.

The movement of lipids from their sites of synthesis to ultimate intracellular destinations must be coordinated with lipid metabolic pathways to ensure overall lipid homeostasis is maintained. Thus, lipids would be predicted to play regulatory roles in the movement of vesicles within cells. Recent work has highlighted how specific lipid metabolic events can affect distinct vesicle trafficking steps and has resulted in our first glimpses of how alterations in lipid metabolism participate in the regulation of intracellular vesicles. Specifically, (i) alterations in sphingolipid metabolism affect the ability of SNAREs to fuse membranes, (ii) sterols are required for efficient endocytosis, (iii) glycerophospholipids and phosphorylated phosphatidylinositols regulate Golgi-mediated vesicle transport, (iv) lipid acylation is required for efficient vesicle transport mediated membrane fission, and (v) the addition of glycosylphosphatidylinositol lipid anchors to proteins orders them into distinct domains that result in their preferential sorting from other vesicle destined protein components in the endoplasmic reticulum. This review describes the experimental evidence that demonstrates a role for lipid metabolism in the regulation of specific vesicle transport events.

Regulation of vesicle trafficking, transcription, and meiosis: lessons learned from yeast regarding the disparate biologies of phosphatidylcholine.

Phosphatidylcholine (PtdCho) is the major phospholipid present in eukaryotic cell membranes generally comprising 50% of the phospholipid mass of most cells and their requisite organelles. PtdCho has a major structural role in maintaining cell and organelle integrity, and thus its synthesis must be tightly monitored to ensure appropriate PtdCho levels are present to allow for its coordination with cell growth regulatory mechanisms. One would also expect that there needs to be coordinated regulation of PtdCho synthesis with its transport from its site of synthesis to cellular organelles to ensure organellar structures and functions are maintained. Each of these processes need to be intimately coordinated with cellular growth decision making processes. To this end, it has recently been revealed that ongoing PtdCho synthesis is required for global transcriptional regulation of phospholipid synthesis. PtdCho is also a major component of intracellular transport vesicles and the synthesis of PtdCho is intimately involved in the regulation of vesicle transport from the Golgi apparatus to the cell surface and the vacuole (yeast equivalent of the mammalian lysosome). This review details some of the more recent advances in our knowledge concerning the role of PtdCho in the regulation of global lipid homeostasis through (i) its restriction of the trafficking of intracellular vesicles that distribute lipids and proteins from their sites of synthesis to their ultimate cellular destinations, (ii) its regulation of specific transcriptional processes that coordinate lipid biosynthetic pathways, and (iii) the role of PtdCho catabolism in the regulation of meiosis. Combined, these regulatory roles for PtdCho ensure vesicular, organellar, and cellular membrane biogenesis occur in a coordinated manner.

Preferential externalization of newly synthesized phosphatidylserine in apoptotic U937 cells is dependent on caspase-mediated pathways.

Externalization of phosphatidylserine (PtdSer) is a common feature of programmed cell death and plays an important role in the recognition and removal of apoptotic cells. In this study with U937 cells, PtdSer synthesis from [(3)H]serine was stimulated and newly synthesized PtdSer was transferred preferentially to cell-free medium vesicles (CFMV) from cells when apoptosis was induced with a topoisomerase I inhibitor, camptothecin (CAM). When CAM-induced apoptosis was blocked by a caspase inhibitor, z-VAD-fmk, stimulation of PtdSer synthesis and movement to CFMV were abolished. In contrast, changes in synthesis and transport of sphingomyelin (SM) or phosphatidylethanolamine (PtdEtn) were minor; total phosphatidylcholine (PtdCho) synthesis was below control levels. All phospholipids appeared in CFMV but PtdSer displayed a 6-fold increase relative to controls compared to 3-fold for SM, 2-fold for PtdCho and 1.8-fold for PtdEtn. Even greater effects on specificity of PtdSer synthesis, movement to CFMV and inhibition by z-VAD-fmk were observed in apoptotic cells induced by UV irradiation or tumor necrosis factor-alpha/cycloheximide treatment. Thus, PtdSer biosynthesis stimulated during apoptosis in U937 cells was specific for this phospholipid and was correlated with caspase-mediated exposure of PtdSer at the cell surface and preferential movement to vesicles during apoptosis.

Cloning, genomic organization, and characterization of a human cholinephosphotransferase.

A cholinephosphotransferase activity catalyzes the final step in the de novo synthesis of phosphatidylcholine via the transfer of a phosphocholine moiety from CDP choline to diacylglycerol. Ethanolaminephosphotransferase activity catalyzes a similar reaction substituting CDP ethanolamine as the phosphobase donor. We report the identification and cloning of a human cDNA (human cholinephosphotransferase (hCPT1)) that codes for a cholinephosphotransferase-specific enzyme. This was demonstrated using in vitro enzyme assays and in vivo measurement of the reconstitution of the phosphatidylcholine and phosphatidylethanolamine biosynthetic pathways in yeast cells devoid of their own endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities. This contrasted with our previously cloned human choline/ethanolaminephosphotransferase cDNA that was demonstrated to code for a dual specificity choline/ethanolaminephosphotransferase. The hCPT1 and human choline/ethanolaminephosphotransferase (hCEPT1) predicted amino acid sequences possessed 60% overall identity and had only one variation in the amino acid residues within the CDP-alcohol phosphotransferase catalytic motif. In vitro assessment of hCPT1 and hCEPT1 derived cholinephosphotransferase activities also revealed differences in diradylglycerol specificities including their capacity to synthesize platelet-activating factor and platelet-activating factor precursor. Expression of the hCPT1 mRNA varied greater than 100-fold between tissues and was most abundant in testis followed by colon, small intestine, heart, prostate, and spleen. This was in marked contrast to the hCEPT1 mRNA, which has been found in similar abundance in all tissues tested to date. Both the hCPT1 and hCEPT1 enzymes were able to reconstitute the synthesis of PC in yeast to levels provided by the endogenous yeast cholinephosphotransferase; however, only hCEPT1-derived activity was able to complement the yeast CPT1 gene in its interaction with SEC14 and affect cell growth.

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

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

Scanning alanine mutagenesis of the CDP-alcohol phosphotransferase motif of Saccharomyces cerevisiae cholinephosphotransferase.

Cholinephosphotransferase (EC 2.7.8.2) catalyzes the formation of a phosphoester bond via the transfer of a phosphocholine moiety from CDP-choline to diacylglycerol forming phosphatidylcholine and releasing CMP. A motif, Asp113-Gly114-(X)2-Ala117-Arg118-(X)8-Gly127+ ++-(X)3-Asp131-(X)3-Asp135, located within the CDP-choline binding region of Saccharomyces cerevisiae cholinephosphotransferase (CPT1 ?/Author: Please confirm that a gene is meant here.) is also found in several other phospholipid synthesizing enzymes that catalyze the formation of a phosphoester bond utilizing a CDP-alcohol and a second alcohol as substrates. To determine if this motif is diagnostic of the above reaction type scanning alanine mutagenesis of the conserved residues within S. cerevisiae cholinephosphotransferase was performed. Enzyme activity was assessed in vitro using a mixed micelle enzyme assay and in vivo by determining the ability of the mutant enzymes to restore phosphatidylcholine synthesis from radiolabeled choline in an S. cerevisiae strain devoid of endogenous cholinephosphotransferase activity. Alanine mutants of Gly114, Gly127, Asp131, and Asp135 were inactive; mutants, Ala117 and Arg118 displayed reduced enzyme activity, and Asp113 displayed wild type activity. The analysis described is the first molecular characterization of a CDP-alcohol phosphotransferase motif and results predict a catalytic role utilizing a general base reaction proceeding through Asp131 or Asp135 via a direct nucleophilic attack of the hydroxyl of diacylglyerol on the phosphoester bond of CDP-choline that does not proceed via an enzyme bound intermediate. Residues Ala117 and Arg118 do not participate directly in catalysis but are likely involved in substrate binding or positioning with Arg118 predicted to associate with a phosphate moiety of CDP-choline.

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: regulation by a high-affinity Zn2+ binding site.

Saccharomyces cerevisiae cells were demonstrated to contain lysophosphatidylcholine (lysoPtdCho) acyltransferase (E.C. 2.3.1.23) activity. The enzyme displayed Km(app) of 69 microM for lysoPtdCho and 152 microM for oleoyl CoA. Enzyme activity was not affected by the addition of 1 mM Mg2+, Mn2+, Ca2+, or 200 mM EDTA. However, Zn2+ inhibited lysoPtdCho acyltransferase activity to 33% control values at 0.1 mM and to 7% at 1.0 mM Zn2+. To further explore the possibility that lysoPtdCho acyltransferase may contain a high-affinity Zn2+ binding site, we tested the strong Zn2+ chelator o-phenanthroline for its ability to inhibit enzyme activity. LysoPtdCho acyltransferase activity was inhibited to 18 and 27%, respectively, those of control values in the presence of 2 and 1 mM o-phenanthroline, implying that a high-affinity Zn2+ binding site exists in lysoPtdCho acyltransferase or in an accessory protein that is essential for protein stability and/or activity. Saccharomyces cerevisiae lysoPtdCho acyltransferase activity displayed a broad lysoPtdCho fatty acyl chain substrate specificity utilizing lysoPtdCho molecules ranging in length from C10-C20 (the entire range tested). In addition, the enzyme was capable of using the ether-linked analog of lysoPtdCho, 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine, as a substrate. The ability of S. cerevisiae to incorporate radiolabeled 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine into phosphatidylcholine in vitro was exploited to demonstrate a direct precursor-product relationship between lysoPtdCho molecules and their incorporation into phosphatidylcholine in vivo. Identical labeling results were obtained in S. cerevisiae cells disrupted for their major transacylase activity, PLB1, demonstrating that the incorporation of lysolipid was via acyltransferase, and not transacylase, activity.

CDP-choline:1,2-diacylglycerol cholinephosphotransferase.

Cholinephosphotransferase transfers a phosphocholine moiety from CDP-choline to diacylglycerol thus forming phosphatidylcholine (PtdCho) and CMP. This reaction defines the ultimate step in the Kennedy pathway for the genesis of de novo synthesized PtdCho. Hence, the intracellular location of cholinephosphotransferase identifies both the site from which de novo synthesized PtdCho is transported to other organelles and the site from which it is assembled with proteins and other lipids for secretion from the cell during the generation of lung surfactant, lipoproteins, and bile. Most subcellular fractionation studies observed the majority of cholinephosphotransferase activity in the endoplasmic reticulum, although the method of subcellular fractionation was found to grossly affect these results with activity alternately dispersed within Golgi, nuclear, and mitochondrial fractions. Coupling subcellular fractionation results with immunofluorescence or electron microscopy studies would resolve the issue of the site of PtdCho synthesis. However, antibodies have yet to be generated to cholinephosphotransferase since its integral membrane-bound nature has prevented its purification from any source and a mammalian cholinephosphotransferase cDNA has also yet to be isolated. However, cholinephosphotransferase genes have recently been isolated from the yeast Saccharomyces cerevisiae. Structure/function analysis of the S. cerevisiae cholinephosphotransferase has allowed for an in depth molecular examination resulting in the identification of the catalytic site. In addition, this analysis has generated the predicted amino acid data necessary to produce antibodies to pursue the site of PtdCho synthesis in this organism, as well as to provide information that should allow for the isolation of mammalian cholinephosphotransferase cDNA(s).

CDP-ethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase.

Ethanolaminephosphotransferase catalyzes the final step of the CDP-ethanolamine pathway for the de novo synthesis of phosphatidylethanolamine (PtdEtn) via transfer of a phosphoethanolamine moiety from CDP-ethanolamine to diacylglycerol for the formation of PtdEtn and CMP. Ethanolaminephosphotransferase is an integral membrane-bound enzyme whose intracellular location defines the site of PtdEtn synthesis by the CDP-ethanolamine pathway. Subcellular fractionation experiments have yet to resolve the precise subcellular location of ethanolaminephosphotransferase, although it is routinely associated with the microsomal fraction. Ethanolaminephosphotransferase has yet to be purified from any source and its cDNA has not been isolated from any mammalian source, thus preventing the generation of antibodies necessary to directly examine its intracellular location through immunofluorescence or electron microscopy approaches. An ethanolaminephosphotransferase gene has recently been isolated from the yeast Saccharomyces cerevisiae and structure/function analyses of the encoded enzyme identified several important characteristics including the catalytic site. The predicted amino acid sequence of the S. cerevisiae ethanolaminephosphotransferase gene should allow for the generation of antibodies required to directly define the site of PtdEtn synthesis in this organism, and it has provided the necessary information to pursue the isolation of a mammalian cDNA.

Activation of mouse sperm phosphatidylinositol-4,5 bisphosphate-phospholipase C by zona pellucida is modulated by tyrosine phosphorylation.

Many cellular responses to the occupancy of membrane receptors include the hydrolysis of phosphatidylinositol-4,5 bisphosphate (PIP2) by phospholipase C (PLC) and the subsequent generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). In the gamete interaction system, sperm respond to binding to the egg's extracellular matrix, the zona pellucida (zp), by exocytosis of the acrosome in a process known as the acrosome reaction (AR). Under physiological conditions, zp binding stimulates ARs only after sperm have undergone a final maturation phase, known as capacitation. One of the zp glycoproteins, ZP3, serves as the ligand for sperm plasma membrane receptors and as the trigger for this regulated exocytosis. Both phosphoinositide-linked and tyrosine kinase-mediated pathways participate in the signalling cascade triggered by sperm-zp interaction. This paper reports that stimulation with solubilized zp increased PIP2-PLC enzymatic activity from mouse sperm. ZP3 is the zp component responsible for this stimulation. The effect was abolished by tyrphostin, suggesting that zp activation of PLC was mediated by tyrosine phosphorylation and that gamma was the PLC isoform involved. We show the presence and distribution of PLC gamma 1 in mouse sperm. Immunostaining studies indicate that PLC gamma 1 is restricted to the sperm head. Sperm capacitation induced translocation of PLC gamma 1 from the soluble to the particulate fraction. These data suggest that PLC gamma 1 constitutes a component in the cascade that couples sperm binding to the egg's extracellular matrix with acrosomal exocytosis, a regulated secretory response upon which fertilization depends absolutely.

Phospholipid and cation activation of chimaeric choline/ethanolamine phosphotransferases.

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.

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by CTP.

In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways. We examined the regulation of phosphatidylcholine synthesis by CTP. The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP. The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway. CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway. The apparent Km of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline. Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase. This was not due to a transcriptional repression mechanism. Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP. These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized. This regulation includes the supple of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway.

The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity.

The Saccharomyces cerevisiae protein SEC14p is required for Golgi function and cell viability in vivo. This requirement is obviated by mutations that specifically inactivate the CDP-choline pathway for phosphatidylcholine biosynthesis. The biochemical basis for the in vivo relationship between SEC14p function and the CDP-choline pathway has remained obscure. We now report that SEC14p effects an in vivo depression of CDP-choline pathway activity by inhibiting choline-phosphate cytidylyltransferase (CCTase; EC 2.7.7.15), the rate-determining enzyme of the CDP-choline pathway. Moreover, this SEC14p-mediated inhibition of CCTase was recapitulated in vitro and was saturable. Finally, whereas the SEC14p-dependent inhibition of CCTase in vitro was markedly reduced under assay conditions that were expected to increase levels of phosphatidylinositol-bound SEC14p, assay conditions expected to increase levels of phosphatidylcholine-bound SEC14p resulted in significant potentiation of CCTase inhibition. The collective data suggest that the phosphatidylcholine-bound form of SEC14p effects an essential repression of CDP-choline pathway activity in Golgi membranes by inhibiting CCTase and that the phospholipid-binding/exchange activity of SEC14p represents a mechanism by which the regulatory activity of SEC14p is itself controlled.

Phosphatidylcholine biosynthesis in Saccharomyces cerevisiae. Regulatory insights from studies employing null and chimeric sn-1,2-diacylglycerol choline- and ethanolaminephosphotransferases.

The Saccharomyces cerevisiae CPT1 and EPT1 genes encode distinct choline- and choline/ethanolaminephosphotransferases, respectively. In vitro, each gene product accounts for 50% of the measurable choline-phosphotransferase activity. Strains containing null mutations in the CPT1 and EPT1 loci were used to investigate the function of each gene product in vivo. The CPT1 gene product was responsible for 95% of phosphatidylcholine (PC) synthesis via the CDP-choline pathway in vivo. The EPT1 gene product accounted for only 5% of PC synthesis in vivo. Chimeric CPT1/EPT1 enzymes with diacylglycerol and CDP-aminoalcohol specificities both similar and distinct from the parental enzymes were used to determine the specific segments of the CPT1/EPT1 gene products required to restore PC synthesis to cpt- cells in vivo. Only chimeras expressing the CDP-aminoalcohol specificity region of CPT1 were capable of PC synthesis via the CDP-choline pathway in vivo. Analysis of phospholipids extracted from wild type, cpt-, and ept- cells labeled with 32Pi indicated an intact CPT1 gene product was required for the pleiotropic regulation of phospholipid synthesis by inositol. Chimeric CPT1/EPT1 enzymes expressed in a cpt- background mapped the regulatory region of the CPT1 gene product required for the inositol-dependent regulation of phospholipid synthesis to the CDP-aminoalcohol binding domain of CPT1. Strains harboring dysfunctional cholinephosphotransferase enzymes also displayed decreased levels of choline uptake, suggesting that a feedback loop exists to coordinate choline uptake with ongoing PC biosynthesis. The data also implicate the CPT1 gene product in PC biosynthesis from an endogenous source of choline derived from turnover of PC via the phosphatidylserine-dependent route for PC synthesis.