Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are two aminophospholipids whose metabolism is interrelated. Both phospholipids are components of mammalian cell membranes and play important roles in biological processes such as apoptosis and cell signaling. PS is synthesized in mammalian cells by base-exchange reactions in which polar head groups of preexisting phospholipids are replaced by serine. PS synthase activity resides primarily on mitochondria-associated membranes and is encoded by two distinct genes. Studies in mice in which each gene has been individually disrupted are beginning to elucidate the importance of these two synthases for biological functions in intact animals. PE is made in mammalian cells by two completely independent major pathways. In one pathway, PS is converted into PE by the mitochondrial enzyme PS decarboxylase. In addition, PE is made via the CDP-ethanolamine pathway, in which the final reaction occurs on the endoplasmic reticulum and nuclear envelope. The relative importance of these two pathways of PE synthesis has been investigated in knockout mice. Elimination of either pathway is embryonically lethal, despite the normal activity of the other pathway. PE can also be generated from a base-exchange reaction and by the acylation of lyso-PE. Cellular levels of PS and PE are tightly regulated by the implementation of multiple compensatory mechanisms.     

Hexadecylphosphocholine inhibits phosphatidylcholine synthesis via both the methylation of phosphatidylethanolamine and CDP-choline pathways in HepG2 cells

We reported in a recent publication that hexadecylphosphocholine (HePC), a lysophospholipid analogue, reduces cell proliferation in HepG2 cells and at the same time inhibits the biosynthesis of phosphatidylcholine (PC) via CDP-choline by acting upon CTP:phosphocholine cytidylyltransferase (CT). We describe here the results of our study into the influence of HePC on other biosynthetic pathways of glycerolipids. HePC clearly decreased the incorporation of the exogenous precursor [1,2,3-3H]glycerol into PC and phosphatidylserine (PS) whilst increasing that of the neutral lipids diacylglycerol (DAG) and triacylglycerol (TAG). Interestingly, the uptake of L-[3-3H]serine into PS and other phospholipids remained unchanged by HePC and neither was the activity of either PS synthase or PS decarboxylase altered, demonstrating that the biosynthesis of PS is unaffected by HePC. We also analyzed the water-soluble intermediates and final product of the CDP-ethanolamine pathway and found that HePC caused an increase in the incorporation of [1,2-14C]ethanolamine into CDP-ethanolamine and phosphatidylethanolamine (PE) and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine cytidylyltransferase activity. Since PE can be methylated to give PC, we studied this process further and observed that HePC decreased the synthesis of PC from PE by inhibiting the PE N-methyltransferase activity. These results constitute the first experimental evidence that the inhibition of the synthesis of PC via CDP-choline by HePC is not counterbalanced by any increase in its formation via methylation. On the contrary, in the presence of HePC both pathways seem to contribute jointly to a decrease in the overall synthesis of PC in HepG2 cells.     

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[³H(G)]serine and [¹⁴C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of “correct” PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.     

Tissue-Specific and Developmental Effects of the Easily Shocked Mutation on Ethanolamine Kinase Activity and Phospholipid Composition in Drosophila melanogaster

The easily shocked (eas) gene of Drosophila melanogaster encodes ethanolamine kinase (EK), the first step in phosphatidylethanolamine (PE) synthesis via the CDP-ethanolamine pathway Flies mutant for eas display a complex neurological phenotype. In this paper we look at the contribution of EK to lipid metabolism during Drosophila development with the goal of linking the eas biochemical defect with the organismal phenotype. Using a chromatography-based assay, EK activity was detected in wild-type flies throughout development. Most of the activity in the adult was present in heads, which is primarily tissue of neural origin. Flies mutant for eas showed severely reduced levels of activity at each stage assayed. Using standard extraction methods and thin layer chromatography, phospholipid composition was assayed in wholeflies and in heads. While PE levels were decreased significantly in both tissues, heads also had significantly less phosphatidylserine (PS). Therefore, decreases in both phospholipids may play a role in producing the aberrant phenotype in eas flies.     

Deficiency in phosphatidylserine decarboxylase activity in the psd1 psd2 psd3 triple mutant of Arabidopsis affects phosphatidylethanolamine accumulation in mitochondria

Phosphatidylserine (PS) decarboxylase is involved in the synthesis of the abundant phospholipid phosphatidylethanolamine (PE), particularly in mitochondria, in many organisms, including yeast (Saccharomyces cerevisiae) and animals. Arabidopsis (Arabidopsis thaliana) contains three genes with sequence similarity to PS decarboxylases, and the respective gene products were functionally characterized after heterologous expression in yeast and Escherichia coli. While the PSD1 protein localizes to mitochondria, PSD2 and PSD3 are found in the endomembrane system. To study the role of PSD genes in plant phospholipid metabolism, Arabidopsis insertional mutants for psd1, psd2, and psd3 were obtained. The single mutants were decreased in PS decarboxylase activity to various extents, but mutant plants showed no obvious growth or morphological phenotype. A triple mutant, psd1 psd2 psd3, was generated that was totally devoid of PS decarboxylase activity. While the phospholipid composition in whole leaves was unchanged, the PE content in isolated mitochondria of psd1 psd2 psd3 was decreased. Therefore, the predominant proportion of PE in Arabidopsis is synthesized by alternative pathways, but a significant amount of mitochondrial PE is derived from the PS decarboxylase reaction. These results imply that, similar to yeast and animal cells, a specific phospholipid transfer from the endoplasmic reticulum to mitochondria exists in plants.     

Identification and characterization of human ethanolaminephosphotransferase1

CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (EPT) catalyzes the transfer of phosphoethanolamine from CDP-ethanolamine to diacylglycerol to produce phosphatidylethanolamine (PE). To date, the dual specificity of choline/ethanolaminephosphotransferase (CEPT) has been recognized as the total activity responsible for the synthesis of PE via the CDP-ethanolamine pathway in human. We report here the identification and characterization of another human cDNA that encodes CDP-ethanolamine-specific human EPT (hEPT1). Through homology search, we found that human selenoprotein I contained the CDP-alcohol phosphatidyltransferase signature, a common motif conserved in phospholipid synthases. Bacterial expression of the cDNA in Escherichia coli demonstrated that the product specifically used CDP-ethanolamine as the phosphobase donor to produce PE with the activation by both Mn(2+) and Mg(2+). RT-PCR and Northern blot analysis revealed that hEPT1 was ubiquitously expressed in multiple tissues, but in brain it was highly expressed in cerebellum. Here, we propose that in addition to previously identified CEPT, hEPT1 is involved in the biosynthesis of PE via the Kennedy pathway.     

Import of phosphatidylserine to and export of phosphatidylethanolamine molecular species from mitochondria

Heavy isotope-labeled ethanolamine and serine as well as exogenous PE and PS species were used to study trafficking of phosphatidylethanolamine (PE) and -serine (PS) molecular species between the endoplasmic reticulum (ER) and mitochondria in HeLa cells. Import of both endogenous and exogenous PS to IMM was a relatively slow process (T1/2 = several hours), but depended on the acyl chains. In particular, the 38:4 and 38:5 species were imported more efficiently compared to the other PS species. Knock-down of Mitofusin 2 or Mitostatin had no detectable effect on PS import to mitochondria, suggesting that the ER–mitochondria contacts regulated by these proteins are not essential. Knock-down of PS synthase 1 inhibited PS decarboxylation, suggesting that import of PS to mitochondria is coupled to its synthesis. Also the export of PE from IMM to microsomes is a relatively slow process, but again depends markedly on the acyl chain structure. Most notably, the polyunsaturated 38:4 and 38:5 PE species were less efficiently exported, which together with rapid import of the PS precursors most probably explains their enrichment in IMM. PE synthesized via the CDP-ethanolamine was also imported to IMM, but most of the PE in this membrane derives from imported PS. In contrast to PS, all PC species made in Golgi/ER translocated similarly and rapidly to IMM. In conclusion, selective translocation of PS species and PS-derived PE species between ER and mitochondria plays a major role in phospholipid homeostasis of these organelles.     

Phospholipid homeostasis in phosphatidylserine synthase-2-deficient mice

Phosphatidylserine (PS) is synthesized in mammalian cells by two distinct serine-exchange enzymes, phosphatidylserine synthase-1 and -2. We recently demonstrated that mice lacking PS synthase-2 develop normally and exhibit no overt abnormalities [Bergo et al., (2002) J. Biol. Chem. 277:47701-47708]. We now show that PS synthase-2 mRNA levels are up to 80-fold higher in livers of embryos than in adults. Despite reduced serine-exchange activity in several tissues of PS synthase-2 deficient mice, the phospholipid composition of mitochondria and microsomes from these tissues is normal. Although PS synthase-2 is highly expressed in neurons, axon extension of cultured sympathetic neurons is not impaired by PS synthase-2 deficiency. We hypothesized that mice compensate for PS synthase-2 deficiency by modifying their phospholipid metabolism. Our data show that the rate of PS synthesis in hepatocytes is not reduced by PS synthase-2 deficiency but PS synthase-1 activity is increased. Moreover, PS degradation is decreased by PS synthase-2 deficiency, probably as a result of decreased PS degradation via phospholipases rather than decreased PS decarboxylation. These experiments underscore the idea that cellular phospholipid composition is tightly controlled and show that PS synthase-2-deficient hepatocytes modify phospholipid metabolism by several compensatory mechanisms to maintain phospholipid homeostasis.     

A phosphatidylserine decarboxylase activity in root cells of oat (Avena sativa) is involved in altering membrane phospholipid composition during drought stress acclimation.

During acclimation to drought stress, the lipid composition of oat root cell membranes is altered. The level of phosphatidylethanolamine (PE), a non-bilayer forming lipid, is increased relative to the bilayer-forming lipid phosphatidylcholine (PC). These changes are believed to increase stress tolerance by increasing the flexibility of the membranes. To elucidate if de novo lipid synthesis is involved in altering membrane lipid composition, oat plants, acclimated or non-acclimated, were incubated in vivo with radioactively labelled lipid precursors. The labelling pattern indicated that de novo synthesis, at least partly, is causing the alterations. In plants, phospholipids can be synthesized by the Kennedy pathway, with addition of activated head groups to diacylglycerol (DAG) or, alternatively, via the CDP-DAG pathway, where phospahtidylserine (PS) is decarboxylated to form PE. To reveal the importance of the respective pathways during acclimation, we studied the effect of a decarboxylase inhibitor and the relative incorporation of [(3)H]-serine and [(14)C]-ethanolamine in vivo. Activities of CTP:ethanolaminephosphate cytidyltransferase (EC 2.7.7.14), phosphatidylserine decarboxylase (EC 4.1.1.65) and phosphatidylserine synthase; CDP-DAG:L-serine o-phosphatidyltransferase (EC 2.7.8.8) were measured and additionally, the presence of a PS decarboxylase (PSD1) in oat was confirmed by immunoblotting. The results suggest that PE synthesis via the Kennedy pathway is downregulated during acclimation and that synthesis by PS decarboxylation, via the CDP-DAG pathway, is increased, mainly through an increased activity of PS synthase.     

The Role of Phosphatidylserine Decarboxylase in Brain Phospholipid Metabolism

In brain, phosphatidylethanolamine can be synthesized from free ethanolamine either by a pathway involving the formation of CDP-ethanolamine and its transfer to diglyceride, or by base-exchange of ethanolamine with existing phospholipids. Although de novo synthesis from serine has also been demonstrated, the metabolic pathway involved is not known. The enzyme phosphatidylserine decarboxylase appears to be involved in the synthesis of much of the phosphatidylethanolamine in liver, but the significance of this route in brain has been challenged. Our in vitro studies demonstrate the existence of phosphatidylserine decarboxylase activity in rat brain and characterize some of its properties. This enzyme is localized in the mitochondrial fraction, whereas the enzymes involved in base-exchange and the cytidine pathway are localized to microsomal membranes. Parallel in vivo studies showed that after the intracranial injection of L-[G-3H]serine, the specific activity of phosphatidylserine was greater in the microsomal fractions than in the mitochondrial fraction, whereas the opposite was true for phosphatidylethanolamine. When L-[U-14C]serine and [1-3H]ethanolamine were simultaneously injected, the 14C/3H ratio in mitochondrial phosphatidylethanolamine was 10 times that in microsomal phosphatidylethanolamine. The results demonstrate that serine is incorporated into the base moiety of phosphatidylethanolamine primarily through the decarboxylation of phosphatidylserine in brain mitochondria. A minimal value of 7% for the contribution of phosphatidylserine decarboxylase to whole-brain phosphatidylethanolamine synthesis can be estimated from the in vivo data.     

Import of phosphatidylethanolamine for the assembly of rat brain mitochondrial membranes

Mitochondria can synthesize phosphatidyl-ethanolamine (PE) through phosphatidylserine decarboxylase (PS decarboxylase) activity or can import this lipid from the endoplasmic reticulum. In this work, we studied the factors influencing the import of PE in brain mitochondria and its utilization for the assembly of mitochondrial membranes. Incubation of rat brain homogenate with [1-³H]ethanolamine resulted in the synthesis and distribution of ³H-PE to subcellular fractions. T-wenty-one percent of labeled PE was recovered in purified mitochondria. The import of PE in mitochondria was studied in a reconstituted system made of microsomes (donor particles) and purified mitochondria (acceptor particles). Ca⁺² and nonspecific lipid transfer protein purified from liver tissue (nsL-TP) enhanced the translocation process. ³H-PE synthesized in membrane associated to mitochondria (MAM) could also translocate to mitochondria in the reconstituted system. Exposure of mitochondria to trinitrobenzensulfonic acid (TNBS) resulted in the reaction of more than 60% of ³H-PE imported from endoplasmic reticulum and of about 25% of ¹⁴C-PE produced in mitochondria by decarboxylation of ¹⁴C-PS. Moreover, the removal of the outer mitochondrial membrane by digitonin treatment, resulted in the loss of ³H-PE, but not ¹⁴C-PE. These results indicate that labeled PE imported in mitochondria is mainly localized in the outer mitochondrial membrane, whereas PE produced by PS decarboxylase activity is confined to the inner mitochondrial membrane. Phospholipase C hydrolyzed 25–30% of both PE radioactivity and mass of the outer mitochondrial membrane indicating an asymmetrical distribution of this lipid across the membrane.Mr. Carlo Ricci is thanked for his skillful technical assistance. This work has been supported by a grant from the Ministry of Education, Rome, Italy.     

The CDP-ethanolamine pathway and phosphatidylserine decarboxylation generate different phosphatidylethanolamine molecular species

In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d(3)-serine and d(4)-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:0-20:5)PtdEtn in McArdle and (18: 0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning.     

Phosphatidylethanolamine Biosynthesis in Mitochondria: PHOSPHATIDYLSERINE (PS) TRAFFICKING IS INDEPENDENT OF A PS DECARBOXYLASE AND INTERMEMBRANE SPACE PROTEINS UPS1P AND UPS2P*

Phosphatidylethanolamine (PE) plays important roles for the structure and function of mitochondria and other intracellular organelles. In yeast, the majority of PE is produced from phosphatidylserine (PS) by a mitochondrion-located PS decarboxylase, Psd1p. Because PS is synthesized in the endoplasmic reticulum (ER), PS is transported from the ER to mitochondria and converted to PE. After its synthesis, a portion of PE moves back to the ER. Two mitochondrial proteins located in the intermembrane space, Ups1p and Ups2p, have been shown to regulate PE metabolism by controlling the export of PE. It remains to be determined where PS is decarboxylated in mitochondria and whether decarboxylation is coupled to trafficking of PS. Here, using fluorescent PS as a substrate in an in vitro assay for Psd1p-dependent PE production in isolated mitochondria, we show that PS is transferred from the mitochondrial outer membrane to the inner membrane independently of Psd1p, Ups1p, and Ups2p and decarboxylated to PE by Psd1p in the inner membrane. Interestingly, Ups1p is required for the maintenance of Psd1p and therefore PE production. Restoration of Psd1p levels rescued PE production defects in ups1Δ mitochondria. Our data provide novel mechanistic insight into PE biogenesis in mitochondria.     

Independent repression of bile acid synthesis and activation of c-Jun N-terminal kinase (JNK) by activated hepatocyte fibroblast growth factor receptor 4 (FGFR4) and bile acids

The fibroblast growth factor (FGF) receptor complex is a regulator of adult organ homeostasis in addition to its central role in embryonic development and wound healing. FGF receptor 4 (FGFR4) is the sole FGFR receptor kinase that is significantly expressed in mature hepatocytes. Previously, we showed that mice lacking mouse FGFR4 (mR4(-/-)) exhibited elevated fecal bile acids, bile acid pool size, and expression of liver cholesterol 7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for canonical neutral bile acid synthesis. To prove that hepatocyte FGFR4 was a negative regulator of cholesterol metabolism and bile acid synthesis independent of background, we generated transgenic mice overexpressing a constitutively active human FGFR4 (CahR4) in hepatocytes and crossed them with the FGFR4-deficient mice to generate CahR4/mR4(-/-) mice. In mice expressing active FGFR4 in liver, fecal bile acid excretion was 64%, bile acid pool size was 47%, and Cyp7a1 expression was 10-30% of wild-type mice. The repressed level of Cyp7a1 expression was resistant to induction by a high cholesterol diet relative to wild-type mice. Expression of CahR4 in mR4(-/-) mouse livers depressed bile acid synthesis below wild-type levels from the elevated levels observed in mR4(-/-). Levels of phosphorylated c-Jun N-terminal kinase (JNK), which is part of a pathway implicated in bile acid-mediated repression of synthesis, was 30% of wild-type levels in mR4(-/-) livers, whereas CahR4 livers exhibited an average 2-fold increase. However, cholate still strongly induced phospho-JNK in mR4(-/-) livers. These results confirm that hepatocyte FGFR4 regulates bile acid synthesis by repression of Cyp7a1 expression. Hepatocyte FGFR4 may contribute to the repression of bile acid synthesis through JNK signaling but is not required for activation of JNK signaling by bile acids.     

Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.     

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.     

Developmental and metabolic effects of disruption of the mouse CTP:phosphoethanolamine cytidylyltransferase gene (Pcyt2)

The CDP-ethanolamine pathway is responsible for the de novo biosynthesis of ethanolamine phospholipids, where CDP-ethanolamine is coupled with diacylglycerols to form phosphatidylethanolamine. We have disrupted the mouse gene encoding CTP:phosphoethanolamine cytidylyltransferase, Pcyt2, the main regulatory enzyme in this pathway. Intercrossings of Pcyt2(+/-) animals resulted in small litter sizes and unexpected Mendelian frequencies, with no null mice genotyped. The Pcyt2(-/-) embryos die after implantation, prior to embryonic day 8.5. Examination of mRNA expression, protein content, and enzyme activity in Pcyt2(+/-) animals revealed the anticipated 50% decrease due to the gene dosage effect but rather a 20 to 35% decrease. [(14)C]ethanolamine radiolabeling of hepatocytes, liver, heart, and brain corroborated Pcyt2 gene expression and activity data and showed a decreased rate of phosphatidylethanolamine biosynthesis in heterozygotes. Total phospholipid content was maintained in Pcyt2(+/-) tissues; however, this was not due to compensatory increases in the decarboxylation of phosphatidylserine. These results establish the necessity of Pcyt2 for murine development and demonstrate that a single Pcyt2 allele in heterozygotes can maintain phospholipid homeostasis.