Thematic Review Series: Glycerolipids. 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. 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. Mammalian cell membranes contain >1,000 different phospholipids. This large mixture of phospholipid species is primarily the result of the distinct fatty acyl chains esterified to the sn-1 and sn-2 positions of the glycerol backbone as well as the different polar head groups attached to the sn-3 position of the glycerol backbone. The amounts of the various phospholipids in a membrane define the fluidity of the membrane and, consequently, the functions of the embedded proteins. Phosphatidylcholine is the most abundant phospholipid in mammalian cell membranes, constituting 40–50% of total phospholipids. The second most abundant mammalian membrane phospholipid is phosphatidylethanolamine (PE), which constitutes 20–50% of total phospholipids. In the brain, ∼45% of total phospholipids are PE, whereas in the liver, only ∼20% of total phospholipids are PE. Phosphatidylserine (PS) is a quantitatively minor membrane phospholipid that makes up 2–10% of total phospholipids. The metabolic interrelationships among PS, PE, and phosphatidylcholine are depicted in Fig. 1. Additional relatively minor mammalian membrane phospholipids include phosphatidylinositol, sphingomyelin, and the mitochondria-specific phospholipid, cardiolipin. Different types of mammalian cells and tissues have characteristic phospholipid compositions. For example, the brain is enriched in the two aminophospholipids PE and PS compared with other tissues. In the brain, and particularly in the retina (1Ford D.A. Monda J.K. Brush R.S. Anderson R.E. Richards M.J. Fliesler S.J. Lipidomic analysis of the retina in a rat model of Smith-Lemli-Opitz syndrome: alterations in docosahexaenoic acid content of phospholipid molecular species.J. 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For example, in the plasma membrane, PS and PE are asymmetrically distributed across the bilayer such that the great majority (>80%) of these aminophospholipids are normally confined to the inner leaflet, whereas phosphatidylcholine and sphingomyelin are enriched on the outer leaflet. Phospholipids were, for many years, thought to play primarily structural roles in biological membranes. A large number of recent studies have revealed, however, that these lipids mediate important regulatory functions in cells, partly because of their ability to be converted into key lipid second messengers such as diacylglycerol, inositol-1,4,5-trisphosphate (12Berridge M.J. Irvine R.F. Inositol trisphosphate, a novel second messenger in cellular signal transduction.Nature. 1984; 312: 315-321Crossref PubMed Google Scholar, 13Nishizuka Y Studies and perspectives of protein kinase C.Science. 1986; 233: 305-312Crossref PubMed Google Scholar), lyso-phosphatidic acid, and arachidonic acid. PS and PE are metabolically related, as depicted in Fig. 1. PS is an important precursor of mitochondrial PE, which is produced by the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) (see below) (14Borkenhagen L.F. Kennedy E.P. Fielding L. Enzymatic formation and decarboxylation of phosphatidylserine.J. Biol. Chem. 1961; 236: 28-32Abstract Full Text PDF Google Scholar). As noted above, in the plasma membrane of mammalian cells PS normally resides almost entirely on the inner leaflet of the bilayer. In the past decade, PS has become a major focus of interest because during the early phases of apoptosis PS becomes externalized on the outside of cells. The surface exposure of PS is believed to be one of the recognition signals by which apoptotic cells are removed by phagocytes (15Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages.J. Immunol. 1992; 148: 2207-2216Crossref PubMed Google Scholar, 16Fadok V.A. de Cathelineau, D. L. Daleke, P. M. Henson, and D. L. Bratton A Loss of phospholipid asymmetry and surface exposure of phosphatidylserine is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar, 17Balasubramanian K. Mirnikjoo B. Schroit A.J. Regulated externalization of phosphatidylserine at the cell surface: implications for apoptosis.J. Biol. Chem. 2007; 282: 18357-18364Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), although the identity of a PS receptor on macrophages remains controversial (18Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. 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Bidirectional phospholipid transporters called scramblases are also located on the plasma membrane, and these proteins can randomize the distribution of PS and other lipids across the bilayer. The scramblases are activated by calcium and do not require ATP for activity (25Zhou Q. Zhao J. Stout J.G. Luhm R.A. Wiedmer T. Sims P.J. Molecular cloning of human plasma membrane phospholipid scramblase: a protein mediating transbilayer movement of plasma membrane phospholipids.J. Biol. Chem. 1997; 272: 18240-18244Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar). Thus, the exposure of PS on the outside of cells undergoing apoptosis is likely to be promoted by reduced activity of the aminophospholipid translocase combined with increased scramblase activity. The induction of PS exposure on cell surfaces is not restricted to apoptotic cells. For example, the exposure of PS on the surface of activated platelets initiates the blood-clotting cascade (17Balasubramanian K. 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Mammalian cells express two distinct serine-exchange activities. The first indication that two mammalian PS synthase genes existed was the partial purification of a rat brain enzymatic activity that synthesized PS by a serine-exchange reaction with PE but not with phosphatidylcholine (55Suzuki T.T. Kanfer J.N. Purification and properties of an ethanolamine-serine base exchange enzyme of rat brain microsomes.J. Biol. Chem. 1985; 260: 1394-1399Abstract Full Text PDF PubMed Google Scholar). An epitope-tagged version of this protein has now been purified to near homogeneity (56Kuge O. Hasegawa K. Ohsawa T. Saito K. Nishijima M. Purification and characterization of Chinese hamster phosphatidylserine synthase 2.J. Biol. Chem. 2003; 278: 42692-42698Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The existence of two mammalian Pss genes was confirmed when mutant Chinese hamster ovary (CHO) cells, which had the capacity to synthesize PS from PE but not from phosphatidylcholine, were generated in the laboratories of Nishijima (PSA-3 cells) (57Kuge O. Nishijima M. Akamatsu Y. Isolation of a somatic cell mutant defective in phosphatidylserine biosynthesis.Proc. Natl. Acad. Sci. USA. 1985; 82: 1926-1930Crossref PubMed Scopus (45) Google Scholar) and Voelker (M9.1.1 cells) (58Voelker D.R. Frazier J.L. Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity.J. Biol. Chem. 1986; 261: 1002-1008Abstract Full Text PDF PubMed Google Scholar). The defective choline-exchange activity was called PS synthase-1, and the residual serine-exchange activity, which uses PE as substrate, was named PS synthase-2. Radiolabeling experiments and in vitro enzyme assays in PS synthase-1-deficient CHO cells revealed that the rate of PS synthesis was 35–55% lower than that in parental cells, and the mass of both PS and PE was reduced correspondingly (57Kuge O. Nishijima M. Akamatsu Y. Isolation of a somatic cell mutant defective in phosphatidylserine biosynthesis.Proc. Natl. Acad. Sci. USA. 1985; 82: 1926-1930Crossref PubMed Scopus (45) Google Scholar, 58Voelker D.R. Frazier J.L. Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity.J. Biol. Chem. 1986; 261: 1002-1008Abstract Full Text PDF PubMed Google Scholar). The growth of cells that lacked PS synthase-1 was severely impaired in the absence of supplementation with PS, PE, or ethanolamine. Thus, mammalian cells express PS synthase-1 activity that synthesizes PS from PC. The second serine-exchange activity, PS synthase-2, is encoded by a distinct gene and catalyzes the exchange of serine with PE to make PS (Fig. 1). cDNAs encoding PS synthase-1 and PS synthase-2 from hamster (59Kuge O. Nishijima M. Akamatsu Y. A Chinese hamster cDNA encoding a protein essential for phosphatidylserine synthase I activity.J. Biol. Chem. 1991; 266: 24184-24189Abstract Full Text PDF PubMed Google Scholar, 60Kuge O. Saito K. Nishijima M. Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells.J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and mouse liver (61Stone S.J. Cui Z. Vance J.E. Cloning and expression of mouse liver phosphatidylserine synthase-1 cDNA: overexpression in rat hepatoma cells inhibits the CDP-ethanolamine pathway for phosphatidylethanolamine biosynthesis.J. Biol. Chem. 1998; 273: 7293-7302Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 62Stone S.J. Vance J.E. Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2.Biochem. J. 1999; 342: 57-64Crossref PubMed Scopus (50) Google Scholar) were cloned. The murine and human PS synthase-1 genes reside on chromosomes 13 and 8, respectively. When PS synthase-2 activity was overexpressed in CHO cells, the ethanolamine-exchange activity was increased by 10-fold, whereas the choline-exchange activity remained unchanged (60Kuge O. Saito K. Nishijima M. Cloning of a Chinese hamster ovary (CHO) cDNA encoding phosphatidylserine synthase (PSS) II, overexpression of which suppresses the phosphatidylserine biosynthetic defect of a PSS I-lacking mutant of CHO-K1 cells.J. Biol. Chem. 1997; 272: 19133-19139Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), supporting the view that PS synthase-2 catalyzes ethanolamine but not choline exchange. Additional evidence that the putative PS synthase-1 cDNA encodes PS synthase-1 was obtained when choline-exchange activity was eliminated by the immunoprecipitation of cell lysates with an antibody raised against a C-terminal peptide corresponding to the predicted PS synthase-1 sequence (63Saito K. Kuge O. Akamatsu Y. Nishijima M. Immunochemical identification of the pssA gene product as phosphatidylserine synthase I of Chinese hamster ovary cells.FEBS Lett. 1996; 395: 262-266Crossref PubMed Scopus (34) Google Scholar). In other experiments, overexpression of PS synthase-2 in PS synthase-1-deficient cells eliminated the requirement for exogenously added PS (62Stone S.J. Vance J.E. Cloning and expression of murine liver phosphatidylserine synthase (PSS)-2: differential regulation of phospholipid metabolism by PSS1 and PSS2.Biochem. J. 1999; 342: 57-64Crossref PubMed Scopus (50) Google Scholar), indicating that PS synthase-2 can substitute for PS synthase-1 in CHO cells. Cells lacking PS synthase-1 activity (i.e., PSA-3 cells) were further mutagenized, resu