Defining the importance of phosphatidylserine synthase-1 (PSS1): unexpected viability of PSS1-deficient mice

Phosphatidylserine (PS) is a quantitatively minor, but physiologically important, phospholipid in mammalian cells. PS is synthesized by two distinct base-exchange enzymes, PS synthase-1 (PSS1) and PS synthase-2 (PSS2), that are encoded by different genes. PSS1 exchanges serine for choline of phosphatidylcholine, whereas PSS2 exchanges ethanolamine of phosphatidylethanolamine for serine. We previously generated mice lacking PSS2 (Bergo, M. O., Gavino, B. J., Steenbergen, R., Sturbois, B., Parlow, A. F., Sanan, D. A., Skarnes, W. C., Vance, J. E., and Young, S. G. (2002) J. Biol. Chem. 277, 47701-47708) and found that PSS2 is not required for mouse viability. We have now generated PSS1-deficient mice. In light of the markedly impaired survival of Chinese hamster ovary cells lacking PSS1 we were surprised that PSS1-deficient mice were viable, fertile, and had a normal life span. Total serine-exchange activity (contributed by PSS1 and PSS2) in tissues of Pss1(-/-) mice was reduced by up to 85%, but except in liver, the PS content was unaltered. Despite the presumed importance of PS in the nervous system, the rate of axonal extension of PSS1-deficient neurons was normal. Intercrosses of Pss1(-/-) mice and Pss2(-/-) mice yielded mice with three disrupted Pss alleles but no double knockout mice. In Pss1(-/-)/Pss2(-/-) and Pss1(-/-)/Pss2(-/-) mice, serine-exchange activity was reduced by 65-91%, and the tissue content of PS and phosphatidylethanolamine was also decreased. We conclude that (i) elimination of either PSS1 or PSS2, but not both, is compatible with mouse viability, (ii) mice can tolerate as little as 10% of normal total serine-exchange activity, and (iii) mice survive with significantly reduced PS and phosphatidylethanolamine content.     

Reconstitution of phosphatidylserine import into rat liver mitochondria

The synthesis translocation and decarboxylation of phosphatidylserine occurs in a cell-free system. The principal membrane components necessary are microsomes (source of phosphatidylserine synthase) and mitochondria (source of phosphatidylserine decarboxylase). The interorganelle translocation of phosphatidylserine can be measured by quantitating the decarboxylation of phosphatidyl[1'-14C]serine initially present in prelabeled microsomal membranes using a 14CO2 trapping assay. The decarboxylation of microsomal phosphatidylserine by intact mitochondria is 1) dependent upon substrate (microsomal membrane) concentration, 2) different from decarboxylation of liposomal phosphatidylserine, 3) resistant to proteases, 4) independent of soluble factors, and 5) unaffected by the addition of partially purified phospholipid exchange proteins but accelerated by purified nonspecific phospholipid exchange protein. The rate-limiting step in the reconstituted translocation-decarboxylation system is not the decarboxylation reaction but the initial translocation event between the microsomal membrane and the outer mitochondrial membrane. These data are interpreted to demonstrate that phosphatidylserine import into the mitochondria can occur via collision complexes formed between the endoplasmic reticulum or vesicles derived therefrom and the outer mitochondrial membrane.     

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.     

The N-terminal half of talin2 is sufficient for mouse development and survival

Using a talin2 gene-trapped embryonic stem cell clone, we have developed a talin2 mutant mouse line that expresses the N-terminal half (1-1295) of talin2 fused with beta-galactosidase. The homozygous mutant mice appear to be normal and healthy. In the testis, talin2 expresses as a shorter form with a unique 30 residues at N-terminus linking to a common C-terminus from 1122 to 2453 of the long form. The resulting talin2 in the mutant testis only contains 204 residues of the wild-type testis talin2. However, it did not seem to affect the morphology of testis or reproduction of male mice. In fact, male and female mutant mice are fertile. Utilizing the expression of talin2(1-1295)/beta-galactosidase fusion protein, we have examined the distribution of talin2 in tissues. In contrast to talin1, talin2 expression is more restricted in tissues and cell types.     

PKC phosphorylation disrupts gap junctional communication at G0/S phase in clone 9 cells

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-g-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-g-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-g-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis.The preincubation of plasma membranes in the buffer without any other addition at 37øC for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-g-S but was insensitive to 5 mM AlF4-, a concentration that inhibited by 60% the enzyme assayed without preincubation.These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF4- was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.     

Effect of growth phase on phospholipid biosynthesis in Saccharomyces cerevisiae.

The effect of growth phase on the membrane-associated phospholipid biosynthetic enzymes CDP-diacylglycerol synthase, phosphatidylserine synthase, phosphatidylinositol synthase, and the phospholipid N-methyltransferases in wild-type Saccharomyces cerevisiae was examined. Maximum activities were found in the exponential phase of cells grown in complete synthetic medium. As cells entered the stationary phase of growth, the activities of the CDP-diacylglycerol synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases decreased 2.5- to 5-fold. The subunit levels of phosphatidylserine synthase and the cytoplasmic-associated enzyme inositol-1-phosphate synthase were not significantly affected by the growth phase. When grown in medium supplemented with inositol-choline, cells in the exponential phase of growth had reduced CDP-diacylglycerol synthase, phosphatidylserine synthase, and phospholipid N-methyltransferase activities, with repressed subunit levels of phosphatidylserine synthase and inositol-1-phosphate synthase compared with cells grown without inositol-choline. Enzyme activity levels remained reduced in the stationary phase of growth of cells supplemented with inositol-choline. The phosphatidylserine synthase and inositol-1-phosphate synthase subunit levels, however, were depressed. Phosphatidylinositol synthase (activity and subunit) was not affected by growth in medium supplemented with or without inositol-choline or the growth phase of the culture. The phospholipid composition of cells in the exponential and stationary phase of growth was also examined. The phosphatidylinositol to phosphatidylserine ratio doubled in stationary-phase cells. The phosphatidylcholine to phosphatidylethanolamine ratio was not significantly affected by the growth phase of cells.     

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.     

Isolation and characterization of a Chinese hamster ovary cell line requiring ethanolamine or phosphatidylserine for growth and exhibiting defective phosphatidylserine synthase activity

A mutant cell line (designated M.9.1.1) requiring ethanolamine for growth was derived from Chinese hamster ovary (CHO-K1) cells using 5-bromodeoxyuridine enrichment. The ethanolamine requirement was readily replaced by 20 microM phosphatidylserine and 10 microM lysophosphatidylethanolamine. When M.9.1.1 cells were supplemented with phosphatidyl[3H]serine it was rapidly taken up, and subsequently decarboxylated to form phosphatidyl[3H]ethanolamine. The incorporation of [3H]serine into phosphatidylserine in the mutant cells was 57% of that in the parental cells. Phosphatidylethanolamine synthesis from [3H]serine in the mutant cells was 35% of that in parental cells. When M.9.1.1 cells were deprived of ethanolamine for 48 h the level of phosphatidylserine decreased 34% and the level of phosphatidylethanolamine decreased 26% compared to parental cells. At the same time the rate of turnover of phosphatidylserine was reduced to half that found in parental cells. Examination of the enzymes of phosphatidylserine metabolism indicated defective phosphatidylserine synthase activity in the mutant. When exogenous phosphatidylcholine was used as the phospholipid substrate for the reaction the apparent kinetic constants were Vmax (mutant) = 5.7 pmol/min/mg protein and Vmax (parental) = 17.5 pmol/min/mg protein. Measurement of the back reaction (ATP-independent incorporation of choline into phospholipid) gave no detectable activity in the mutant cells. The data indicate that the phosphatidylcholine-dependent synthesis of phosphatidylserine is the primary lesion in M.9.1.1.     

Phospholipid synthesis is decreased in neuronal tissue in a mouse model of Sandhoff disease

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes for the beta-subunit of beta-hexosaminidase A and B, resulting in ganglioside GM(2) accumulation in the brain. We now demonstrate that phospholipid metabolism is altered in both cultured neurons and in brain tissue from a mouse model of Sandhoff disease, the Hexb-/- mouse. Metabolic labelling using [methyl-(14)C]choline and l-[3-(3)H]serine demonstrated reduced incorporation of [methyl-(14)C]choline into phospholipids in brain tissue but not in liver or spleen. Phospholipid mass was also reduced in brain. The activities of CTP : phosphocholine cytidylyltransferase (CCT) and phosphatidylserine synthase were also reduced in brain tissue from Hexb-/- mice, probably because of post-translational modification as no changes were observed in levels of enzyme expression. The relevance of these findings to Sandhoff disease in human patients is strengthened by observations made over 30 years ago on autopsy tissue of Tay Sachs and Sandhoff disease patients, in which reduced phospholipid levels were observed. We suggest that changes in phospholipid metabolism are not simply because of loss of neuronal tissue as a result of degeneration but rather may cause degeneration, and we discuss the possible effects that changes in phospholipid metabolism could play in the neuropathophysiology of Sandhoff disease.     

Properties of particulate and solubilized phosphatidylserine synthase activity from Saccharomyces cerevisiae. Inhibitory effect of choline in the growth medium

When radiolabeled serine is incubated with a particulate fraction from Saccharomyces cerevisiae, radioactivity is incorporated initially into phosphatidylserine and gradually appears in phosphatidylethanolamine. Because decarboxylation of phosphatidylserine is blocked by hydroxylamine, phosphatidylserine synthase can be assayed separately. The yeast phosphatidylserine synthase activity 1) exhibits a divalent cation requirement; 2) is stimulated by exogenous CDP-diolein (apparent Km = 0.17 mM); 3) has an apparent Km = 4 mM for L-serine; 4) has a neutral pH optimum; 5) is inhibited by p-hydroxymercuribenzoate; and 6) is reversible in the presence of 5'-CMP, but not 2'-CMP, 3'-CMP, or 5'-AMP. The phospholipid-synthesizing activity is solubilized with Triton X-100 and the enzymatic parameters have been compared with the particulate form of the enzyme. Detergent extracts catalyze the conversion of exogenous purified [31P]CDP-diglyceride to [32P]phosphatidylserine in the presence of Mn2+ and L-serine. Enzyme preparations from cells grown in the presence of choline, that have reduced phospholipid methylation activity (Waechter, C. J., Steiner, M. R., and Lester, R. L. (1969) J. Biol. Chem. 244, 3419-3422), also have substantially less phosphatidylserine synthase activity compared to identical preparations grown in the absence of choline. When choline, phosphocholine, CDP-choline, and phosphatidylcholine are present in vitro, there is no direct inhibitory effect on phosphatidylserine synthase activity. While the inclusion of choline in the growth medium caused a significant reduction in phosphatidylserine synthase activity, it did not appreciably effect the apparent Km values for L-serine and CDP-diglyceride. These results are consistent with choline-grown cells containing less phosphatidylserine synthase activity because of lower amounts of enzyme present or perhaps less active enzyme due to covalent modification.     

N-myristoyltransferase 1 is essential in early mouse development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.     

Regulation of phosphatidylserine synthase from Saccharomyces cerevisiae by phospholipid precursors.

The addition of ethanolamine or choline to inositol-containing growth medium of Saccharomyces cerevisiae wild-type cells resulted in a reduction of membrane-associated phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) activity in cell extracts. The reduction of activity did not occur when inositol was absent from the growth medium. Under the growth conditions where a reduction of enzyme activity occurred, there was a corresponding qualitative reduction of enzyme subunit as determined by immunoblotting with antiserum raised against purified phosphatidylserine synthase. Water-soluble phospholipid precursors did not effect purified phosphatidylserine synthase activity. Phosphatidylserine synthase (activity and enzyme subunit) was not regulated by the availability of water-soluble phospholipid precursors in S. cerevisiae VAL2C(YEp CHO1) and the opi1 mutant. VAL2C(YEp CHO1) is a plasmid-bearing strain that over produces phosphatidylserine synthase activity, and the opi1 mutant is an inositol biosynthesis regulatory mutant. The results of this study suggest that the regulation of phosphatidylserine synthase by the availability of phospholipid precursors occurs at the level of enzyme formation and not at the enzyme activity level. Furthermore, the regulation of phosphatidylserine synthase is coupled to inositol synthesis.     

Regulation of CDP-diacylglycerol synthesis and utilization by inositol and choline in Schizosaccharomyces pombe.

CDP-diacylglycerol (CDP-DG) is an important branchpoint intermediate in eucaryotic phospholipid biosynthesis and could be a key regulatory site in phospholipid metabolism. Therefore, we examined the effects of growth phase, phospholipid precursors, and the disruption of phosphatidylcholine (PC) synthesis on the membrane-associated phospholipid biosynthetic enzymes CDP-DG synthase, phosphatidylglycerolphosphate (PGP) synthase, phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase in cell extracts of the fission yeast Schizosaccharomyces pombe. In complete synthetic medium containing inositol, maximal expression of CDP-DG synthase, PGP synthase, PI synthase, and PS synthase in wild-type cells occurred in the exponential phase of growth and decreased two- to fourfold in the stationary phase of growth. In cells starved for inositol, this decrease in PGP synthase, PI synthase, and PS synthase expression was not observed. Starvation for inositol resulted in a twofold derepression of PGP synthase and PS synthase expression, while PI synthase expression decreased initially and then remained constant. Upon the addition of inositol to inositol-starved cells, there was a rapid and continued increase in PI synthase expression. We examined expression of these enzymes in cho2 and cho1 mutants, which are blocked in the methylation pathway for synthesis of PC. Choline starvation resulted in a decrease in PS synthase and CDP-DG synthase expression in cho1 but not cho2 cells. Expression of PGP synthase and PI synthase was not affected by choline starvation. Inositol starvation resulted in a 1.7-fold derepression of PGP synthase expression in cho2 but not cho1 cells when PC was synthesized. PS synthase expression was not depressed, while CDP-DG synthase and PI synthase expression decreased in cho2 and cho1 cells in the absence of inositol. These results demonstrate that (i) CDP-DG synthase, PGP synthase, PI synthase, and PS synthase are similarly regulated by growth phase; (ii) inositol affects the expression of PGP synthase, PI synthase, and PS synthase; (iii) disruption of the methylation pathway results in aberrant patterns of regulation of growth phase and phospholipid precursors. Important differences between S. pombe and Saccharomyces cerevisiae with regard to regulation of these enzymes are discussed.