Phosphatidylserine Synthase Activity Research Articles

Solubilization, purification, and characterization of the hexameric form of phosphatidylserine synthase from Candida albicans

Phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals against systemic candidiasis. Rational drug design or small molecule screening are effective ways to identify specific inhibitors of Cho1, but both will be facilitated by protein purification. Due to the transmembrane nature of Cho1, methods were needed to solubilize and purify the native form of Cho1. Here, we used six non-ionic detergents and three styrene maleic acids (SMAs) to solubilize an HA-tagged Cho1 protein from the total microsomal fractions. Blue native PAGE and immunoblot analysis revealed a single band corresponding to Cho1 in all detergent-solubilized fractions, while two bands were present in the SMA2000-solubilized fraction. Our enzymatic assay suggests that digitonin-or DDM-solubilized enzyme has the most PS synthase activity. Pull-downs of HA-tagged Cho1 from the digitonin-solubilized fraction reveal an apparent MW of Cho1 consistent with a hexamer. Furthermore, negative-staining electron microscopy analysis and AlphaFold2 structure prediction modeling suggest the hexamer is composed of a trimer of dimers. We purified Cho1 protein to near-homogeneity as a hexamer using affinity chromatography and TEV protease treatment, and optimized Cho1 enzyme activity for manganese and detergent concentrations, temperature (24 °C), and pH (8.0). The purified Cho1 has a Km for its substrate CDP-diacylglycerol of 72.20μM with a Vmax of 0.079nmol/(μg∗min) while exhibiting a sigmoidal kinetic curve for its other substrate serine, indicating cooperative binding. Purified hexameric Cho1 can potentially be used in downstream structure determination and small drug screening.

Solubilization and purification of phosphatidylserine synthase from Candida albicans

Systemic infections by Candida spp. are associated with high mortality rates, and this is related, in part, to limitations in current antifungals. In this regard, the phosphatidylserine (PS) synthase from Candida albicans, encoded by the CHO1 gene, has been identified as a potential drug target for new antifungals. The reasons include: 1) Cho1 is required for oral and systemic Candida infection in the mouse model and is essential for viability in the fungal pathogen Cryptococcus neoformans; 2) this gene and gene product is absent in humans, and 3) it is a highly conserved protein among many human fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Small molecule screening is one way to develop specific inhibitors. This requires protein purification of the drug target. Due to the transmembrane nature of Cho1, therefore, we aim to solubilize and purify the native form of the Cho1 protein. Initially, four nonionic detergents (Digitonin, n‐Dodecyl‐b‐D‐ Maltopyranoside [DDM], n‐Tetradecyl‐b‐D‐Maltopyranoside [TDM], and Triton X‐100) were used to solubilize an HA‐tagged Cho1 protein from the total microsomal fraction. Analysis by Blue Native PAGE (BN‐PAGE) and immunoblotting revealed a single band corresponding to Cho1 protein in these four solubilized fractions. A radiochemical enzymatic assay suggests that the digitonin‐ and DDM‐solubilized fractions have more PS synthase activity than the TDM‐ and Triton X‐100‐ solubilized fractions. Using a pulldown assay for the HA‐tagged Cho1 in the digitonin‐solubilized fraction reveals a single band in BN‐PAGE, and a subsequent 2D gel electrophoresis confirms the purity of this protein following silver staining. The comparative mobility by BN‐PAGE gives an apparent MW of Cho1 of ~178 kDa. Based on the amino acid sequence, this would suggest a hexameric form of the CandidaCho1. This is quite different from the structure of other membrane phospholipid synthases, which all appear to be dimers. Preliminary negative staining and TEM single particle analysis suggests the presence of a hexamer population of Cho1 complex in the pulldown sample. The hexametric Cho1 is predicted to be assembled as a trimer of dimers. As expected, a subsequent 2D particle classification on the TEM images has shown different assemblies of Cho1 dimers, mostly trimers of dimers, strengthening the claim that Cho1 forms native trimers of dimers.In order to achieve large‐scale purification for small molecule screening, three protein affinity tags (His8‐, GST‐, & MBP‐tag) were added to the Cho1 protein, and a TEV protease recognition sequence was also added between Cho1 protein and the tags. All three different versions of Cho1 protein showed expression, but His8‐tagged Cho1 stands out among the others because it retains protein activity and is compatible with digitonin. Though nonspecific bands exist in the initial His‐tag affinity purification, a subsequent TEV protease treatment and a second His‐tag affinity purification were able to clear the nonspecific bands and result in a very homogeneous band of Cho1 protein. In sum, we are able to achieve an over‐500‐fold and microgram‐scale purification of Cho1. We plan to further characterize the pure Cho1 protein and use it in future small molecule screening.

Mapping the Substrate-Binding Sites in the Phosphatidylserine Synthase in Candida albicans.

The fungal phosphatidylserine (PS) synthase, a membrane protein encoded by the CHO1 gene, is a potential drug target for pathogenic fungi, such as Candida albicans. However, both substrate-binding sites of C. albicans Cho1 have not been characterized. Cho1 has two substrates: cytidyldiphosphate-diacylglycerol (CDP-DAG) and serine. Previous studies identified a conserved CDP-alcohol phosphotransferase (CAPT) binding motif, which is present within Cho1. We tested the CAPT motif for its role in PS synthesis by mutating conserved residues using alanine substitution mutagenesis. PS synthase assays revealed that mutations in all but one conserved amino acid within the CAPT motif resulted in decreased Cho1 function. In contrast, there were no clear motifs in Cho1 for binding serine. Therefore, to identify the serine binding site, PS synthase sequences from three fungi were aligned with sequences of a similar enzyme, phosphatidylinositol (PI) synthase, from the same fungi. This revealed a motif that was unique to PS synthases. Using alanine substitution mutagenesis, we found that some of the residues in this motif are required for Cho1 function. Two alanine substitution mutants, L184A and R189A, exhibited contrasting impacts on PS synthase activity, and were characterized for their Michaelis-Menten kinetics. The L184A mutant displayed enhanced PS synthase activity and showed an increased V max. In contrast, R189A showed decreased PS synthase activity and increased K m for serine, suggesting that residue R189 is involved in serine binding. These results help to characterize PS synthase substrate binding, and should direct rational approaches for finding Cho1 inhibitors that may lead to better antifungals.

Identification of the substrate‐binding sites in the phosphatidylserine synthase from Candida albicans

Systemic infections by Candida spp. have high mortality rates, and this is related, in part, to limitations in current antifungals. Thus, there is a need for novel antifungals. The phosphatidylserine (PS) synthase (annotated Cho1) from C. albicans has been identified as a potential drug target because [1] it is required for virulence in mouse models of Candidiasis, [2] it is absent in humans, and [3] it is highly conserved among fungal pathogens. Inhibitors of Cho1 could be used as lead compounds for drug development. Rational drug design is one way to develop specific inhibitors, but neither the substrate-enzyme interactions nor the substrate-binding sites have been well-defined for Cho1. Therefore, we aim to biochemically identify and characterize the substrate-binding sites of Cho1, which binds cytidine-diphosphate-diacylglycerol (CDP-DAG) and serine, and catalyzes their condensation to form PS. For the CDP-DAG-binding site, a conserved CDP-alcohol-binding (CAPT) motif is present in Cho1. We tested this site for its role in PS synthesis by mutating the conserved residues using standard alanine-scanning mutagenesis, and using two assays to measure the impact of these mutations on PS synthesis. The first assay measures the impact of PS synthesis in whole cells based on their growth. PS is used to synthesize phosphatidylethanolamine (PE), and in the absence of PS, C. albicans must acquire exogenous ethanolamine to make PE by a salvage pathway. Thus, mutants lacking PS synthase activity grow very poorly in media lacking ethanolamine. The second assay measures PS synthesis directly in crude membranes based on incorporation of [3H]serine into [3H]PS. These experiments revealed that, with the exception of one residue (R133), all alanine substitutions of the conserved amino acids within the fungal CAPT motif showed decreased Cho1 function. A subsequent R133E mutant displayed reduced activity compared to R133A, indicating residue R133 interacts with CDP-DAG in a dispensable manner. For the serine-binding site, by using computational tools, we predicted potential serine-binding residues. Using alanine-scanning mutagenesis, we found that some of these predicted residues are required for Cho1 function. Two of the alanine substitution mutants, L184A and R189A, were characterized regarding their Michaelis-Menten kinetics. The mutant L184A, which displayed enhanced activity both for growth and in vitro PS synthase activity, showed an increased Vmax and an unchanged Km. The gain-of-function mechanism of this mutation is currently unknown. On the contrary, R189A, which showed decreased growth and in vitro PS synthase activity, increased the Km for serine but not Vmax, indicating that the residue R189 is involved in serine-binding. We plan to continue to determine if other potential residues directly bind serine, as this could be useful in future rational inhibitor design studies.

Phosphatidate phosphatase regulates membrane phospholipid synthesis via phosphatidylserine synthase

The yeast Saccharomyces cerevisiae serves as a model eukaryote to elucidate the regulation of lipid metabolism. In exponentially growing yeast, a diverse set of membrane lipids are synthesized from the precursor phosphatidate via the liponucleotide intermediate CDP-diacylglycerol. As cells exhaust nutrients and progress into the stationary phase, phosphatidate is channeled via diacylglycerol to the synthesis of triacylglycerol. The CHO1-encoded phosphatidylserine synthase, which catalyzes the committed step in membrane phospholipid synthesis via CDP-diacylglycerol, and the PAH1-encoded phosphatidate phosphatase, which catalyzes the committed step in triacylglycerol synthesis are regulated throughout cell growth by genetic and biochemical mechanisms to control the balanced synthesis of membrane phospholipids and triacylglycerol. The loss of phosphatidate phosphatase activity (e.g., pah1Δ mutation) increases the level of phosphatidate and its conversion to membrane phospholipids by inducing Cho1 expression and phosphatidylserine synthase activity. The regulation of the CHO1 expression is mediated through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. Consequently, phosphatidate phosphatase activity regulates phospholipid synthesis through the transcriptional regulation of the phosphatidylserine synthase enzyme.

Yeast PAH1-encoded phosphatidate phosphatase controls the expression of CHO1-encoded phosphatidylserine synthase for membrane phospholipid synthesis

The PAH1-encoded phosphatidate phosphatase (PAP), which catalyzes the committed step for the synthesis of triacylglycerol in Saccharomyces cerevisiae, exerts a negative regulatory effect on the level of phosphatidate used for the de novo synthesis of membrane phospholipids. This raises the question whether PAP thereby affects the expression and activity of enzymes involved in phospholipid synthesis. Here, we examined the PAP-mediated regulation of CHO1-encoded phosphatidylserine synthase (PSS), which catalyzes the committed step for the synthesis of major phospholipids via the CDP-diacylglycerol pathway. The lack of PAP in the pah1Δ mutant highly elevated PSS activity, exhibiting a growth-dependent up-regulation from the exponential to the stationary phase of growth. Immunoblot analysis showed that the elevation of PSS activity results from an increase in the level of the enzyme encoded by CHO1 Truncation analysis and site-directed mutagenesis of the CHO1 promoter indicated that Cho1 expression in the pah1Δ mutant is induced through the inositol-sensitive upstream activation sequence (UASINO), a cis-acting element for the phosphatidate-controlled Henry (Ino2-Ino4/Opi1) regulatory circuit. The abrogation of Cho1 induction and PSS activity by a CHO1 UASINO mutation suppressed pah1Δ effects on lipid synthesis, nuclear/endoplasmic reticulum membrane morphology, and lipid droplet formation, but not on growth at elevated temperature. Loss of the DGK1-encoded diacylglycerol kinase, which converts diacylglycerol to phosphatidate, partially suppressed the pah1Δ-mediated induction of Cho1 and PSS activity. Collectively, these data showed that PAP activity controls the expression of PSS for membrane phospholipid synthesis.

Phosphatidylserine synthesis at membrane contact sites promotes its transport out of the ER

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments.

Phosphatidate Phosphatase Plays Role in Zinc-mediated Regulation of Phospholipid Synthesis in Yeast

In the yeast Saccharomyces cerevisiae, the synthesis of phospholipids is coordinately regulated by mechanisms that control the homeostasis of the essential mineral zinc (Carman, G.M., and Han, G. S. (2007) Regulation of phospholipid synthesis in Saccharomyces cerevisiae by zinc depletion. Biochim. Biophys. Acta 1771, 322-330; Eide, D. J. (2009) Homeostatic and adaptive responses to zinc deficiency in Saccharomyces cerevisiae. J. Biol. Chem. 284, 18565-18569). The synthesis of phosphatidylcholine is balanced by the repression of CDP-diacylglycerol pathway enzymes and the induction of Kennedy pathway enzymes. PAH1-encoded phosphatidate phosphatase catalyzes the penultimate step in triacylglycerol synthesis, and the diacylglycerol generated in the reaction may also be used for phosphatidylcholine synthesis via the Kennedy pathway. In this work, we showed that the expression of PAH1-encoded phosphatidate phosphatase was induced by zinc deficiency through a mechanism that involved interaction of the Zap1p zinc-responsive transcription factor with putative upstream activating sequence zinc-responsive elements in the PAH1 promoter. The pah1Δ mutation resulted in the derepression of the CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol pathway enzyme) and loss of the zinc-mediated regulation of the enzyme. Loss of phosphatidate phosphatase also resulted in the derepression of the CKI1-encoded choline kinase (Kennedy pathway enzyme) but decreased the synthesis of phosphatidylcholine when cells were deficient of zinc. This result confirmed the role phosphatidate phosphatase plays in phosphatidylcholine synthesis via the Kennedy pathway.

Phosphorylation of Yeast Phosphatidylserine Synthase by Protein Kinase A: IDENTIFICATION OF SER46 AND SER47 AS MAJOR SITES OF PHOSPHORYLATION

The CHO1-encoded phosphatidylserine synthase from Saccharomyces cerevisiae is phosphorylated and inhibited by protein kinase A in vitro. CHO1 alleles bearing Ser(46) --> Ala and/or Ser(47) --> Ala mutations were constructed and expressed in a cho1Delta mutant lacking phosphatidylserine synthase. In vitro, the S46A/S47A mutation reduced the total amount of phosphorylation by 90% and abolished the inhibitory effect protein kinase A had on phosphatidylserine synthase activity. The enzyme phosphorylation by protein kinase A, which was time- and dose-dependent and dependent on the concentration of ATP, caused a electrophoretic mobility shift from a 27-kDa form to a 30-kDa form. The two electrophoretic forms of phosphatidylserine synthase were present in exponential phase cells, whereas only the 27-kDa form was present in stationary phase cells. In vivo labeling with (32)P(i) and immune complex analysis showed that the 30-kDa form was predominantly phosphorylated when compared with the 27-kDa form. However, the S46A/S47A mutations abolished the protein kinase A-mediated electrophoretic mobility shift. The S46A/S47A mutations also caused a 55% reduction in the total amount of phosphatidylserine synthase in exponential phase cells and a 66% reduction in the amount of enzyme in stationary phase cells. In phospholipid composition analysis, cells expressing the S46A/S47A mutant enzyme exhibited a 57% decrease in phosphatidylserine and a 40% increase in phosphatidylinositol. These results indicate that phosphatidylserine synthase is phosphorylated on Ser(46) and Ser(47) by protein kinase A, which results in a higher amount of enzyme for the net effect of stimulating the synthesis of phosphatidylserine.

Role of PS Product Inhibition in Accumulation of Phosphatidylserine (PS) in Neuronal Membranes

PS is a major membrane component in mammalian tissues. Neuronal tissues are highly enriched with docosahexaenoic acid (DHA, 22:6n‐3) and PS in comparison to non‐neuronal tissues. Such concentration of DHA and PS is known to be important for proper neuronal functions. In mammalian cells, PS synthase (PSS) 1 and 2 catalyze PS synthetic activity, which is inhibited by the product PS through the feedback mechanism. We have previously found that DHA containing PS species (DHA‐PS) highly accumulates in neuronal membranes mainly due to the substrate preference in PS synthesis towards DHA phospholipid species. To understand the biochemical mechanisms underlying enrichment of PS in neuronal membranes, we hypothesized that fatty acyl composition‐dependent product inhibition in PS synthesis also plays a role in PS accumulation. We found that DHA‐PS has weaker inhibitory effect compared to oleic acid (OA, 18:1n‐9) containing PS species (OA‐PS) when tested with mouse liver microsomes. Consistently, DHA‐PS inhibited PSS activity less effectively than OA‐PS when flag‐PSS2 were expressed in mammalian cells, successfully immuno‐purified, and subsequently used as an enzyme source. DHA‐PS as a weaker inhibitor of PSS together with the high specificity towards DHA‐PS synthesis may contribute to the high PS accumulation in neuronal membranes.Intramural Program of National Institutes of Health

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Expression, Purification, and Characterization of Phosphatidylserine Synthase from Escherichia coli K12 in Bacillus subtilis

Although phosphatidylserine synthase (PSS) from Escherichia coli is an ideal enzyme for phospholipid production, its application in the food industry has been limited because of the low PSS yield. In this study, the pss gene was cloned from E. coli K(12) and expressed in Bacillus subtilis DB104, and the recombinant PSS was characterized subsequently. PSS was purified to 39.59-fold, and the highest activity was detected as 13.62 U/mg. The enzyme was found to be stable in a pH range of 6.5-9.5, with optimal pH values of 8.0 for hydrolysis and 7.0 for transphosphatidylation, respectively. The optimal temperature for PSS activity was 35 degrees C. The enzyme activity could be detected after 1 h of heating at 65 degrees C. Among the detected detergents and metal ions, Triton X-100, Ca(2+), Mn(2+), and Co(2+) could improve PSS activity. The transformation of phosphatidylcholine to phosphatidylserine under PSS catalyzation was carried out in a biphasic system, which confirmed the actual catalyzing ability of the recombinant protein.

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.

Lysophosphatidylcholine Metabolism in Saccharomyces cerevisiae: THE ROLE OF P-TYPE ATPases IN TRANSPORT AND A BROAD SPECIFICITY ACYLTRANSFERASE IN ACYLATION

We recently described a new route for the synthesis of phosphatidylethanolamine (PtdEtn) from exogenous lyso-PtdEtn, which we have termed the exogenous lysolipid metabolism (ELM) pathway. The ELM pathway for lyso-PtdEtn requires the action of plasma membrane P-type ATPases Dnf1p and Dnf2p and their requisite beta-subunit, Lem3p, for the active uptake of lyso-PtdEtn. In addition, the acyl-CoA-dependent acyltransferase, Ale1p, mediates the acylation of the imported lysolipid to form PtdEtn. We now report that these components of the lyso-PtdEtn ELM pathway are also active with lyso-1-acyl-2-hydroxyl-sn-glycero-3-phosphocholine (PtdCho) as a substrate. Lyso-PtdCho supports the growth of a choline auxotrophic pem1Delta pem2Delta strain. Uptake of radiolabeled lyso-PtdCho was impaired by the dnf2Delta and lem3Delta mutations. Introduction of a lem3Delta mutation into a pem1Delta pem2Delta background impaired the ability of the resulting strain to grow with lyso-PtdCho as the sole precursor of PtdCho. After import of lyso-PtdCho, the recently characterized lyso-PtdEtn acyltransferase, Ale1p, functioned as the sole lyso-PtdCho acyltransferase in yeast. A pem1Delta pem2Delta ale1Delta strain grew with lyso-PtdCho as a substrate but showed a profound reduction in PtdCho content when lyso-PtdCho was the only precursor of PtdCho. Ale1p acylates lyso-PtdCho with a preference for monounsaturated acyl-CoA species, and the specific LPCAT activity of Ale1p in yeast membranes is >50-fold higher than the basal rate of de novo aminoglycerophospholipid biosynthesis from phosphatidylserine synthase activity. In addition to lyso-PtdCho, lyso-PtdEtn, and lyso-phosphatidic acid, Ale1p was also active with lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol as substrates. These results establish a new pathway for the net synthesis of PtdCho in yeast and provide new tools for the study of PtdCho synthesis, transport, and remodeling.

Respiratory Deficiency Mediates the Regulation of CHO1-encoded Phosphatidylserine Synthase by mRNA Stability in Saccharomyces cerevisiae

The CHO1-encoded phosphatidylserine synthase (CDP-diacylglycerol:l-serine O-phosphatidyltransferase, EC 2.7.8.8) is one of the most highly regulated phospholipid biosynthetic enzymes in the yeast Saccharomyces cerevisiae. CHO1 expression is regulated by nutrient availability through a regulatory circuit involving a UAS(INO) cis-acting element in the CHO1 promoter, the positive transcription factors Ino2p and Ino4p, and the transcriptional repressor Opi1p. In this work, we examined the post-transcriptional regulation of CHO1 by mRNA stability. CHO1 mRNA was stabilized in mutants defective in deadenylation (ccr4Delta), mRNA decapping (dcp1), and the 5'-3'-exonuclease (xrn1), indicating that the CHO1 transcript is primarily degraded through the general 5'-3' mRNA decay pathway. In respiratory-sufficient cells, the CHO1 transcript was moderately stable with a half-life of 12 min. However, the CHO1 transcript was stabilized to a half-life of >45 min in respiratory-deficient (rho(-) and rho(o)) cells, the cox4Delta mutant defective in the cytochrome c oxidase, and wild type cells treated with KCN (a cytochrome c oxidase inhibitor). The increased CHO1 mRNA stability in response to respiratory deficiency caused increases in CHO1 mRNA abundance, phosphatidylserine synthase protein and activity, and the synthesis of phosphatidylserine in vivo. Respiratory deficiency also caused increases in the activities of CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, and the phospholipid methyltransferases. Phosphatidylinositol synthase and choline kinase activities were not affected by respiratory deficiency. This work advances our understanding of phosphatidylserine synthase regulation and underscores the importance of mitochondrial respiration to the regulation of phospholipid synthesis in S. cerevisiae.

Functional Characterization of the Fission Yeast Phosphatidylserine Synthase Gene, pps1 , Reveals Novel Cellular Functions for Phosphatidylserine

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.

Lipid dependence and activity control of phosphatidylserine synthase from Escherichia coli

The activity of phosphatidylserine synthase from Escherichia coli depends significantly on the nature and level of the lipids in the matrix, at which the enzyme is operating. To elucidate the role of anionic lipids in the regulation of PtdSer synthase, its activity was studied in mixed micelles containing phosphatidylglycerol (one charge) or diphosphatidylglycerol (two charges), the two main anionic membrane lipids in E. coli. Membrane association and activity of PtdSer synthase were increased by the two lipids, indicating their essential role in the positive regulation mechanism of the phosphatidylethanolamine level in the E. coli membrane.

A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Regulation of the DPP1-encoded Diacylglycerol Pyrophosphate (DGPP) Phosphatase by Inositol and Growth Phase: INHIBITION OF DGPP PHOSPHATASE ACTIVITY BY CDP-DIACYLGLYCEROL AND ACTIVATION OF PHOSPHATIDYLSERINE SYNTHASE ACTIVITY BY DGPP

The regulation of the Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate (DGPP) phosphatase by inositol supplementation and growth phase was examined. Addition of inositol to the growth medium resulted in a dose-dependent increase in the level of DGPP phosphatase activity in both exponential and stationary phase cells. Activity was greater in stationary phase cells when compared with exponential phase cells, and the inositol- and growth phase-dependent regulations of DGPP phosphatase were additive. Analyses of DGPP phosphatase mRNA and protein levels, and expression of beta-galactosidase activity driven by a P(DPP1)-lacZ reporter gene, indicated that a transcriptional mechanism was responsible for this regulation. Regulation of DGPP phosphatase by inositol and growth phase occurred in a manner that was opposite that of many phospholipid biosynthetic enzymes. Regulation of DGPP phosphatase expression by inositol supplementation, but not growth phase, was altered in opi1Delta, ino2Delta, and ino4Delta phospholipid synthesis regulatory mutants. CDP-diacylglycerol, a phospholipid pathway intermediate used for the synthesis of phosphatidylserine and phosphatidylinositol, inhibited DGPP phosphatase activity by a mixed mechanism that caused an increase in K(m) and a decrease in V(max). DGPP stimulated the activity of pure phosphatidylserine synthase by a mechanism that increased the affinity of the enzyme for its substrate CDP-diacylglycerol. Phospholipid composition analysis of a dpp1Delta mutant showed that DGPP phosphatase played a role in the regulation of phospholipid metabolism by inositol, as well as regulating the cellular levels of phosphatidylinositol.

Light exposure activates retina ganglion cell lysophosphatidic acid acyl transferase and phosphatidic acid phosphatase by a c-fos-dependent mechanism.

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.