The pgpA and pgpB genes of Escherichia coli are not essential: evidence for a third phosphatidylglycerophosphate phosphatase.

To further define the genes and gene products responsible for the in vivo conversion of phosphatidylglycerophosphate to phosphatidylglycerol in Escherichia coli, we disrupted two genes (pgpA and pgpB) which had previously been shown to encode gene products which carried out this reaction in vitro (T. Icho and C. R. H. Raetz, J. Bacteriol. 153:722-730, 1983). Strains with either gene or both genes disrupted had the same properties as the original mutants isolated with mutations in these genes, i.e., reduced in vitro phospholipid phosphatase activities, normal growth properties, and an increase in the level of phosphatidylglycerophosphate (1.6% versus less than 0.1% in wild-type strains). These results demonstrate that these genes are not required for either normal cell growth or the biosynthesis of phosphatidylglycerol in vivo. In addition, the total phosphatidylglycerophosphate phosphatase activity in the doubly disrupted mutant was reduced by only 50%, which indicates that there is at least one other gene that encodes such an activity and thus accounts for the lack of a dramatic effect on the biosynthesis of anionic phospholipids in these mutant strains. The phosphatidic acid and lysophosphatidic acid phosphatase activities of the pgpB gene product were also significantly reduced in gene-interrupted mutants, but the detection of residual phosphatase activities in these mutants indicated that additional genes encoding such phosphatases exist. The lack of a significant phenotype resulting from disruption of the pgpA and pgpB genes indicates that these genes may be required only for nonessential cell function and leaves the biosynthesis of phosphatidylglycerophosphate as the only step in E. coli phospholipid biosynthesis for which a gene locus has not been identified.     

Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli

The phospholipids of Escherichia coli consist mainly of phosphatidylethanolamine, phosphatidylglycerol (PG), and cardiolipin. PG makes up ～25% of the cellular phospholipid and is essential for growth in wild-type cells. PG is synthesized on the inner surface of the inner membrane from cytidine diphosphate-diacylglycerol and glycerol 3-phosphate, generating the precursor phosphatidylglycerol-phosphate (PGP). This compound is present at low levels (～0.1% of the total lipid). Dephosphorylation of PGP to PG is catalyzed by several PGP-phosphatases. The pgpA and pgpB genes, which encode structurally distinct PGP-phosphatases, were identified previously. Double deletion mutants lacking pgpA and pgpB are viable and still make PG, suggesting the presence of additional phosphatase(s). We have identified a third PGP-phosphatase gene (previously annotated as yfhB but renamed pgpC) using an expression cloning strategy. A mutant with deletions in all three phosphatase genes is not viable unless covered by a plasmid expressing either pgpA, pgpB, or pgpC. When the triple mutant is covered with the temperature-sensitive plasmid pMAK705 expressing any one of the three pgp genes, the cells grow at 30 but not 42 °C. As growth slows at 42 °C, PGP accumulates to high levels, and the PG content declines. PgpC orthologs are present in many other bacteria.     

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpB gene and dual subcellular localization of the pgpB product.

The phosphatidyl glycerophosphate B phosphatase of Escherichia coli has a multiple substrate specificity and a peculiar dual subcellular localization in the envelope. Its phosphatidyl glycerophosphate phosphatase activity is higher in the cytoplasmic membrane, while phosphatidic acid and lysophosphatidic acid phosphatase activities are higher in the outer membrane. The DNA sequencing of the pgpB gene revealed a protein of 251 amino acids which had at least five hydrophobic membrane-spanning regions. About 37 hydrophilic residues in the middle of the sequence had considerable homology with the C-terminal conserved region of the ras family genes in eucaryotes. A protein of 28,000 daltons was expressed from the pgpB gene under a tac promoter in a runaway replication plasmid. This overproduced protein also revealed the dual subcellular localization.     

Identification of multiple genes encoding membrane proteins with undecaprenyl pyrophosphate phosphatase (UppP) activity in Escherichia coli

The bacA gene product of Escherichia coli was recently purified to near homogeneity and identified as an undecaprenyl pyrophosphate phosphatase activity (El Ghachi, M., Bouhss, A., Blanot, D., and Mengin-Lecreulx, D. (2004) J. Biol. Chem. 279, 30106-30113). The enzyme function is to synthesize the carrier lipid undecaprenyl phosphate that is essential for the biosynthesis of peptidoglycan and other cell wall components. The inactivation of the chromosomal bacA gene was not lethal but led to a significant, but not total, depletion of undecaprenyl pyrophosphate phosphatase activity in E. coli membranes, suggesting that other(s) protein(s) should exist and account for the residual activity and viability of the mutant strain. Here we report that inactivation of two additional genes, ybjG and pgpB, is required to abolish growth of the bacA mutant strain. Overexpression of either of these genes, or of a fourth identified one, yeiU, is shown to result in bacitracin resistance and increased levels of undecaprenyl pyrophosphate phosphatase activity, as previously observed for bacA. A thermosensitive conditional triple mutant delta bacA,delta ybjG,delta pgpB in which the expression of bacA is impaired at 42 degrees C was constructed. This strain was shown to accumulate soluble peptidoglycan nucleotide precursors and to lyse when grown at the restrictive temperature, due to the depletion of the pool of undecaprenyl phosphate and consequent arrest of cell wall synthesis. This work provides evidence that two different classes of proteins exhibit undecaprenyl pyrophosphate phosphatase activity in E. coli and probably other bacterial species; they are the BacA enzyme and several members from a superfamily of phosphatases that, different from BacA, share in common a characteristic phosphatase sequence motif.     

Export of mitochondrially synthesized lysophosphatidic acid

We have previously demonstrated that the properties of mitochondrial glycerophosphate acyltransferase are in keeping with the asymmetric distribution of fatty acids found in naturally occurring cell glycerophospholipids. We are now examining if mitochondria can export lysophosphatidic acid and if it is converted to other phospholipids by the microsomes. Rat liver mitochondria were incubated for 3 min with [2-3H]-sn-glycerol 3-phosphate, palmityl-CoA, and N-ethylmaleimide in the acyltransferase assay medium. In the absence of bovine serum albumin in the medium, greater than 80% of the phospholipids sedimented with the mitochondria. In the presence of the albumin, the lysophosphatidic acid was present entirely in the supernatant fluid. The very little phosphatidic acid that was formed sedimented with the mitochondria. Addition of microsomes to the supernatant fluid followed by a further incubation of 5 min converted 61% of the lysophosphatidic acid to phosphatidic acid which sedimented with the microsomes. When mitochondria and microsomes were incubated together in the assay medium containing albumin and N-ethylmaleimide, the product contained more phosphatidic and less lysophosphatidic acid. When the subcellular components were reisolated by differential centrifugation, 70% of the phosphatidic acid sedimented with the microsomes and the lysophosphatidic acid stayed in the postmicrosomal supernatant. Thus, under appropriate conditions mitochondrially produced lysophosphatidic acid can leave the organelles and this phospholipid can be converted to phosphatidic acid by the microsomes.     

Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae.

Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis. In lipid particles of the s1c1 disruptant YMN5 (M. M. Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid. Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles. Lipid particles of a second mutant strain, TTA1 (T. S. Tillman and R. M. Bell, J. Biol. Chem. 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro. Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1. These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p. Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid. As a consequence, an s1c1 gat1 double mutant is also viable. Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid. Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.     

Purification and properties of phosphatidic acid phosphatase from porcine thymus membranes.

We purified phosphatidic acid phosphatase (EC 3.1.3.4) 2300-fold from porcine thymus membranes. The enzyme was solubilized with beta-octyl glucoside and Triton X-100 and fractionated with ammonium sulfate. The purification was then achieved by chromatography in the presence of Triton X-100 with Sephacryl S-300, hydroxylapatite, heparin-Sepharose, and Affi-Gel Blue. The final enzyme preparation gave a single band of M(r) = 83,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The native enzyme, on the other hand, was eluted at M(r) = 218,000 in gel filtration chromatography with Superose 12 in the presence of Triton X-100. The enzyme was judged to be specific to phosphatidic acid, since excess amounts of dicetylphosphate or lysophosphatidic acid did not inhibit the enzyme activity. In this respect, the enzyme was inhibited by 1,2-diacylglycerol but not by 1- or 2-monoacylglycerol and triacylglycerol. The enzyme required Triton X-100 or deoxycholate for its activity. Although the enzyme appeared to be an integral membrane protein, we could not detect its phospholipid dependencies. The activity was independent of Mg2+, and other cations were strongly inhibitory. The specific enzyme activity was 15 mumol/min/mg of protein when assayed using phosphatidic acid as Triton X-100 mixed micelles. The Km for the surface concentration of phosphatidic acid was 0.30 mol%. The enzyme was inhibited by sphingosine and chloropromazine, and less potently, by propranolol and NaF. The enzyme was insensitive to thio-reactive reagents like N-ethylmaleimide.     

Facilitated utilization of endogenously synthesized lysophosphatidic acid by 1-acylglycerophosphate acyltransferase from Escherichia coli

Complete separation of glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase from Escherichia coli was obtained by sequential extraction with Triton X-100. Solubilized glycerophosphate acyltransferase was reconstituted by the cholate dispersion and gel filtration method in small unilamellar vesicles. 1-Acylglycerophosphate acyltransferase could not be solubilized from the membranes and was used in endogenous membrane fragments after detergent removal. Mixing of the two preparations and subsequent incubation in the presence of glycerol 3-phosphate, palmitoyl-CoA and oleoyl-CoA resulted in the efficient synthesis of phosphatidic acid. Inclusion of exogenous lysophosphatitic acid in the assay medium resulted in a dilution of the newly synthesized lysophosphatidate. By contrast, the synthesis of phosphatidic acid from glycerol 3-phosphate by the acyltransferases present in native membrane vesicles was barely influenced by the presence of exogenous lysophosphatidic acid. When comparing the utilization of membrane-associated 14C-labeled and newly generated 3H-labeled lysophosphatidic acid, the latter appeared to be the preferred substrate. These results indicate that lysophosphatidic acid, synthesized by glycerophosphate acyltransferase, is utilized by 1-acylglycerophosphate acyltransferase without prior mixing with the total membrane-associated pool of lysophosphatidic acid, and suggest a close proximity of the two enzymes in native E. coli membranes. This property of the acyltransferases is lost upon separation and reconstitution of enzyme activities.     

Lysophosphatidic acids are new substrates for the phosphatase domain of soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that has a C-terminus epoxide hydrolase domain and an N-terminus phosphatase domain. The endogenous substrates of epoxide hydrolase are known to be epoxyeicosatrienoic acids, but the endogenous substrates of the phosphatase activity are not well understood. In this study, to explore the substrates of sEH, we investigated the inhibition of the phosphatase activity of sEH toward 4-methylumbelliferyl phosphate by using lecithin and its hydrolyzed products. Although lecithin itself did not inhibit the phosphatase activity, the hydrolyzed lecithin significantly inhibited it, suggesting that lysophospholipid or fatty acid can inhibit it. Next, we investigated the inhibition of phosphatase activity by lysophosphatidyl choline, palmitoyl lysophosphatidic acid, monopalmitoyl glycerol, and palmitic acid. Palmitoyl lysophosphatidic acid and fatty acid efficiently inhibited phosphatase activity, suggesting that lysophosphatidic acids (LPAs) are substrates for the phosphatase activity of sEH. As expected, palmitoyl, stearoyl, oleoyl, and arachidonoyl LPAs were efficiently dephosphorylated by sEH (Km, 3-7 μM; Vmax, 150-193 nmol/min/mg). These results suggest that LPAs are substrates of sEH, which may regulate physiological functions of cells via their metabolism.     

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

Phospholipase D Activity of Rat Brain Neuronal Nuclei

Abstract: Phospholipase D activity of rat brain neuronal nuclei, measured with exogenous phosphatidylcholine as substrate, was characterized. The measured activity of neuronal nuclei was at least 36-fold greater than the activity in glia nuclei. The pH optimum was 6.5, and unsaturated but not saturated fatty acids stimulated the enzyme. The optimal concentration of sodium oleate for stimulation of the enzyme activity was 1.2 m M in the presence of 0.75 m M phosphatidylcholine. This phospholipase D activity was cation independent. In the absence of NaF, used as a phosphatidic acid phosphatase inhibitor, the principal product was diglyceride; whereas in the presence of NaF, the principal product was phosphatidic acid. The phospholipase D, in addition to having hydrolytic activity, was able to catalyze a transphosphatidylation reaction. Maximum phosphatidylethanol formation was seen with 0.2–0.3 M ethanol. GTPγS, ATPγS, BeF₂, AIF₃, phosphatidic acid, and phosphatidylethanol inhibited the neuronal nuclei phospholipase D activity. The addition of the cytosolic fraction of brain, liver, kidney, spleen, and heart to the incubation mixtures resulted in inhibition of the phospholipase D activity. Phospholipase D activity was detectable in nuclei prepared from rat kidney, spleen, heart, and liver.     

In vivo studies on stereospecificity of the monoglyceride kinase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and CDP-diglyceride synthase of Streptococcus mutans BHT using the stereoisomers of the ether lipid, dodecylglycerol

Streptococcus mutans BHT metabolizes radioactive 3-dodecyl-sn-glycerol (sn-3-DDG) almost exclusively to lysophosphatidic acid, phosphatidic acid and 1,3-diradyl-sn-glycerol, whereas the cells of this organism metabolize 1-dodecyl-sn-glycerol (sn-1-DDG) to all of the glycerol lipids of S. mutans BHT, with the largest amounts incorporated into phosphatidylglycerol and diradylglycerol (mostly the 1,2- but also the 1,3-isomer). (The common names of lipids, such as phosphatidic acid, are used in the broader sense to mean that the lipid may contain alkyl as well as acyl groups.) The addition of an equivalent amount of nonradioactive sn-3-DDG to radioactive sn-1-DDG causes more of the radioactivity to accumulate at phosphatidic acid. These results indicate that the monoglyceride kinase (EC 2.7.1.94), lysophosphatidic acid acyltransferase (EC 2.3.1.40) and the monoglyceride acyltransferase (EC 2.3.1.22) enzymatic reactions are not stereospecific, and that the CDP-diglyceride synthase (EC 2.7.7.41) and phosphatidic acid phosphatase (EC 3.1.3.4) metabolic steps are stereospecific in S. mutans BHT. The synthesis of phosphatidic acid and lysophosphatidic acid from sn-3-DDG provides a unique method for synthesizing these glycerol lipids with the uncommon stereochemical configuration in which the phosphate moiety is in the sn-1 position.     

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.     

Isolation and localization of a cytosolic 10 S triacylglycerol biosynthetic multienzyme complex from oleaginous yeast

Triacylglycerol is one of the major storage forms of metabolic energy in eukaryotic cells. Biosynthesis of triacylglycerol is known to occur in membranes. We report here the isolation, purification, and characterization of a catalytically active cytosolic 10 S multienzyme complex for triacylglycerol biosynthesis from Rhodotorula glutinis during exponential growth. The complex was characterized and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, and acyl carrier protein. The 10 S triacylglycerol biosynthetic complex rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, and diacylglycerol acyltransferase from the complex were microsequenced. Antibodies were raised against the synthetic peptides corresponding to lysophosphatidic acid acyltransferase and phosphatidic acid phosphatase sequences. Immunoprecipitation and immunolocalization studies show the presence of a cytosolic multienzyme complex for triacylglycerol biosynthesis. Chemical cross-linking studies revealed that the 10 S multienzyme complex was held together by protein-protein interactions. These results demonstrate that the cytosol is one of the sites for triacylglycerol biosynthesis in oleaginous yeast.     

Isolation of a cDNA encoding human lysophosphatidic acid phosphatase that is involved in the regulation of mitochondrial lipid biosynthesis.

In this study, we isolated cDNA encoding lysophosphatidic acid (LPA) phosphatase (LPAP). The amino acid sequence deduced from the cDNA encoding LPAP had 421 residues including a putative signal peptide and was homologous to acid phosphatase, especially at the active site. Human LPAP had 28.5% amino acid identity to human prostatic acid phosphatase. Northern blot analysis showed a ubiquitous expression of LPAP, which was marked in kidney, heart, small intestine, muscle, and liver. Human chromosome map obtained by fluorescence in situ hybridazation showed that the gene for LPAP was localized to chromosome 1 q21. The mutant in which histidine was replaced with alanine at the active site and the putative signal peptide-deleted LPAP had no LPA phosphatase activity. In addition, the putative signal peptide-deleted LPAP showed no mitochondrial localization. The site of intracellular localization of endogenous LPAP was also mitochondria in MDCK cells and differentiated C2C12 cells. The LPAP homologous phosphatase, human prostatic acid phosphatase, also has LPA phosphatase activity. LPAP-stable transfected NIH 3T3 cells showed less phosphatidic acid, phosphatidylglycerol, and cardiolipin. These results suggested that LPAP regulates lipid metabolism in mitochondria via the hydrolysis of LPA to monoacylglycerol.     

Swiss 3T3 cells preferentially incorporate sn-2-arachidonoyl monoacylglycerol into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.

The sn-1-stearoyl-2-arachidonoyl phospholipids of animal cells appear to be formed by special mechanisms. To determine whether monoacylglycerol (MG) incorporation pathways are involved we incubated quiescent Swiss 3T3 cells with [3H]glycerol-labeled sn-2-arachidonoyl MG, then analyzed the radioactive cell lipids that accumulated. We also examined cell homogenates to identify enzyme activities that might promote the incorporation of sn-2-arachidonoyl MG into other cell lipids. The cell incubation experiments demonstrated rapid labeling of several lipids, including diacylglycerol, lysophosphatidic acid, phosphatidic acid, and phosphatidylinositol. They also demonstrated selective labeling of sn-1-stearoyl-2-arachidonoyl species of phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. The cell homogenate experiments identified an sn-2-acyl MG acyltransferase activity, an MG kinase activity that phosphorylates sn-2-arachidonoyl MG in preference to sn-2-oleoyl MG, and a stearoyl-specific acyl transferase activity that converts sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidic acid. The results also showed that this stearoyl transferase could act with other enzymes to convert sn-2-arachidonoyl lysophosphatidic acid into sn-1-stearoyl-2-arachidonoyl phosphatidylinositol. The combined results indicate that Swiss 3T3 cells incorporate sn-2-arachidonoyl MG into phospholipids by at least two different pathways, including one that specifically forms sn-1-stearoyl-2-arachidonoyl phosphatidylinositol.     

Triacylglycerol synthesis in lipid particles from baker's yeast (Saccharomyces cerevisiae).

Triacylglycerol synthesis has been studied in a lipid particle preparation of baker's yeast (Saccharomyces cerevisiae), and compared with the synthesis in other subcellular fractions. Fatty acid-CoA ligase (AMP) (EC 6.2.1.3) activity and sn-glycerol 3-phosphate acyltransferase activity (EC 2.3.1.15) were present in all the subcellular fractions tested but the highest specific activities of both enzymes were observed with the lipid particle fraction. The products of the glycerol 3-phosphate acylation indicate that triacyglycerol synthesis proceeds through the phosphatidic acid pathway. However, only a small and nearly constant amount of lysophosphatidic acid was found with the lipid particle fraction while the other subcellular fraction produced lysophosphatidic and phosphatidic acid with a more pronounced precursor/product relationship. Triacylglycerol synthesis from endogenous diacylglycerol present in the lipid particle was also demonstrated.     

Characterization of dolichol and dolichyl phosphate phosphatase from soya beans (Glycine max).

A series of polyprenols, ranging in length from 15 to 22 isoprene units, has been isolated from soya beans (Glycine max) and purified by high-pressure liquid chromatography. N.m.r., i.r. and mass spectra of the compounds indicated that they are alpha-saturated polyprenols of the dolichol type. The amount present in dry seeds was about 9 mg/100 g, whereas dolichyl phosphate (Dol-P) was present only in trace amounts. Dol-P phosphatase activity was detected in the microsomal fraction of 5-day-old germinating soya-bean cotyledons. The Dol-P phosphatase activity was linear with respect to time and protein concentration and exhibited a broad pH optimum (pH 7-9). Triton X-100 was necessary for significant enzyme activity. Enzyme activity was slightly enhanced by EDTA, whereas dithiothreitol was without effect. An apparent Km of 5 microM was determined for Dol-P. Bivalent metal ions were not required for enzyme activity. A number of phosphorylated compounds tested as enzyme substrates (including a number of nucleoside phosphates, glucose 6-phosphate, sodium beta-glycerophosphate and Na4P2O7) did not compete with [1-3H]Dol-P as substrate. A number of phospholipids were also tested for their ability to act as Dol-P phosphatase substrates. At 1 mM concentration, phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid and lysophosphatidic acid each inhibited enzymic activity. However, at 0.1 mM concentration, phosphatidylcholine and phosphatidylethanolamine were slightly stimulatory, whereas phosphatidic acid and lysophosphatidic acid were still inhibitory. Phosphatidic acid showed competitive inhibition.     

Membrane-bound phosphatases in Escherichia coli: sequence of the pgpA gene.

One of the phosphatidyl glycerophosphate phosphatase genes of Escherichia coli, pgpA, was cloned, and its DNA sequence was determined. Its 507-base-pair open reading frame was consistent with the 18,000-molecular-weight product identified by a maxicell experiment. Between its possible promoter and methionine initiation codon, a repetitive extragenic palindromic sequence was found.     

Loss of plastidic lysophosphatidic acid acyltransferase causes embryo-lethality in Arabidopsis

Phosphatidic acid is a key intermediate for chloroplast membrane lipid biosynthesis. De novo phosphatidic acid biosynthesis in plants occurs in two steps: first the acylation of the sn-1 position of glycerol-3-phosphate giving rise to lysophosphatidic acid; second, the acylation of the sn-2 position of lysophosphatidic acid to form phosphatidic acid. The second step is catalyzed by a lysophosphatidic acid acyltransferase (LPAAT). Here we describe the identification of the ATS2 gene of Arabidopsis encoding the plastidic isoform of this enzyme. Introduction of the ATS2 cDNA into E. coli JC 201, which is temperature-sensitive and carries a mutation in its LPAAT gene plsC, restored this mutant to nearly wild type growth at high temperature. A green-fluorescent protein fusion with ATS2 localized to the chloroplast. Disruption of the ATS2 gene of Arabidopsis by T-DNA insertion caused embryo lethality. The development of the embryos was arrested at the globular stage concomitant with a transient increase in ATS2 gene expression. Apparently, plastidic LPAAT is essential for embryo development in Arabidopsis during the transition from the globular to the heart stage when chloroplasts begin to form.