Reconstitution of sugar phosphate transport systems of Escherichia coli

Studies with Escherichia coli cells showed that the transport systems encoded by glpT (sn-glycerol 3-phosphate transport) and uhpT (hexose phosphate transport) catalyze a reversible 32Pi:Pi exchange. This reaction could be used to monitor the glpT or uhpT activities during reconstitution. Membranes from suitably constructed strains were extracted with octylglucoside in the presence of lipid and glycerol, and proteoliposomes were formed by dilution in 0.1 M KPi (pH 7). Both reconstituted systems mediated a 32Pi:Pi exchange which was blocked by the appropriate heterologous substrate, sn-glycerol 3-phosphate (G3P) or 2-deoxyglucose 6-phosphate (2DG6P), with an apparent Ki near 50 microM. In the absence of an imposed cation-motive gradient, Pi-loaded proteoliposomes also transported the expected physiological substrate; Michaelis constants for the transport of G3P or 2DG6P were near 20 microM. The heterologous exchange showed a maximal velocity of 130 nmol/min/mg protein via the glpT system and 11 nmol/min/mg protein for the uhpT system. This difference was expected because the G3P transport activity had been reconstituted from a strain carrying multiple copies of the glpT gene. Taken together, these results suggest that anion exchange may be the molecular basis for transport by the glpT and uhpT proteins.     

A second transport system for sn-glycerol-3-phosphate in Escherichia coli.

Strains containing phage Mucts inserted into glpT were isolated as fosfomycin-resistant clones. These mutants did not transport sn-glycerol-3-phosphate, and they lacked GLPT, a protein previously shown to be a product of the glpT operon. By plating these mutants on sn-glycerol-3-phosphate at 43 degrees C, we isolated revertants that regained the capacity to grow on G3P. Most of these revertants did not map in glpT and did not regain GLPT. These revertants exhibited a highly efficient uptake system for sn-glycerol-3-phosphate within an apparent Km of 5 micron. In addition, three new proteins (GP 1, 2, and 3) appeared in the periplasm of these revertants. None of these proteins were antigentically related to GLPT. However, like GLPT, GP1 exhibits abnormal behavior on sodium dodecyl sulfate-polyacrylamide gels. GP 2 is an efficient binding protein. The new uptake system showed different characteristics than the system that is coded for by the glpT operon. It was inhibited neither by phosphate nor fosfomycin. So far, none of the systems that transport organic acids in Escherichia coli could be implicated in the new sn-glycerol-3-phosphate uptake activity. The mutation ugp+, which was responsible for the appearance of the new transport system and the appearance of GP 1, 2, and 3 in the periplasm was cotransducible with araD by phage P1 transduction and was recessive in merodiploids.     

Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate.     

Characterization of a periplasmic protein related to sn-glycerol-3-phosphate transport in escherichia coli

The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn-glycerol-3-phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn-glycerol 3-phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn-glycerol-3-phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2- to 3-fold over a fully induced wild-type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris-HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4). In contrast to the typical transport-related periplasmic-binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn-glycerol-3-phosphate. In vivo new appearance of transport activity for sn-glycerol-3-phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell-division-dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity. Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn-glycerol-3-phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn-glycerol-3-phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles. Iodination of whole cells with [¹²⁵I] followed by osmotic shock reveals that several shock-releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium.     

Simultaneous uptake of galactose and glucose by Azotobacter vinelandii.

Azotobacter vinelandii growing on galactosides induced two distinct permeases for glucose and galactose. The apparent Vmax and Km of the galactose permease were 16 nmol galactose/min per 10(10) cells and 0.5 mM, respectively. The apparent Vmax and Km of the glucose permease were 7.8 nmol glucose/min per 10(10) cells and 0.04 mM, respectively. Excess glucose had no effect on the galactose uptake. However, excess galactose inhibited glucose transport. The galactosides-induced glucose permease also exhibited different uptake kinetics from that induced by glucose.     

Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene

The influx of Mg2+ in Salmonella typhimurium LT-2 was studied by both kinetic and genetic techniques. Wild-type cells grown in a high MgSO4 concentration (10 mM) exhibited a Km of 15 microM for Mg2+ influx, with a Vmax of 0.25 nmol of Mg2+ per min per 10(8) cells. The apparent Km decreased to 3 microM, and the Vmax increased 60% after growth in a low MgSO4 concentration (10 microM). Co2+ was a simple competitive inhibitor (Ki = 30 microM) of Mg2+ influx in cells grown in high Mg2+ concentrations but blocked only a portion of the Mg2+ influx in cells grown in low Mg2+ concentrations. Co2+ influx exhibited kinetics similar to those of Mg2+ influx (Km = 30 microM; Vmax = 0.5 nmol of Co2+ per min per 10(8) cells) but was not affected by growth conditions. Co2+ influx was competitively inhibited by both Mg2+ and Mn2+. Mutations affecting Mg2+ uptake were isolated by selection for spontaneous resistance to toxic levels of Co2+. One class of mutants designated corA mapped at 84 min near metE with the following gene order: corA, metE, zie-3161::Tn10, pepQ. A second class designated corB mapped at 98 min near pyrB. Mg2+ influx was decreased in a corA mutant strain (relative to that of the wild type) when grown in high Mg2+ concentrations but was restored when grown in low Mg2+ concentrations. Co2+ transport was completely abolished by the corA mutation under all growth conditions. Recombinant plasmids carrying the corA region from either Escherichia coli K-12 or S. typhimurium complemented the corA mutation in S. typhimurium, restoring uptake of both Co2+ and Mg2+ and conferring sensitivity to Co2+. The S. typhimurium corA gene was localized to a restriction fragment of approximately 1.5 kilobases.     

Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.     

The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions

The Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn-glycerol-3-phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino-terminal end of the permease is attached to alkaline phosphatase (phoA) and to beta-galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra-cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the beta-galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two-dimensional model for the permease. The model proposes that GlpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot-spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.     

Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The GlpT system for sn-glycerol-3-phosphate transport in Escherichia coli is shown to catalyze a rapid efflux of Pi from the internal phosphate pools in response to externally added Pi or glycerol-3-phosphate. A glpR mutation, which results in constitutive expression of the GlpT system, is responsible for this rapid Pi efflux and the arsenate sensitivity of several laboratory strains, including the popular strain C600. Glucose and other phosphotransferase system sugars inhibit Pi efflux by repressing glpT expression.     

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.     

Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in Escherichia coli.

An Escherichia coli periplasmic protein (GlpT) related to sn-glycerol-3-phosphate transport was synthesized in a cell-free system directed by hybrid plasmic ColE1-glpT DNA. The in vitro product cross-reacted with antisera against the purified protein. The ColE1-glpT DNA-directed cell-free system was induced by sn-glycerol-3-phosphate and phosphonomycin and was dependent on cyclic AMP. The in vitro-synthesized protein showed the characteristics of a multimeric protein, as did the purified periplasmic protein. The main proportion of the newly synthesized product had a higher molecular weight than the mature protein found in the periplasm of cells and showed a more positive charge in two-dimensional gel electrophoresis. Thus, a proportion of this protein is presumed to be synthesized in vitro as a precursor. The cell-free system yielded a second protein that is likely to be also coded for by the glpT operon. This protein had a molecular weight of approximately 33,000 in sodium dodecyl sulfate-acrylamide gel electrophoresis and behaved like an intrinsic membrane protein.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism.

sn-Glycerol 3-phosphorothioate was found to be bacteriocidal to strains of Escherichia coli which have a functional sn-glycerol 3-phosphate transport system. This effect was manifest in strains 7 and 8, which are constitutive mutants for the utilization and transport of sn-glycerol 3-phosphate (glpRc2). Strain E15, which is considered to be wild type for the glycerol phosphate functional units, was affected by the phosphorothioate analog only under conditions that are known to induce the transport system for sn-glycerol 3-phosphate. In addition, another strain of E. coli, strain 6, which is isogenic with strain E15 but has an impaired sn-glycerol 3-phosphate transport system (glpT13), was not affected by similar concentrations of sn-glycerol 3-phosphorothioate. Transport studies in which [3H]glycerol phosphate and its phosphorothioate analog were used demonstrated that the latter compound was taken up via the specific active transport system for sn-glycerol 3-phosphate; the Km values were 9 and 11 microM, respectively. The rates of macromolecular synthesis were found to be inhibited severely by sn-glycerol 3-phosphorothioate at a concentration at which sn-glycerol 3-phosphate had no effect (5 microM). At a lower concentration of the analog (0.5 microM), the rates of protein synthesis and RNA synthesis (52 and 58% below control values after 90 min, respectively) were more sensitive than the rates of DNA synthesis and cell wall synthesis (18% below control values after 3 h for DNA; transient decrease in the cell wall values after 90 min). The levels of the nucleoside triphosphates were not affected by the presence of the phospholipid precursor or its analog at a concentration of 5 microM. The phospholipid composition was significantly altered in the presence of bacteriocidal concentrations (5 microM) of sn-glycerol 3-phosphorothioate. The amount of phosphatidylglycerol in the membranes decreased from 13.5 to 3.5%. Concomitant with this decrease in phosphatidylglycerol content was a fourfold increase in the 32P content of cardiolipin (from 6.8 to 24.2%), whereas the phosphatidylethanolamine content showed only a minor reduction (8%) after 3 h. The rates of synthesis of all of the phospholipids decreased in the presence of 5 microM sn-glycerol 3-phosphorothioate, with the most significant effects observed for phosphatidylglycerol (63% after 3 h). Phosphatidylglycerol showed increased rates of turnover after 90 min (21%) and 3 h (11%), with concomitant increases in the levels of cardiolipin of more than twofold. Our data suggest that a considerably greater proportion of phosphatidylglycerol turnover may be recover in cardiolipin than is metabolized via other pathways (e.g., the membrane-derived oligosaccharide pathway).     

Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity.

Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.     

Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture

Inorganic phosphate, amino acids and sugars are of obvious importance in lung metabolism. We investigated sodium-coupled transports with these organic and inorganic substrates in type II alveolar epithelial cells from adult rat after one day in culture. Alveolar type II cells actively transported inorganic phosphate and alanine, a neutral amino acid, by sodium-dependent processes. Cellular uptakes of phosphate and alanine were decreased by about 80% by external sodium substitution, inhibited by ouabain (30 and 41%, respectively) and displayed saturable kinetics. Two sodium-phosphate cotransport systems were characterized: a high-affinity one (apparent Km = 18 microM) with a Vmax of 13.5 nmol/mg protein per 10 min and a low-affinity one (apparent Km = 126 microM) with a Vmax of 22.5 nmol/mg protein per 10 min. Alanine transport had an apparent Km of 87.9 microM and a Vmax of 43.5 nmol/mg protein per 10 min. By contrast, cultured alveolar type II cells did not express sodium-dependent hexose transport. Increasing time in culture decreased Vmax values of the two phosphate transport systems on day 4 while sodium-dependent alanine uptake was unchanged. This study demonstrated the existence of sodium-dependent phosphate and amino acid transports in alveolar type II cells similar to those documented in other epithelial cell types. These sodium-coupled transports provide a potent mechanism for phosphate and amino acid absorption and are likely to play a role in substrate availability for cellular metabolism and in regulating the composition of the alveolar subphase. The decrease in phosphate uptake with time in culture is parallel to decrease in surfactant synthesis reported in cultured alveolar type II cells, suggesting that phosphate availability for surfactant synthesis may be accomplished by a sodium-dependent phosphate uptake.     

Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12.

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a scrB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3-,5'-phosphate receptor protein. Sucrose uptake (apparent Km = 10 microM) was dependent on the scrA gene product and on the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). The active enzyme was shown to be a dimer of Mr 110,000.     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

Purification and properties of a periplasmic protein related to sn-glycerol-3-phosphate transport in Escherichia coli.

Protein GLPT, a periplasmic protein previously recognized as closely related to the active transport of sn-glycerol-3-phosphate in Escherichia coli was isolated by the cold osmotic shock procedure. It was purified by Sephadex chromatography and isoelectric focussing. The purified protein does not exhibit any detectable binding activity toward sn-glycerol-3-phosphate. It has no activity as a glycerol phosphatase nor as a glycerol kinase. Polyacrylamide gel electrophoresis in the presence of dodecylsulfate of the protein subsequent to treatment in urea, boiling in dodecylsulfate and crosslinking indicates that it occurs as an oligomeric protein composed of four identical subunits of 40 000 molecular weight. Membrane vesicles of wild-type strains that contain protein GLPT in whole cells loose it during vesicle preparation. However, they still exhibit high transport activity toward sn-glycerol-3-phosphate. Membrane vesicles prepared from glp T mutants that may or may not contain protein GLPT do not transport sn-glycerol-3-phospahte. We conclude from these results that protein GLPT does not participate in the energy-dependent active transport through the cytoplasmic membrane but could be involved in facilitating the diffusion of sn-glycerol-3-phosphate through the outer layers of E. coli.     

Biosynthesis in Escherichia coli of sn-glycerol 3-phosphate, a precursor of phospholipid. Palmitoyl-CoA inhibition of the biosynthetic sn-glycerol-3-phosphate dehydrogenase.

Homogeneous biosynthetic sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of Escherichia coli was potently inhibited by palmitoyl-CoA and other long chain acyl-CoA thioesters. The concentration dependence of this inhibition was not cooperative. Enzyme activity was inhibited 50% at 1 microM palmitoyl-CoA; thus, this inhibition occurred at concentrations below the critical micellar concentration of palmitoyl-CoA. Palmitoyl-CoA was a reversible, noncompetitive inhibitor with respect to both NADPH and dihydroxyacetone phosphate. Palmitoyl-CoA did not affect the quaternary structure of the enzyme. This inhibition could be prevented or reversed by the addition of phospholipid vesicles prepared from E. coli phospholipids. Palmitoyl-CoA did not alter the kinetics of inhibition by sn-glycerol 3-phosphate, which is a proven physiological regulator of this enzyme. Decanoyl-CoA, dodecanoyl-CoA, myristoyl-CoA, palmitoyl-(1,N6-etheno)CoA, stearoyl-CoA, and oleoyl-CoA inhibited sn-glycerol-3-phosphate dehydrogenase at concentrations below their critical micellar concentrations. Palmitate inhibited sn-glycerol-3-phosphate dehydrogenase activity 50% at 200 microM. Palmitoyl-carnitine, deoxycholate, taurocholate, and dodecyl sulfate were more potent inhibitors than Triton X-100, Tween-20, or Tween-80. Palmitoyl-acyl carrier protein at concentrations up to 50 microM had no effect on sn-glycerol-3-phosphate dehydrogenase activity. The possible physiological role of long chain fatty acyl-CoA thioesters in the regulation of sn-glycerol 3-phosphate and phospholipid biosynthesis in E. coli is discussed.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.