首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 46 毫秒
1.
When sn-glycerol-3-phosphate (G3P) is taken up exclusively by the pho regulon-dependent Ugp transport system, it can be used as the sole source of Pi but not as the sole source of carbon. We had previously suggested that the inability of G3P to be used as a carbon source under these conditions is due to trans inhibition of G3P uptake by internal Pi derived from the degradation of G3P (P. Brzoska, M. Rimmele, K. Brzostek, and W. Boos, J. Bacteriol. 176:15-20, 1994). Here we report 31P nuclear magnetic resonance measurements of intact cells after exposure to G3P as well as to Pi, using different mutants defective in pst (high-affinity Pi transport), ugp (pho-dependent G3P transport), glpT (glp-dependent G3P transport), and glpD (aerobic G3P dehydrogenase). When G3P was transported by the Ugp system and when metabolism of G3P was allowed (glpD+), Pi accumulated to about 13 to 19 mM. When G3P was taken up by the GlpT system, the preexisting internal Pi pool (whether low or high) did not change. Both systems were inversely controlled by internal Pi. Whereas the Ugp system was inhibited, the GlpT system was stimulated by elevated internal Pi.  相似文献   

2.
Auer M  Kim MJ  Lemieux MJ  Villa A  Song J  Li XD  Wang DN 《Biochemistry》2001,40(22):6628-6635
The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.  相似文献   

3.
sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H. Schweizer, M. Argast, and W. Boos, J. Bacteriol. 150:1154-1163, 1982). In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi. Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system. This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P. Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system. Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P. However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system. Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM. Cells had to be starved for Pi in order to observe this inhibition. We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate.  相似文献   

4.

Background

Fosfomycin is widely used to treat urinary tract and pediatric gastrointestinal infections of bacteria. It is supposed that this antibiotic enters cells via two transport systems, including the bacterial Glycerol-3-phosphate Transporter (GlpT). Impaired function of GlpT is one mechanism for fosfomycin resistance.

Methods

The interaction of fosfomycin with the recombinant and purified GlpT of Escherichia coli reconstituted in liposomes has been studied. IC50 and the half-saturation constant of the transporter for external fosfomycin (Ki) were determined by transport assay of [14C]glycerol-3-phosphate catalyzed by recombinant GlpT. Efficacy of fosfomycin on growth rates of GlpT defective bacteria strains transformed with recombinant GlpT was measured.

Results

Fosfomycin, externally added to the proteoliposomes, poorly inhibited the glycerol-3-phosphate/glycerol-3-phosphate antiport catalyzed by the reconstituted transporter with an IC50 of 6.4 mM. A kinetic analysis revealed that the inhibition was completely competitive, that is, fosfomycin interacted with the substrate-binding site and the Ki measured was 1.65 mM. Transport assays performed with proteoliposomes containing internal fosfomycin indicate that it was not very well transported by GlpT. Complementation study, performed with GlpT defective bacteria strains, indicated that the fosfomycin resistance, beside deficiency in antibiotic transporter, could be due to other gene defects.

Conclusions

The poor transport observed in a reconstituted system together with the high value of Ki and the results of complementation study well explain the usual high dosage of this drug for the treatment of the urinary tract infections.

General significance

This is the first report regarding functional analysis of interaction between fosfomycin and GlpT.  相似文献   

5.
Here we report the successful three-dimensional crystallization of GlpT, the glycerol-3-phosphate transporter from Escherichia coli inner membrane. GlpT possesses 12 transmembrane alpha-helices and is a member of the major facilitator superfamily. It mediates the exchange of glycerol-3-phosphate for inorganic phosphate across the membrane. Approximately 20 phospholipid molecules per protein, identified as negatively charged phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, were required for the monodispersity of purified GlpT. Analytical size-exclusion chromatography proved to be efficient in identifying detergents for GlpT monodispersity. Nine such detergents were later used for GlpT crystallization. Screening for crystal nucleation was carried out with a variety of polyethylene glycols as the precipitant over a wide pH range. Subsequent identification of a rigid protein core by limited proteolysis and mass spectroscopy resulted in better-ordered crystals. These crystals exhibited order to 3.7 A resolution in two dimensions. However, the stacking in the third dimension was partially disordered. This stacking problem was overcome by using a detergent mixture and manipulating the ionic interactions in the crystallization solution. The resulting GlpT crystals diffracted isotropically to 3.3 A resolution and were suitable for structure determination by X-ray crystallography.  相似文献   

6.
Active transport of substrates across cytoplasmic membranes is of great physiological, medical and pharmaceutical importance. The glycerol-3-phosphate (G3P) transporter (GlpT) of the E. coli inner membrane is a secondary active antiporter from the ubiquitous major facilitator superfamily that couples the import of G3P to the efflux of inorganic phosphate (Pi) down its concentration gradient. Integrating information from a novel combination of structural, molecular dynamics simulations and biochemical studies, we identify the residues involved directly in binding of substrate to the inward-facing conformation of GlpT, thus defining the structural basis for the substrate-specificity of this transporter. The substrate binding mechanism involves protonation of a histidine residue at the binding site. Furthermore, our data suggest that the formation and breaking of inter- and intradomain salt bridges control the conformational change of the transporter that accompanies substrate translocation across the membrane. The mechanism we propose may be a paradigm for organophosphate:phosphate antiporters.  相似文献   

7.
8.
Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.  相似文献   

9.
ATP hydrolysis catalysed by the H+-ATPase of intact mitochondria can be induced by addition of ATP in the presence of valinomycin and KCl. This leads to an increase in intramitochondrial Pi and therefore allows investigation of potential Pi efflux pathways in intact mitochondria. Combining this approach with the direct measurement of both internal and external Pi, we have attempted to determine whether Pi efflux occurs via an atractyloside-sensitive transporter, by the classical operation of the Pi/H+ and Pi/dicarboxylate carriers, and/or by other mechanisms. Initial experiments re-examined the evidence that led to the current view that one efflux pathway for Pi is an atractyloside-sensitive ATP/ADP,0.5Pi transporter. No evidence was found in support of this efflux pathway. Rather, atractyloside-sensitivity of the low rate of Pi efflux observed in previous studies (oligomycin present) was accounted for by ATP entry on the well known ATP/ADP transport system followed by hydrolysis of ATP and subsequent Pi efflux. Thus, under these conditions, where ATP hydrolysis is not completely inhibited, Pi efflux becomes atractyloside sensitive most likely because this inhibitor blocks ATP entry, not because it directly inhibits Pi efflux. Substantial efflux of Pi from rat liver mitochondria is observed on generation of high levels of matrix Pi by ATP hydrolysis induced by valinomycin and K+ (oligomycin absent). A portion of this efflux can be inhibited by thiol-specific reagents at concentrations that normally inhibit the Pi/H+ and Pi/dicarboxylate carriers. However, a significant fraction of efflux continues even in the presence of p-chloromercuribenzoate, N-ethylmaleimide plus n-butylmalonate or mersalyl. The mersalyl-insensitive Pi efflux, which is also insensitive to carboxyatractyloside, is a saturable process, thus suggesting carrier mediation. During this efflux the mitochondrial inner membrane retains considerable impermeability to other low-molecular-weight anions (i.e., malate, 2-oxoglutarate). In conclusion, results presented here rule out an atractyloside-sensitive ATP/ADP,0.5Pi transport system as a mechanism for Pi efflux in rat liver mitochondria. Rather Pi efflux appears to occur on the classical Pi/H+ transport system as well as via a mersalyl-insensitive saturable process. The inhibitor-insensitive Pi efflux may occur on a portion of the Pi/H+ carrier molecules that exist in a state different from that normally catalysing Pi influx. Alternatively, a separate Pi efflux carrier may exist.  相似文献   

10.
Rapid turnover of mannitol-1-phosphate in Escherichia coli.   总被引:3,自引:1,他引:2       下载免费PDF全文
The phosphate moiety of D-mannitol-1-phosphate in Escherichia coli is subject to rapid turnover and is in close equilibrium with Pi and the phosphorus of fructose-1,6-bisphosphate. These three compounds account for the bulk of 32P label found in cells after several minutes of uptake of 32Pi and mannitol-1-phosphate represents some 30% of this label. Mannitol-1-phosphate occurs in E. coli grown on a variety of carbon sources, in the absence of D-mannitol, and is synthesized de novo even in mutants lacking mannitol-1-phosphate dehydrogenase. The mannitol moiety of mannitol-1-phosphate was not affected during the total chase of the P moiety, which exchanged with a half-life of about 30 s. These findings suggest that the rapid equilibration of the phosphorus is a function of an enzyme, possibly a component of the phosphotransferase system, capable of forming a complex that allows the exchange of the phosphate without the equilibration of the mannitol moiety with free mannitol.  相似文献   

11.
Inorganic phosphate (Pi) homeostasis in multi-cellular eukaryotes depends not only on Pi influx into cells, but also on Pi efflux. Examples in plants for which Pi efflux is crucial are transfer of Pi into the xylem of roots and release of Pi at the peri-arbuscular interface of mycorrhizal roots. Despite its importance, no protein has been identified that specifically mediates phosphate efflux either in animals or plants. The Arabidopsis thaliana PHO1 gene is expressed in roots, and was previously shown to be involved in long-distance transfer of Pi from the root to the shoot. Here we show that PHO1 over-expression in the shoot of A. thaliana led to a two- to threefold increase in shoot Pi content and a severe reduction in shoot growth. (31) P-NMR in vivo showed a normal initial distribution of intracellular Pi between the cytoplasm and the vacuole in leaves over-expressing PHO1, followed by a large efflux of Pi into the infiltration medium, leading to a rapid reduction of the vacuolar Pi pool. Furthermore, the Pi concentration in leaf xylem exudates from intact plants was more than 100-fold higher in PHO1 over-expressing plants compared to wild-type. Together, these results show that PHO1 over-expression in leaves leads to a dramatic efflux of Pi out of cells and into the xylem vessel, revealing a crucial role for PHO1 in Pi efflux.  相似文献   

12.
A phosphate-linked antiporter activity of the glucose-6-phosphate transporter (G6PT) has been recently described in liposomes including the reconstituded transporter protein. We directly investigated the mechanism of glucose-6-phosphate (G6P) transport in rat liver microsomal vesicles. Pre-loading with inorganic phosphate (Pi) did not stimulate G6P or Pi microsomal inward transport. Pi efflux from pre-loaded microsomes could not be enhanced by G6P or Pi addition. Rapid G6P or Pi influx was registered by light-scattering in microsomes not containing G6P or Pi. The G6PT inhibitor, S3483, blocked G6P transport irrespectively of experimental conditions. We conclude that hepatic G6PT functions as an uniporter.  相似文献   

13.
CPDS (6,6'-dithiodinicotinic acid), a non permeant thiol agent which affects several mitochondrial functions in a way different to that of mersalyl [18-19] revealed striking differences between the phosphate translocating systems of pig heart and rat liver mitochondria. Pi entry was measured either by swelling in 0.12 M ammonium phosphate or by rapid centrifugation in 32Pi medium. Pi efflux was measured after preloading of mitochondria with 32Pi, by exchange against Pi or malate; the "ATP-FCCP" system has been tested previously [19]. In pig heart mitochondria, Pi entry seems to proceed exclusively via the Pi/OH- carrier; CPDS completely inhibits this transport and the energy-linked functions. In contrast n-butyl-malonate does not affect the Pi-entry and the energy-linked functions. The Pi efflux is not affected either by CPDS or mersalyl, which do not produce a swelling in the "ATP-uncoupler system". In rat liver mitochondria, CPDS inhibits only the Pi/OH- carrier; both CPDS and n-butylmalonate are necessary to inhibit completely Pi entry. CPDS as well as mersalyl provokes a swelling in the presence of the "APT-uncoupler system". The results suggest two distinct functions of phosphate transport in both types of mitochondria.  相似文献   

14.
The metabolism of 2-deoxy-D-galactose has been studied in AS-30D rat ascites hepatoma cells in suspension. Using 2-deoxy-D-(1-14C)galactose and an alkaline ethanol deproteinization procedure, the quantitatively identified metabolites included 2-deoxy-D-galactose 1-phosphate comprising 99.3%, and UDP-2-deoxy-D-galactose and UDP-2-deoxy-D-glucose, together amounting to 0.4% of the total metabolites. After incubation for 5 h in the presence of 2-deoxy-D-galactose (1 mmo1/1), the content of 2-deoxy-D-galactose 1-phosphate reached 35 mmo1x(kg cells)-1. The rate of phosphorylation of 2-deoxy-D-galactose was rapid during the first 30 min and decreased to approximately 20% of this rate during the subsequent hours. The rapid trapping of Pi in the form of 2-deoxy-D-galactose 1-phosphate resulted in a depression of free intracellular Pi in spite of a concomitant increase in net 32Pi uptake from the medium and a decrease of ATP and other 5'-nucleotides. The rates of glucose utilization and lactate production were depressed by more than 80% in the presence of 2-deoxy-D-galactose (1 mmo1/1). Interruption of Pi trapping by removal of 2-deoxy-D-galactose from the medium reversed the depressions of Pi and ATP and resulted in a rapid but incomplete relief of glycolysis inhibition. Crossover analysis of glycolytic intermediates indicated an inhibition at the 6-phosphofructokinase step. The depression of glucose utilization may be mediated by the increased level of glucose 6-phosphate, a potent inhibitor of hexokinase. An additional inhibitory effect of a metabolite of 2-deoxy-D-galactose at the 6-phosphofructokinase step was indicated by crossover analysis after reversal of Pi and ATP depressions in the presence of a high intracellular content of 2-deoxy-D-glactose 1-phosphate. The quantitative analysis of the metabolites of 2-deoxy-D-galactose demonstrated the predominance of the monophosphate and the negligible formation of UPD derivatives of this sugar analog in AS-30D hepatoma cells. This provides a system for the investigation of a galactose analog as a phosphate-trapping agent in the virtual absence of uridylate trapping.  相似文献   

15.
The glycerol-3-phosphate transporter (GlpT) is a member of the major facilitator superfamily (MFS). GlpT is an organic phosphate/inorganic phosphate antiporter. It shares a similar fold with other MFS transporters (e.g. LacY and EmrD) consisting of 12 transmembrane (TM) helices which form two domains (each of six TM helices) surrounding a central ligand-binding cavity. The TM helices (especially the cavity-lining helices) contain a large number of proline and glycine residues, which may aid in the conformational changes believed to underline the transport mechanism. Molecular dynamics simulations in a phospholipid bilayer have been used to compare the conformational properties of the isolated TM helices with those in the intact GlpT protein. Analysis of these simulations focuses on the role of proline-induced flexibility in the TM helices. Our results are consistent with the proposed rocker switch mechanism for transport by GlpT. In particular, the simulations highlight the cavity-lining helices (H4, H5, H10 and H11) as being significantly flexible, suggesting that the transport mechanism may involve intra-helix motions in addition to pseudo-rigid body motions of the N- and C-terminal domains relative to one another.  相似文献   

16.
Resting cells of Staphylococcus aureus displayed a phosphate (Pi) exchange that was induced by growth with glucose 6-phosphate (G6P) or sn-glycerol 3-phosphate (G3P). Pi-loaded membrane vesicles from these cells accumulated 32Pi, 2-deoxyglucose 6-phosphate (2DG6P) or G3P by an electroneutral exchange that required no external source of energy. On the other hand, when vesicles were loaded with morpholinopropane sulfonic acid (MOPS), only transport of 32Pi (and L-histidine) was observed, and in that case transport depended on addition of an oxidizable substrate (DL-lactate). In such MOPS-loaded vesicles, accumulation of the organic phosphates, 2DG6P and G3P, could not be observed until vesicles were preincubated with both Pi and DL-lactate to establish an internal pool of Pi. This trans effect demonstrates that movement of 2DG6P or G3P is based on an antiport (exchange) with internal Pi. Reconstitution of membrane protein allowed a quantitative analysis of Pi-linked exchange. Pi-loaded proteoliposomes and membrane vesicles had comparable activities for the homologous 32Pi: Pi exchange (Kt's of 2.2 and 1.4 mM; Vmax's of 180 and 83 nmol Pi/min per mg protein), indicating that the exchange reaction was recovered intact in the artificial system. Other work showed that heterologous exchange from either G6P- or G3P-grown cells had a preference for 2DG6P (Kt = 27 microM) over G3P (Kt = 1.3 mM) and Pi (Kt = 2.2 mM), suggesting that the same antiporter was induced in both cases. We conclude that 32Pi: Pi exchange exhibited by resting cells reflects operation of an antiporter with high specificity for sugar 6-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Two types of fosfomycin-resistant mutants of Bacillus subtilis were isolated. Mutants of the first type (GlpT mutants) were resistant to at least 200 microgram of fosfomycin per ml and failed to take up exogenous glycerol 3-phosphate. Mutants of the second type were resistant to lower concentrations of fosfomycin and transported glycerol-3-phosphate as efficiently as wild-type bacteria. The glpT mutations, but not the mutations in the second type of fosfomycin-resistant mutants, map in the cysA-aroI region of the B. subtilis chromosome.  相似文献   

18.
Streptococcus pyogenes accumulated thiomethyl-beta-galactoside as the 6-phosphate ester due to the action of the phosphoenolpyruvate:lactose phosphotransferase system. Subsequent addition of glucose resulted in rapid efflux of the free galactoside after intracellular dephosphorylation (inducer expulsion). Efflux was shown to occur in the apparent absence of the galactose permease, but was inhibited by substrate analogs of the lactose enzyme II and could not be demonstrated in a mutant of S. lactis ML3 which lacked this enzyme. The results suggest that the enzymes II of the phosphotransferase system can catalyze the rapid efflux of free sugar under appropriate physiological conditions.  相似文献   

19.
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.  相似文献   

20.
K Yano  S Nakashima  Y Nozawa 《FEBS letters》1983,161(2):296-300
Exposure of rabbit neutrophils to formyl-methionyl-leucyl-phenylalanine (FMLP) induced the efflux of 45Ca2+ from pre-labeled cells which was almost complete within 30 s. On the other hand, FMLP-induced 45Ca2+ influx did not become apparent until 60 s after stimulation. When [3H]arachidonic acid-labeled neutrophils were stimulated with FMLP, the radioactivities in phosphatidylinositol 4,5-biphosphate (TPI) and phosphatidylinositol 4-phosphate (DPI) significantly decreased in parallel with the induction of 45Ca2+ efflux. In contrast, degradation of polyphosphoinositides in [3H]glycerol-labeled neutrophils was not significant until 60 s. Taken together, these results indicate that the early degradation of polyphosphoinositides, especially of those rich in arachidonic acid is closely associated with the initial efflux of calcium in FMLP-stimulated rabbit neutrophils. The study of resynthesis of polyphosphoinositides by measuring 32Pi incorporation into these lipids is also presented.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号