The human erythrocyte sugar transporter presents two sugar import sites

The human erythrocyte sugar transporter presents sugar import (e2) and sugar export (e1) sites simultaneously. This study asks whether the sugar transporter exposes only one or multiple import sites. We approached this question by analysis of cytochalasin B binding to the human erythrocyte sugar export site in the presence of sugars that bind to the sugar import site. Extracellular maltose does not enter human erythrocytes. High concentrations of maltose (1-100 mM) inhibit cytochalasin B binding to human red cells. Low concentrations (25-500 microM) increase the level of erythrocyte cytochalasin B binding. Maltose modulation of cytochalasin B binding is mediated by altered affinity of sugar export sites for cytochalasin B. Similar results are obtained with other cell-impermeant inhibitors of sugar uptake. Extracellular D-glucose (a transported sugar) stimulates cytochalasin B binding at low D-glucose concentrations (10-250 microM), but this effect is lost at higher concentrations. Intracellular D-glucose inhibits cytochalasin B binding. Low concentrations of extracellular maltose and other nontransported inhibitors stimulate 3-O-methylglucose uptake in erythrocytes. Higher sugar concentrations (1-100 mM) inhibit transport. These data support the hypothesis that the erythrocyte sugar transporter presents two sugar import sites and at least one sugar export site. This conclusion is consistent with the proposed oligomeric structure of the sugar transporter, a complex of four GluT1 proteins in which each subunit presents a translocation pathway.     

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.     

The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.     

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.     

Determinants of ligand binding affinity and cooperativity at the GLUT1 endofacial site

Cytochalasin B (CB) and forskolin (FSK) inhibit GLUT1-mediated sugar transport in red cells by binding at or close to the GLUT1 endofacial sugar binding site. Paradoxically, very low concentrations of each of these inhibitors produce a modest stimulation of sugar transport [ Cloherty, E. K., Levine, K. B., and Carruthers, A. ((2001)) The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites. Biochemistry 40 ((51)) 15549-15561]. This result is consistent with the hypothesis that the glucose transporter contains multiple, interacting, endofacial binding sites for CB and FSK. The present study tests this hypothesis directly and, by screening a library of cytochalasin and forskolin analogues, asks what structural features of endofacial site ligands determine binding site affinity and cooperativity. Like CB, FSK competitively inhibits exchange 3-O-methylglucose transport (sugar uptake in cells containing intracellular sugar) but noncompetitively inhibits sugar uptake into cells lacking sugar at 4 °C. This refutes the hypothesis that FSK binds at GLUT1 endofacial and exofacial sugar binding sites. Some forskolin derivatives and cytochalasins inhibit equilibrium [(3)H]-CB binding to red cell membranes depleted of peripheral proteins at 4 °C. Others produce a moderate stimulation of [(3)H]-CB binding when introduced at low concentrations but inhibit binding as their concentration is increased. Yet other analogues modestly stimulate [(3)H]-CB binding at all inhibitor concentrations applied. These findings are explained by a carrier that presents at least two interacting endofacial binding sites for CB or FSK. We discuss this result within the context of models for GLUT1-mediated sugar transport and GLUT1 quaternary structure, and we evaluate the major determinants of ligand binding affinity and cooperativity.     

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.     

ATP regulation of the human red cell sugar transporter

Purified human red blood cell sugar transport protein intrinsic tryptophan fluorescence is quenched by D-glucose and 4,6-ethylidene glucose (sugars that bind to the transport), phloretin and cytochalasin B (transport inhibitors), and ATP. Cytochalasin B-induced quenching is a simple saturable phenomenon with Kd app of 0.15 microM and maximum capacity of 0.85 cytochalasin B binding sites per transporter. Sugar-induced quenching consists of two saturable components characterized by low and high Kd app binding parameters. These binding sites appear to correspond to influx and efflux transport sites, respectively, and coexist within the transporter molecule. ATP-induced quenching is also a simple saturable process with Kd app of 50 microM. Indirect estimates suggest that the ratio of ATP-binding sites per transporter is 0.87:1. ATP reduces the low Kd app and increases the high Kd app for sugar-induced fluorescence quenching. This effect is half-maximal at 45 microM ATP. ATP produces a 4-fold reduction in Km and 2.4-fold reduction in Vmax for cytochalasin B-inhibitable D-glucose efflux from inside-out red cell membrane vesicles (IOVs). This effect on transport is half-maximal at 45 microM ATP. AMP, ADP, alpha, beta-methyleneadenosine 5'-triphosphate, and beta, gamma-methyleneadenosine 5'-triphosphate at 1 mM are without effect on efflux of D-glucose from IOVs. ATP modulation of Km for D-glucose efflux from IOVs is immediate in onset and recovery. ATP inhibition of Vmax for D-glucose exit is complete within 5-15 min and is only partly reversed following 30-min incubation in ATP-free medium. These findings suggest that the human red cell sugar transport protein contains a nucleotide-binding site(s) through which ATP modifies the catalytic properties of the transporter.     

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.     

Cytochalasin B as a probe of protein structure and substrate recognition by the galactose/H+ transporter of Escherichia coli.

Cytochalasin B is a potent inhibitor of mammalian passive glucose transporters. The recent demonstration of sequence similarities between these proteins and several bacterial proton-linked sugar transporters suggested that cytochalasin B might be a useful tool for investigation of the galactose/H+ symport protein (GalP) of Escherichia coli. Equilibrium binding studies using membranes from a GalP-constitutive (GalPc) strain of E. coli revealed a single set of high affinity binding sites for cytochalasin B with a Kd of 0.8-2.2 microM. Binding was inhibited by D-glucose, but not by L-glucose. UV irradiation of the membranes in the presence of [4-3H]cytochalasin B photolabeled principally a protein of apparent Mr 38,000, corresponding to the GalP protein. Labeling was inhibited by greater than 80% in the presence of 500 mM D-glucose or D-galactose, the major substrates of the GalP system. The extent of inhibition of photolabeling by different sugars and sugar analogues showed that the substrate specificity of GalP closely resembles that of the mammalian passive glucose transporters. Structural similarity to the latter was revealed by tryptic digestion of [4-3H]cytochalasin B-photolabeled GalP, which yielded a radiolabeled fragment of apparent Mr 17,000-19,000, similar to that previously reported for the human erythrocyte glucose transporter.     

Dissection of discrete kinetic events in the binding of antibiotics and substrates to the galactose-H+ symport protein,GalP, ofEscherichia coli

GalP is the membrane protein responsible for H⁺-driven uptake of D-galactose intoEscherichia coli. It is suggested to be the bacterial equivalent of the mammalian glucose transporter, GLUT1, since these proteins share sequence homology, recognise and transport similar substrates and are both inhibited by cytochalasin B and forskolin. The successful over-production of GalP to 35–55% of the total inner membrane protein ofE. coli has allowed direct physical measurements on isolated membrane preparations. The binding of the antibiotics cytochalasin B and forskolin could be monitored from changes in the inherent fluorescence of GalP, enabling derivation of a kinetic mechanism describing the interaction between the ligands and GalP. The binding of sugars to GalP produces little or no change in the inherent fluorescence of the transporter. However, the binding of transported sugars to GalP produces a large increase in the fluorescence of 8-anilino-1-naphthalene sulphonate (ANS) excited via tryptophan residues. This has allowed a binding step, in addition to two putative translocation steps, to be measured. From all these studies a basic kinetic mechanism for the transport cycle under non-energised conditions has been derived. The ease of genetical manipulation of thegalP gene inE. coli has been exploited to mutate individual amino acid residues that are predicted to play a critical role in transport activity and/or the recognition of substrates and antibiotics. Investigation of these mutant proteins using the fluorescence measurements should elucidate the role of individual residues in the transport cycle as well as refine the current model.Abbreviations GalP galactose-H⁺ transporter - AraE arabinose-H⁺ transporter - GLUT1 human erythrocyte glucose transporter requests for offprints: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2UH, UK     

EFFECTS OF CYTOCHALASIN B ON THE UPTAKES OF MONOSACCHARIDES BY RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal uptakes of a number of simple carbohydrates were strongly inhibited by cytochalasin B (K₁= 7-9 × 10⁻⁸M). Phloretin (K₁= 2-4 × 10⁻⁶M) and phloridzin (K₁= 3-4 × 10⁻⁴M) were less inhibitory. Cytochalasin B competitively inhibited the uptakes of carbohydrates with saturable transport kinetics. Inhibition of sugar uptake was immediate on addition of cytochalasin B but was promptly reversed upon removal of the drug. Cytochalasin B had no effect on the efflux of D-glucos-amine or of the phosphorylated sugar, and did not affect intrasynaptosomal hexokinase(s). The synapto-somal uptakes of L-glucose, D-mannitol, L-fucose and the N -acetylated amino sugars were non-saturable and uninhibited by cytochalasin B. In the case of sugars which enter synaptosomes by both saturable and non-saturable processes, cytochalasin B could be used to selectively inhibit the saturable uptake components. The resultant non-saturable cytochalasin-insensitive uptake rates obtained were found to be widely different among the sugars studied, and must be corrected for in order to estimate accurate kinetic constants of the saturable processes.     

Cytochalasin inhibition of hexose transport by platelets

Previously we described a two-transporter model (T1, T2) for galactose uptake by platelets (Horne, M.K. and Hart, J.S. (1986) Biochim. Biophys. Acta 856, 448-456). In the current work we have sought corroborative evidence for this model by studying the effects of cytochalasins on this transport system. Of the various cytochalasins tested, cytochalasin B was the most potent inhibitor (I) of galactose transport, whereas cytochalasin A was less inhibitory and dihydrocytochalasin B and cytochalasin E had no inhibitory effect. The same order of potency was observed for the inhibition of L-glucose diffusion into platelets. The mechanism of cytochalasin B inhibition was investigated in detail. Inhibition of T1 was competitive and required a higher concentration of cytochalasin B (Ki1 approximately 1.7 microM) than inhibition of T2, which was of a mixed type (Ki2 approximately 0.8 microM). The effect of cytochalasin B on T2 could be accounted for by a membrane alteration which enhanced the affinity of the transporter for galactose while simultaneously preventing passage of the TSI complex into the cell. Since a similar effect on membrane permeability would also explain cytochalasin B inhibition of L-glucose diffusion, it is hypothesized that cytochalasin B binds to a membrane structure shared by T2 and the passage for L-glucose. The differences in cytochalasin B sensitivity and mechanism of inhibition manifested by T1 and T2 support our original hypothesis that galactose is indeed transported by kinetically distinct agencies and suggest that these may be physically distinct as well.     

Comparison of the equilibrium exchange of nucleosides and 3-O-methylglucose in human erythrocytes and of the effects of cytochalasin B, phloretin and dipyridamole on their transport

Because of similarities in the physical and molecular properties of the nucleoside and sugar transporters of human erythrocytes and the photoaffinity labeling of the sugar transporter by 8-azidoadenosine (Jarvis et al. (1986) J. Biol. Chem. 261, 11077-11085), we have directly compared the equilibrium exchange of uridine and 3-O-methylglucose in these cells as measured by rapid kinetic techniques under identical experimental conditions. Both the Michaelis-Menten constant and maximum velocity were about 100-fold higher for 3-O-methylglucose exchange than for uridine exchange so that the first order rate constants for both transporters were about the same. When calculated on the basis of the number of nucleoside and sugar carriers per red cell estimated by equilibrium binding of nitrobenzylthioinosine and cytochalasin B, respectively, the turnover numbers for the sugar and nucleoside carriers with 3-O-methylglucose and uridine, respectively, as substrates were quite similar. Various sugars up to concentrations of 108 mM had no effect on the exchange of 500 microM uridine or adenosine, and uridine up to a concentration of 50 mM had no effect on the exchange of 10 mM 3-O-methylglucose. Adenosine, on the other hand, inhibited 3-O-methylglucose exchange in a concentration dependent manner, though not very effectively (IC50 approximately equal to 3 mM). Both uridine and 3-O-methylglucose exchange were inhibited in a concentration dependent manner by cytochalasin B, phloretin and dipyridamole, but cytochalasin B and phloretin were 100-times more effective in inhibiting 3-O-methylglucose than uridine exchange, whereas the opposite was the case for the inhibition by dipyridamole.     

Cytochalasin B does not serve as a marker of glucose transport in rabbit erythrocytes

Glucose inhibitable cytochalasin B binding to erythrocyte membranes has been used as a marker of the glucose transporter. Glucose transport and cytochalasin B binding in rabbit erythrocytes differ from those activities found in human erythrocytes. We evaluated the uptake of 3-0-methylglucose and found similar Km (4.81 +/- 1.20 mM (SEM) and 6.59 +/- 0.72 mM) though significantly different Vmax (5.2 +/- 0.7 nM . min-1/10(9) cells and 234 +/- 13 nM X min -1/10(9) cells, p less than 0.001) for rabbit and human erythrocytes, respectively. Equilibrium binding of cytochalasin B to human erythrocyte membranes demonstrates a high affinity cytochalasin B binding site (Kd 38.6 +/- 22.7 nM) which is displaced by glucose. No comparable glucose inhibitable cytochalasin B site exists for rabbit erythrocyte membranes. Photoaffinity labeling of cytochalasin B confirms the presence of a glucose inhibitable cytochalasin B binding site in human, but not rabbit erythrocyte membranes. Cytochalasin B binding is a useful method in the identification of the glucose transporter in human cells, but the technique may be less useful in other species.     

Design and analysis of protein binding experiments

The design and analysis of protein binding experiments for obtaining precise parameter estimates for a one-site and a two-site model treating fu, the fraction unbound as the experimentally determined quantity was investigated. Total drug concentrations were chosen at which the binding isotherm is determined to yield the most information about the parameters under study. The D-optimization information criterion was used to achieve this although other criteria are also discussed. For both the one-site and the two-site models the number of design points was always equal to the number of parameters being estimated. The results arrived at when dealing with constant variance and unconstrained total drug concentration were rather unique in that in most of the cases studied, all the optimal design points were away from the boundary conditions. For constant relative variance and unconstrained total drug concentrations, one of the design points was always placed at the smallest possible value of fu, the fraction unbound. For the one-site model the second point was always given by K(-1) + nP. The optimal designs arrived at lead to lower theoretical coefficients of variation in the parameters than the corresponding conventional ones. Simulated experiments supported these theoretical findings for both the one-site and the two-site models. For the one-site model, results from nonlinear regression were compared with Scatchard analysis and the optimal designs were also optimal in Scatchard space. We also show that using Scatchard analysis with the conventional strategy leads to poorly determined estimates particularly when the number of observations is low.     

Glucose transport in developing Ehrlich ascites tumor cells: parallel changes in the rate of glucose uptake and cytochalasin B binding activity during tumor development and methotrexate treatment

The ability of Ehrlich ascites tumor cells to take up glucose increased progressively during the course of tumor development. Simultaneously as the rate of uptake rose, the density of a class of glucose-reversible binding sites for cytochalasin B on the cell surface also increased. In its stereospecificity requirement toward competing sugars and in its sensitivity to phloretin and diethylstilbestrol, this class of binding sites resembled the putative glucose carriers identified in various other cell systems and may represent the glucose transporter in Ehrlich ascites cells. Work with methotrexate (MTX) substantiated this view. Methotrexate arrested tumor growth, inhibited glucose uptake, and reduced the number of cytochalasin B binding sites. In both MTX-treated and untreated cells, the magnitude of changes in number of cytochalasin B binding sites closely paralleled and sufficiently accounted for the magnitude of changes in glucose uptake. Qualitative changes in the turnover and affinity for substrate of the putative glucose carrier need not be invoked.     

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments.