Evidence for an inducible glucose transport system in Kluyveromyces lactis

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly . In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably because of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.     

Facilitated diffusion of 6-deoxy-d-glucose by the oxidative yeast,Kluyveromyces lactis

The inducible glucose transport system of the yeast, Kluyveromyces lactis, was studied using the nonmetabolizeable glucose analogue, 6-deoxyglucose. The free sugar analogue is transported into glucose-grown cells via a facilitated diffusion system as determined by the nonconcentrative uptake of the sugar analogue, by the failure of energy inhibitors to reduce the rate of transport and by exchange diffusion across the membrane. Free 6-deoxyglucose is also transported into succinate-grown cells passively. Induction experiments revealed that 6-deoxyglucose serves as a gratuitous inducer for the glucose transport system in this yeast.     

Effect of carbon source during growth on sensitivity of Pseudomonas fluorescens to actinomycin D.

Sensitivity to actinomycin D(AD) varies in Pseudomonas fluorescens cells grown in glucose or succinate minimal salts medium. Growth is inhibited in succinate minimal medium by much lower concentrations of AD than in glucose minimal medium. Uptake of selected radioactive metabolites is inhibited by AD in cells incubated for 2 h in succinate medium containing AD but glucose-grown cells were not sensitive. EDTA treatment promotes increased sensitivity to AD in succinate-grown cells but does not alter sensitivity in glucose-grown cells. Succinate-grown cells bound 2-3 times as much 3H-AD as glucose-grown cells. Glucose-grown cells had much higher lipopolysaccharide levels in the envelope than succinate-grown cells. It is proposed that the lipopolysaccharide masks the binding sites and, therefore, is responsible for the difference in binding of AD by the glucose- and succinate-grown cells. The availability of the binding sites is also reflected in the sensitivity of the cells to the antibiotic.     

Glucose transport in nongrowing yeast

The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer.     

Evidence that activation of 2-deoxy-D-glucose transport in rat thymocyte suspensions results from enhanced coupling between transport and hexokinase activity.

1. Suspensions of rat thymocytes accumulate free 2-deoxy-D-glucose (2-dGlc) within the cytosol to a concentration approx. 25-fold above the external concentration. This active accumulation was enhanced by 40 nM-phorbol 12-myristate 13-acetate (phorbol). 2. The Km for zero-trans uptake in control cells was 2.3 +/- 0.14 mM and Vmax. was 0.41 +/- 0.08 mumol/min per 10(10) cells (n = 6). In cells treated with phorbol (40 nM) the Km for zero-trans uptake was 1.2 +/- 0.13 mM and Vmax. 0.46 +/- 0.03 mumol/min per 10(10) cells (n = 6). The Km was decreased significantly by phorbol (P less than 0.01). 3. Phorbol-dependent activation of thymocytes delayed exit of free 2-dGlc into sugar-free solution and prevented exchange exit. Activation had no effect on 3-O-methyl D-glucoside (3-OMG) exit. 4. Coupling of 2-dGlc transport to hexokinase activity was determined by observing the effects of various concentrations of unlabelled cytosolic 2-dGlc on influx of labelled 2-dGlc into the hexose phosphate pool. In control cells this coupling was 0.81 +/- 0.02 and in phorbol-activated cells it was 0.92 +/- 0.01 (P less than 0.01). 5. The high-affinity inhibitor of hexokinase, mannoheptulose, inhibited uptake of 2-dGlc in both control and phorbol-treated cells. These data are consistent with a model for activation of sugar transport in which hexokinase activity is integrated with the sugar transporter at the endofacial surface. The results suggest that phorbol increases the degree of coupling transport with hexokinase activity, thereby leading to an increase in the rate of uptake of 2-dGlc, a decrease in exit of free 2-dGlc from the cytosol and an increase in free 2-dGlc accumulation.     

Pyrimidine biosynthesis in Pseudomonas stutzeri ATCC 17588

The de novo pyrimidine biosynthetic enzymes in the denitrifying bacterium Pseudomonas stutzeri ATCC 17588 were assayed and their activities were lower in glucose-grown cells than in succinate-grown cells. When P. stutzeri was grown in the presence of uracil, the de novo enzyme activities in succinate-grown cells were lowered while they remained largely unchanged in glucose-grown cells. A uracil auxotroph of P. stutzeri, deficient for aspartate transcarbamoylase activity, was isolated and its auxotrophic requirement was met by only uracil and cytosine. The inability of pyrimidine ribonucleosides to meet the auxotrophic requirement was related to the limited ability of P. stutzeri to transport uridine and cytidine. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be repressible by a uracil-related compound in succinate-grown P. stutzeri cells. Regulation of pyrimidine synthesis in P. stutzeri was similar to that observed for other pseudomonads classified within rRNA homology group I.     

The influence of ATP on sugar uptake mediated by the constitutive glucose carrier of Saccharomyces cerevisiae

The glucose carrier of Saccharomyces cerevisiae transports the phosphorylatable sugars glucose, mannose, fructose and 2-deoxy-D-glucose (2-dGlc) and the non-phosphorylatable sugar 6-deoxy-D-glucose (6-dGlc). Reduction of the ATP concentration by, for example, incubating cells with antimycin A, results in a decrease in uptake of 2-dGlc and fructose. These uptake velocities can be increased again by raising the ATP level. These results establish a role of ATP in sugar transport. Transport of glucose and mannose is less affected by changes in the ATP concentration than 2-dGlc and fructose uptake, while the 6-dGlc transport is independent of the amount of ATP in the cells. Also, reduction of the kinase activity by incubation with xylose diminished transport of 2-dGlc and fructose, while the uptake of glucose and mannose remained unchanged. It is discussed that these results are due to transport-associated phosphorylation with ATP as substrate and the hexokinases and the glucokinase as phosphorylating enzymes.     

Transport and accumulation of 2-deoxy-D-glucose in wild-type and hexokinase-deficient cultured Chinese-hamster ovary (CHO) cells.

Hexokinase-deficient mutants and wild-type Chinese-hamster ovary cells have been used to investigate the role of hexokinase in uptake and accumulation of 2-D-deoxyglucose (2-dGlc). The evidence for a specific sugar transport system in both types of cells is that there is similar saturable phloretin-sensitive uptake of 2-dGlc and 3-O-methyl-D-glucose (3-OMG) in both types of cell. In wild-type cells, 2-dGlc is accumulated to a tissue:medium ratio of 10- and in the mutant only 3-fold; 3-OMG is not accumulated by either mutant or wild-type cells. The evidence that hexokinase affects the membrane transport process is that the rate of exit of free 2-dGlc from wild-type cells is 5-fold less than from mutant cells, whereas there is no difference in the rate of loss of 3-OMG between mutant and wild-type cells.     

Contribution of different organs to increased glucose consumption after endotoxin administration

Glucose utilization of different organs (spleen, liver, ileum, kidney, skin, lung, and testis) was investigated in vivo in conscious rats 3, 24, or 48 h after treatment with 100 micrograms of endotoxin/100 g of body weight. Glucose uptake was determined by the 2-deoxyglucose technique, which was validated by demonstrating that endotoxin treatment did not alter either the intracellular retention of the phosphorylated metabolites (P-2-dGlc) of the tracer or the discrimination against 2-deoxyglucose in pathways of glucose metabolism. At 3 h after endotoxin the accumulation of P-2-dGlc was markedly increased in the liver (4.8-fold), spleen and skin (2.9-fold), lung (2.4-fold), and ileum and kidney (2.1-fold), as compared to time-matched controls. This effect was sustained in the liver at 24 and 48 h, was diminishing but still significant in spleen, ileum, and kidney, and absent in skin and lung. Accumulation of P-2-dGlc in the testis remained unchanged after endotoxin. Glucose uptake by individual organs and their contribution to whole body glucose utilization in control and endotoxin-treated rats were compared based on P-2-dGlc accumulation data. Organs rich in mononuclear phagocytes (liver and spleen) exhibited a marked and prolonged increase in glucose uptake after endotoxin. Yet the bulk of the increment in the whole body glucose disappearance rate (Rd) was due to three large tissues (skin, intestine, and muscle, accounting for more than 80% of the total P-2-dGlc accumulation in soft tissues), which showed a more moderate and transient increase in glucose utilization.     

Glucose transport in membrane vesicles from Azotobacter vinelandii,: Properties of the glucose carrierin situ

The active transport of d-glucose by membrane vesicles prepared from Azotobactervinelandii strain O is coupled to the oxidation of l-malate. The glucose carrier, but not the energy coupling system of the vesicles, is induced by growth of the cells on d-glucose medium. Vesicles isolated from A. vinelandii grown in the presence of sucrose or acetate accumulate glucose at less than 7% of the rate observed for vesicles from glucose-grown cells. Nevertheless, vesicles from sucrose- or acetate-grown cells transport sucrose or calcium, respectively, in the presence of malate.The transport system expressed in vesicles from glucose-cultured cells is highly specific for d-glucose. Studies of glucose analog uptake and of the competitive effect of analogs reveal that: (i) The glucose carrier is stereospecific. (ii) The affinity of hexoses for the transport system is inversely related to the bulk of substituents on the pyranose ring, especially at the C-1 and C-2 positions, (iii) The most effective competitors, 6-deoxyglucose and 2-deoxyglucose, exhibit affinities only 10–20% that of d-glucose for the transport system, (iv) Phloretin, but not phlorizin, is a competitive inhibitor of glucose transport, having an apparent K_i of 9 μm at pH 7.0. These latter findings suggest a similarity of the glucose transport system of fxA. vinelandii and those of eukaryotes with regard to the glucose carrier.     

The Pasteur effect and catabolite repression in an oxidative yeast,Kluyveromyces lactis

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.     

Characterization of 2-deoxyglucose and 6-deoxyglucose transport in Kluyveromyces marxianus: evidence for two different transport mechanisms

Transport of 2-deoxy-d-glucose (2-dGlc) and 6-deoxy-d-glucose (6-dGlc) is studied in Kluyveromyces marxianus, grown under different conditions. It is shown that early stationary phase cells contain only one glucose transporter, with low affinity for 6-dGlc and high affinity for 2-dGlc. This transporter is recognized by glucose and fructose. In late stationary phase cells, two transport systems are operative for 6-dGlc, one with a high and one with a low affinity. The high-affinity system appears to be a glucose-galactose carrier, catalyzing uphill transport, energized by coupling sugar transport to translocation of protons. Induction (or derepression) of the high-affinity 6-dGlc transport seems to be coupled, in an as yet unknown way, to citrate consumption and a strong alkalinization of the medium during growth. It is concluded that glucose transport in K. marxianus can proceed by at least two mechanisms: a glucose-fructose carrier, probably having phosphotransferase characteristics, and a derepressible glucose/galactose-proton symporter.     

Glucose uptake by Cellulomonas fimi

Glucose uptake by whole-cell suspension of the facultative anaerobe Cellulomonas fimi, which was two-fold higher under aerobic conditions than under N₂ or H₂, was inhibited by inhibitors of electron transport and ATP synthesis and, particularly, by proton and metal ion ionophores. A variety of sugars, including 2-deoxyglucose, did not inhibit glucose uptake but cellobiose was a non-competitive inhibitor. Cells grown on cellobiose medium transported glucose at one half the rate of glucose-grown cells. Cellulomonas fimi has a highly specific active system for glucose transport.     

Glucose-induced Crypticity Toward Succinate Metabolism in Saccharomyces lactis

Saccharomyces lactis grown on glucose adapted very slowly to growth on succinate. This initial inability of glucose-grown cells to grow on succinate was paralleled by their inability to oxidize succinate. The possibility that repression by glucose of respiratory chain components was responsible for these observations was examined. Glucose-grown cells were able to respire glucose, ethyl alcohol, and lactate and were able to initiate growth on ethyl alcohol as rapidly as succinate-grown cells. Respiratory enzyme levels were essentially the same in cells grown on succinate or on glucose. Spectroscopic analysis revealed that glucose-grown cells possessed a full complement of cytochrome bands. Since by these criteria glucose-grown S. lactis appears to possess a competent respiratory system, the penetration of succinate-2,3-(14)C into succinate- and glucose-grown cells was examined directly. Glucose-grown cells exhibited a strong permeability barrier to succinate. Comparison of glucose oxidation by S. lactis and by S. cerevisiae suggests that the crypticity to succinate does not depend upon a strong Crabtree effect in S. lactis.     

High glucose concentrations inhibit glucose phosphorylation, but not glucose transport, in human endothelial cells.

Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.     

Separation of transport and metabolic steps in the uptake of succinate byKluyveromyces fragilis

Characteristics of succinate uptake were determined in glucose-, glycerol-, and succinate-grown cells of the succinate-utilizingKluyveromyces fragilis. Glucose represses the uptake when present during growth and inhibits it when present during uptake. Absence of glucose or presence of succinate or glycerol derepresses the uptake. The transport and subsequent metabolic steps can be efficiently separated by inhibiting the latter with carboxin.     

2-Deoxy-D-glucose uptake in cultured human muscle cells

Hexose uptake was studied with cultured human muscle cells using 2-deoxy-D-[1-3H]glucose. At a concentration of 0.25 and 4 mM, phosphorylation rather than transport was the rate-limiting step in the uptake of 2-deoxy-D-glucose. This was not due to inhibition of the hexokinase activity by either ATP depletion or 2-deoxyglucose 6-phosphate accumulation. In cellular homogenates, hexokinase showed a lower Km value for glucose as compared to 2-deoxyglucose. Intact cells preferentially phosphorylated glucose instead of 2-deoxyglucose. Therefore, transport instead of phosphorylation may be rate limiting in the uptake of glucose by cultured human muscle cells. These data suggest caution in using 2-deoxyglucose for measuring glucose transport.     

Patterns of resistance to 2-deoxy-D-glucose in pig kidney cells

Variants resistant to 2-deoxy-D-glucose have been isolated from a clonal line of pig kidney cells by serial cultivation in the presence of inhibitor. Hexokinase activity may be affected directly in this system, since the oxidation of glucose to 6-phosphogluconate by extracts from sensitive and resistant cells is blocked by the addition of 2-deoxy-glucose to the reaction mixture. This blockage was removed by the addition of glucose-6-phosphate to the system, but not by ATP. Resistant cells were found to accumulate significantly less 2-deoxyglucose-6-phosphate than sensitive cells. The rate of phosphorylation of 2-deoxyglucose, however, was higher in extracts from the resistant line. Alkaline phosphatase does not account for the reduced level of 2-deoxyglucose-6-phosphate since this enzyme is not detectable in sensitive or resistant pig kidney cells. Increased acid phosphatase activity was observed in resistant cells, but extracts with high acid phosphatase activity proved incapable of hydrolyzing either 2-deoxyglucose-6-phosphate or glucose-6-phosphate. In comparative growth studies, cells resistant to 2-deoxyglucose proliferated more extensively than sensitive cells in a low glucose nutrient. They removed glucose more effectively from this medium, and were less stimulated by the addition of intermediates from the tricarboxylic acid cycle. The evidence suggests that resistance to 2-deoxyglucose in the cells under study may be based on the ability of the resistant cells to proliferate at concentrations of glucose too low to support the growth of sensitive cells.     

Characterization and regulation of galactose transport in Neurospora crassa

Two galactose uptake systems were found in the mycelia of Neurospora crassa. In glucose-grown mycelia, galactose was transported by a low-affinity (Km = 400 mM) constitutive system which was distinct from the previously described glucose transport system I (R. P. Schneider and W. R. Wiley, J. Bacteriol. 106:479--486, 1971). In carbon-starved mycelia or mycelia incubated with galactose, a second galactose transport activity appeared which required energy, had a high affinity for galactose (Km = 0.7 mM), and was shown to be the same as glucose transport system II. System II also transported mannose, 2-deoxyglucose, xylose, and talose and is therefore a general monosaccharide transport system. System II was derepressed by carbon starvation, completely repressed by glucose, mannose, and 2-deoxyglucose, and partially repressed by fructose and xylose. Incubation with galactose yielded twice as much activity as starvation. This extra induction by galactose required protein synthesis, and represented an increase in activity of system II rather than the induction of another transport system. Glucose, mannose, and 2-deoxyglucose caused rapid degradation of preexisting system II; fructose and xylose caused a slower degradation of activity.     

Fructose transport in Neurospora crassa.

A specific fructose uptake system (Km = 0.4 mM) appeared in Neurospora crassa when glucose-grown mycelia were starved. Fructose uptake had kinetics different from those of intramycelial fructose phosphorylation, and uptake appeared to be carrier mediated. The only sugar which competitively inhibited fructose uptake was L-sorbose (Ki = 9 mM). Glucose, 2-deoxyglucose, mannose, and 3-O-methyl glucose were noncompetitive inhibitors of fructose uptake. Incubation of glucose-grown mycelia with glucose, 2-deoxyglucose, or mannose prevented derepression of the fructose transport system, whereas incubation with 3-O-methyl glucose caused the appearance of five times as much fructose uptake activity as did starvation conditions.