Role of de novo protein synthesis in the interconversion of glucose transport systems in the yeast Pichia ohmeri

Glucose-repressed cells of the yeast Pichia ohmeri IGC 2879 transported glucose by facilitated diffusion. Derepression led to the formation of a glucose/proton symport and the simultaneous reduction of the facilitated diffusion capacity by about 70%. Cycloheximide prevented this interconversion indicating its dependence on de novo protein synthesis (proteosynthetic interconversion). In buffer with 2% glucose the glucose/proton symport suffered irreversible inactivation while the facilitated diffusion system was simultaneously restored. This reverse interconversion process did not require de novo protein synthesis as indicated by its lack of sensitivity to cycloheximide (degradative interconversion). Thus the glucose/proton symport system appeared to consist of about 70% of the facilitated diffusion proteins turned silent through association with additional protein(s) the latter being sensitive to glucose-induced repression and glucose-induced inactivation.     

Reversible loss of affinity induced by glucose in the maltose-H+ symport of Saccharomyces cerevisiae

Glucose represses and inactivates maltose transport in Saccharomyces cerevisiae. The inactivation has been described as an irreversible process involving proteolysis. We have studied the inactivation of the maltose-H+ symport in this yeast and have observed that the mechanism of inactivation depends on the physiological conditions. In resting cells there was a decrease in transport capacity. The rate of decrease was enhanced nonspecifically by the presence of a sugar, glucose being more effective than maltose. In growing cells, glucose induced a decrease in affinity of the H+-symport which could be recovered by starvation, even in the presence of cycloheximide; there was no loss in capacity or, if present, this loss could be explained fully by the dilution due to repression during growth on glucose. We submit that in growing cells inactivation consists in a reversible modification of the permease not involving proteolysis.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556.

Regulation of transport of D-glucose and D-fructose was studied in Kluyveromyces marxianus grown in continuous culture. Both substrates could be transported by at least two different transport systems, low-affinity transport and high-affinity proton-sugar symport. The low-affinity transporter, specific for both glucose and fructose, was constitutively present and was apparently not regulated by carbon catabolite repression. Regulation of the activity of the glucose- and fructose-specific proton symport systems appeared to proceed mainly through catabolite repression. Activation of symport did not need the presence of specific inductor molecules in the medium. Nevertheless, the capacities of the proton-sugar symporters varied in cells grown on a wide variety of carbon sources. The possibility that the control of proton symport activity is related to the presence of specific intracellular metabolites is discussed.     

Transport of fructose by a proton symport in a brewing yeast

Abstract Saccharomyces cerevisiae IGC4261, a brewing strain, transported fructose and sorbose but not glucose by a high-affinity, low-capacity proton symport. The symport was not subject to glucose repression and coexisted with the facilitated diffusion system for glucose, fructose, sorbose and other sugars. Transport by the symport was accumulative. The stoichiometry was one proton per molecule of fructose. Maltose acted as a non-competitive inhibitor.     

Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis.

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.     

Occurrence and taxonomic aspects of proton movements coupled to sugar transport in the yeast genusKluyveromyces

The occurrence of proton symport mechanisms for the transport of glucose, galactose, fructose, raffinose and sucrose in 21 yeast strains representing the species of the genusKluyveromyces was surveyed. Proton symport of one or more sugars occurred in 57% of the strains. Similarly, all the sugars investigated were transported by symports by several strains. Symport systems for non-utilisable sugars were rare. Starvation of cells frequently resulted in the appearance of a symport absent in non-starved glucose-grown cells, indicating that repression of proton symports by glucose and subsequent derepression by starvation is a general phenomenon in members ofKluyveromyces. The addition of a sugar to cell suspensions resulted in acidification in 80% of cases, indicating the activity of a membrane-bound ATPase. Acidification was also observed with a number of sugars that cannot be utilised by the particular species. Interesting correlations between the number of proton symports and the abundance of other phenotypic characteristics in members of the genus emerged. Most members of the infertile group of species showing an increase in the number of small chromosomes, inability to produce well-developed pseudomycelium, linoleic and linolenic acid, a decrease in the number of carbon compounds utilised and inability to utilise ethylamine also had no proton symports, whereas most members of the interfertile species produced one or more proton symports. It was concluded that the distribution of the number of proton symports amongstKluyveromyces species coincided with that of other positive characteristics and may therefore be of taxonomic value.     

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco.

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.     

Genes involved in control of galactose uptake in Lactobacillus brevis and reconstitution of the regulatory system in Bacillus subtilis

The heterofermentative lactic acid bacterium Lactobacillus brevis transports galactose and the nonmetabolizable galactose analogue thiomethyl-beta-galactoside (TMG) by a permease-catalyzed sugar:H(+) symport mechanism. Addition of glucose to L. brevis cells loaded with [(14)C]TMG promotes efflux and prevents accumulation of the galactoside, probably by converting the proton symporter into a uniporter. Such a process manifests itself physiologically in phenomena termed inducer expulsion and exclusion. Previous evidence suggested a direct allosteric mechanism whereby the phosphocarrier protein, HPr, phosphorylated at serine-46 [HPr(Ser-P)], binds to the galactose:H(+) symporter to uncouple sugar transport from proton symport. To elucidate the molecular mechanism of inducer control in L. brevis, we have cloned the genes encoding the HPr(Ser) kinase, HPr, enzyme I, and the galactose:H(+) symporter. The sequences of these genes were determined, and the relevant phylogenetic trees are presented. Mutant HPr derivatives in which the regulatory serine was changed to either alanine or aspartate were constructed. The cloned galP gene was integrated into the chromosome of Bacillus subtilis, and synthesis of the mutant HPr proteins in this organism was shown to promote regulation of GalP, as expected for a direct allosteric mechanism. We have thus reconstituted inducer control in an organism that does not otherwise exhibit this phenomenon. These results are consistent with the conclusion that inducer exclusion and expulsion in L. brevis operates via a multicomponent signal transduction mechanism wherein the presence of glycolytic intermediates such as fructose 1,6-bisphosphate (the intracellular effector), derived from exogenous glucose (the extracellular effector), activates HPr(Ser) kinase (the sensor) to phosphorylate HPr on Ser-46 (the messenger), which binds to the galactose:H(+) symporter (the target), resulting in uncoupling of sugar transport from proton symport (the response). This cascade allows bacteria to quickly respond to changes in external sugar concentrations. Understanding the molecular mechanism of inducer control advances our knowledge of the link between metabolic and transport processes in bacteria.     

Allosteric regulation of the glucose:H+ symporter of Lactobacillus brevis: cooperative binding of glucose and HPr(ser-P).

Lactobacillus brevis transports glucose and the nonmetabolizable glucose analog 2-deoxyglucose via a proton symport mechanism that is allosterically inhibited by the seryl-phosphorylated derivative of HPr, the small phosphocarrier protein of the phosphotransferase system. We have demonstrate that S46DHPr, a mutant analog of HPr which conformationally resembles HPr(ser-P) but not free HPr, specifically binds to membranes derived from glucose-grown L. brevis cells if and only if a substrate of the glucose permease is also present.     

Misregulation of maltose uptake in a glucose repression defective mutant of Saccharomyces cerevisiae leads to glucose poisoning

In hex2 mutants of Saccharomyces cerevisiae, which are defective in glucose repression of several enzymes, growth is inhibited if maltose is present in the medium. After adding [14C]maltose to cultures growing with ethanol, maltose metabolism was followed in both hex2 mutant and wild-type cells. The amount of radioactivity incorporated was much higher in hex2 than in wild-type cells. Most of the radioactivity in hex2 cells was located in the low molecular mass fraction. Pulse-chase experiments showed that 2 h after addition of maltose, hex2 cells hydrolysed maltose to glucose, which was partially excreted into the medium. 31P-NMR studies gave evidence that turnover of sugar phosphates was completely abolished in hex2 cells after 2 h incubation with maltose. 13C-NMR spectra confirmed these results: unlike those for the wild-type, no resonances corresponding to fermentation products (ethanol, glycerol) were found for hex2 cells, whereas there were resonances corresponding to glucose. Although maltose is taken up by proton symport, the internal pH in the hex2 mutant did not change markedly during the 5 h after adding maltose. The intracellular accumulation of glucose seems to explain the inhibition of growth by maltose, probably by means of osmotic damage and/or unspecific O-glycosylation of proteins. Neither maltose permease nor maltase was over-expressed, and so these enzymes were not the cause of glucose accumulation. Hence, the coordination of maltose uptake, hydrolysis to glucose and glycolysis of glucose is not regulated simply by the specific activity of the catabolic enzymes involved.(ABSTRACT TRUNCATED AT 250 WORDS)     

A member of the sugar transporter family, Stl1p is the glycerol/H+ symporter in Saccharomyces cerevisiae

Glycerol and other polyols are used as osmoprotectants by many organisms. Several yeasts and other fungi can take up glycerol by proton symport. To identify genes involved in active glycerol uptake in Saccharomyces cerevisiae we screened a deletion mutant collection comprising 321 genes encoding proteins with 6 or more predicted transmembrane domains for impaired growth on glycerol medium. Deletion of STL1, which encodes a member of the sugar transporter family, eliminates active glycerol transport. Stl1p is present in the plasma membrane in S. cerevisiae during conditions where glycerol symport is functional. Both the Stl1 protein and the active glycerol transport are subject to glucose-induced inactivation, following identical patterns. Furthermore, the Stl1 protein and the glycerol symporter activity are strongly but transiently induced when cells are subjected to osmotic shock. STL1 was heterologously expressed in Schizosaccharomyces pombe, a yeast that does not contain its own active glycerol transport system. In S. pombe, STL1 conferred the ability to take up glycerol against a concentration gradient in a proton motive force-dependent manner. We conclude that the glycerol proton symporter in S. cerevisiae is encoded by STL1.     

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae

The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.     

Variable H+/substrate stoicheiometries in Rhodotorula gracilis are caused by a pH-dependent protonation of the carrier(s).

Two carrier-mediated systems transport sugars in the yeast Rhodotorula gracilis depending on the pH. One system, with higher affinity for sugars, catalyses a symport of protons with sugar, whereas the other system, having lower affinity, is independent of protons. This was shown in three different ways. (1) At low pH, where only the high-affinity system works, a H+/sugar stoicheiometry of 1 was found. An increase of the pH and of the sugar concentration, which allowed the low-affinity system to operate, brought about a drop of the stoicheiometry to values below 1. (2) During H+ symport the influx of positive charge was electrically compensated by an equivalent efflux of K+ from the cells. At high pH and high sugar concentrations this stoicheiometry of K+ and sugar decreased concomitant with the H+/sugar stoicheiometry. (3) At pH 7.5 both transport systems were operating, as shown by biphasic saturation kinetics. Under these conditions only the high-affinity transport was found to be electrogenic. These results agree with the theory of an electrogenic H+/sugar symport where changes in the affinity for substrate are brought about by reversible protonation and deprotonation of the carrier.     

Transport of xylose and glucose in the xylose-fermenting yeast Pichia stipitis

Summary A low-affinity and a high-affinity sylose proton symport operated simultaneously in both starved and non-starved cells of Pichia stipitis. Glucose competed with xylose for transport by the low-affinity system and inhibited xylose transport by the high-affinity system non-competitively. The low affinity system was subject to substrate inhibition when glucose but not when xylose was the substrate. The differences between the characteristics of monosaccharide transport by Pichia stipitis and its imperfect state, Candida shehatae, are discussed.     

Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt.

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.     

Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism

In Kluyveromyces marxianus grown on a glucose-containing synthetic medium four different sugar transporters have been identified. In cells, harvested during the exponential phase, only the constitutive glucose/fructose carrier, probed with 6-deoxy-D-glucose or sorbose, appeared to be active. In cells from the stationary phase three proton symporters can be active, recognizing 6-deoxyglucose (a glucose/galactose carrier), sorbose (a fructose carrier) and galactosides (lactose carrier), respectively. These symporters appeared to be sensitive to catabolite inactivation. This process is induced by incubating cells in the presence of glucose, fructose or mannose. Catabolite inactivation was not influenced by the inhibitor of protein synthesis, anisomycin. Derepression of the proton/sorbose and the proton/galactoside symporters proceeded readily when cells were incubated in a medium without glucose. Activation of the proton/galactose symporter needed, in addition, the presence of specific molecules (inducers) in the medium. The activation of each of these active transport systems was inhibited by anisomycin, showing the involvement of protein synthesis.     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.     

Glucose uptake and metabolism in the Trichinella spiralis nurse cell

Isolated Trichinella spiralis nurse cells transport a significantly greater amount of glucose/mg of protein than the normal skeletal muscle cell line (L6). V(max) and K(m) estimations revealed that nurse cells have a much higher saturation point than L6 cells for glucose. The effects of numerous physiological conditions (Na(+) concentration, pH, and temperature) on nurse cell glucose uptake were investigated. It was determined that sodium concentration had no effect on glucose uptake. Low (<6.5) and high (>7.3) pH and low (5 degrees C) temperatures significantly effected glucose uptake. The two hormones, insulin and epinephrine, appeared to have little, if any, influence on the rate of glucose uptake by nurse cells. Glucose uptake was inhibited in the presence of 6-carbon carbohydrates. The H(+)/glucose symport inhibitors, dicyclohexylcarbodiimide (DCCD) and Carbonyl cyanide 4-trifluoromethoxyphenlhydrazone (FCCP), and the facilitated diffusion inhibitor phloretin also inhibited glucose uptake. Oubain, a Na(+)/glucose symport inhibitor, did not inhibit glucose uptake. These data, in conjunction with Western blot analyses, revealed that the transport of glucose occurs via H(+)/glucose symport and facilitated diffusion, perhaps through the glucose transport proteins GLUT 1 and/or 4. It was also demonstrated that nurse cells are capable of synthesising glycogen. It appears that glycogen is in a constant state of flux and physiological conditions, such as glucose concentration, significantly influence the synthesis of this macromolecule. We conclude that these results are consistent with the hypothesis that nurse cells, at least maintained in vitro, are metabolically highly active but show significant divergence from normal muscle cells in several fundamental aspects of sugar metabolism.     

Lactose permease H+-lactose symporter: mechanical switch or Brownian ratchet?

Lactose permease structure is deemed consistent with a mechanical switch device for H(+)-coupled symport. Because the crystallography-assigned docking position of thiodigalactoside (TDG) does not make close contact with several amino acids essential for symport; the switch model requires allosteric interactions between the proton and sugar binding sites. The docking program, Autodock 3 reveals other lactose-docking sites. An alternative cotransport mechanism is proposed where His-322 imidazolium, positioned in the central pore equidistant (5-7 A) between six charged amino acids, Arg-302 and Lys-319 opposing Glu-269, Glu-325, Asp-237, and Asp-240, transfers a proton transiently to an H-bonded lactose hydroxyl group. Protonated lactose and its dissociation product H(3)O+ are repelled by reprotonated His-322 and drift in the electrostatic field toward the cytosol. This Brownian ratchet model, unlike the conventional carrier model, accounts for diminished symport by H322N mutant; how H322 mutants become uniporters; why exchanging Lys-319 with Asp-240 paradoxically inactivates symport; how some multiple mutants become revertant transporters; the raised export rate and affinity toward lactose of uncoupled mutants; the altered specificity toward lactose, melibiose, and galactose of some mutants, and the proton dissociation rate of H322 being 100-fold faster than the symport turnover rate.