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.     

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.     

Inactivation of active glucose transport in Candida wickerhamii is triggered by exocellular glucose

Abstract Under conditions of derepression the yeast Candida wickerhamii formed a high-affinity glucose proton symport. Glucose and glucose analogues induced inactivation of the glucose proton symport and its interconversion into a low-affinity facilitated diffusion system. The specific inactivation rate increased with the concentration of the inactivating sugar and did not obey saturation kinetics. This dependence was still pronounced at sugar concentrations far above saturation of the glucose transport systems. This suggested that the inactivation and interconversion mechanism was triggered by interaction of the inactivating sugar with receptor sites located on the cell surface.     

Characterization of the second external alternative dehydrogenase from mitochondria of the respiratory yeast Kluyveromyces lactis

The mitochondria of the respiratory yeast Kluyveromyces lactis are able to reoxidize cytosolic NADPH. Previously, we characterized an external alternative dehydrogenase, KlNde1p, having this activity. We now characterize the second external alternative dehydrogenase of K. lactis mitochondria, KlNde2p. We examined its role in cytosolic NADPH reoxidation by studying heterologous expression of KlNDE2 in Saccharomyces cerevisiae mutants and by constructing Δklnde1 and Δklnde2 mutants. KlNde2p uses NADH or NADPH as substrates, its activity in isolated mitochondria is not regulated by exogenously added calcium and it is not down-regulated when the cells grow in glucose versus lactate. KlNde2p shows lower affinity for NADPH than KlNde1p. Both enzymes show similar pH optimum.     

Modulation of glucose transport in human red blood cells by ATP

Depletion of energy stores of human red cells decreases the maximum transport capacity, $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$, for glucose transport to a value one-third or less of that found in red cells from freshly drawn blood. There is no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. Hemolysis and resealing of red cells with ATP or ADP reverses the decrease in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$. The maximum effect occurs at concentrations of ATP in the normal range for red cells, however, there is little effect from ADP concentrations in its normal range in freshly drawn red cells. Hemolysis and resealing with ATP gives an increase in $\text{J}{}_{\mathrm{<mtext>m</mtext>}}$ and an increase in differential labeling by photolytic labeling with tritiated cytochalasin B. Most of the activation is lost after a second hemolysis-reseal without ATP but about 25% of the activation remains.     

The glucose signaling network in yeast

Background

Most cells possess a sophisticated mechanism for sensing glucose and responding to it appropriately. Glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae represent an important paradigm for understanding how extracellular signals lead to changes in the gene expression program in eukaryotes.

Scope of review

This review focuses on the yeast glucose sensing and signaling pathways that operate in a highly regulated and cooperative manner to bring about glucose-induction of HXT gene expression.

Major conclusions

The yeast cells possess a family of glucose transporters (HXTs), with different kinetic properties. They employ three major glucose signaling pathways—Rgt2/Snf3, AMPK, and cAMP-PKA—to express only those transporters best suited for the amounts of glucose available. We discuss the current understanding of how these pathways are integrated into a regulatory network to ensure efficient uptake and utilization of glucose.

General significance

Elucidating the role of multiple glucose signals and pathways involved in glucose uptake and metabolism in yeast may reveal the molecular basis of glucose homeostasis in humans, especially under pathological conditions, such as hyperglycemia in diabetics and the elevated rate of glycolysis observed in many solid tumors.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Sugar transport in Coprinus cinereus

Two transport systems for glucose were detected: a high affinity system with a K_m of 27 μM, and a low affinity system with a K_m of 3.3 mM. The high affinity system transported glucose, 2-deoxy-d-glucose (K_m = 26 μM), 3-O-methylglucose (K_m = 19 μM), d-glucosamine (K_m = 652 μM), d-fructose (K_m = 2.3 mM) and l-sorbose (K_m = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45°C). The low affinity system transported glucose, 2-deoxy-d-glucose (K_m = 7.5 mM), and 3-O-methylglucose (K_m = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30–50°C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-d-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present in sporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation in glucose-free medium. The half-time for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5–7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-d-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity.     

Effects of lipids on the transport activity of the reconstituted glucose transport system from rat adipocyte

The glucose transport system, isolated from rat adipocyte membrane fractions, was reconstituted into phospholipid vesicles. Vesicles composed of crude egg yolk phospholipids, containing primarily phosphatidylcholine (PC) and phosphatidylethanolamine (PE), demonstrated specific d-glucose uptake. Purified vesicles made of PC and PE also supported such activity but PC or PE by themselves did not. The modulation of this uptake activity has been studied by systematically altering the lipid composition of the reconstituted system with respect to: (1) polar headgroups; (2) acyl chains, and (3) charge. Addition of small amounts (20 mol%) of PS, phosphatidylinositol (PI), cholesterol, or sphingomyelin significantly reduced glucose transport activity. A similar effect was seen with the charged lipid, phosphatidic acid. In the case of PS, this effect was independent of the acyl chain composition. Polar headgroup modification of PE, however, did not appreciably affect transport activity. Free fatty acids, on the other hand, increased or decreased activity based on the degree of saturation and charge. These results indicate that glucose transport activity is sensitive to specific alterations in both the polar headgroup and acyl chain composition of the surrounding membrane lipids.     

Depletion of casein kinase I leads to a NAD(P)/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

Background

In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag⁻ phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing.

Methods

A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses.

Results

Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced.

Conclusions

The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)⁺/NAD(P)H redox balance state.

General significance

The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.     

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.     

Endotoxin-induced alterations in glucose transport in isolated adipocytes

2-Deoxyglucose and $\text{3-O-}\text{methyglucose}$ were used to assess endotoxin-induced changes in glucose transport in rat adipocytes. 6 h after Escherichia coli endotoxin injection insulin-stimulated 2-deoxyglucose uptake was significantly depressed ($\text{V}\text{decreased}\text{, K}{}_{\mathrm{<mtext>m</mtext>}}\text{unaltered}$), phosphorylation of 2-deoxyglucose was seemingly unimpaired; basal 3-methylglucose entry was significantly increased, insulin-stimulated uptake was unaltered. Insulin significantly reduced $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ in control and endotoxin-treated cells. Cytochalasin B-insensitive uptake of both 2-deoxyglucose and 3-methylglucose, a small fraction of total transport, increased significantly in endotoxic cells. Endotoxin reduced spermine- and insulin-stimulated 2-deoxyglucose uptake to a similar extent. Results are consistent with the hypotheses that (1) a site of endotoxin-induced insulin resistance is at the cell membrane level and may reflect a decrease in number or activity of effective carrier units, rather than alterations in affinity, (2) endotoxin does not compromise the hexokinase system, (3) the cell membrane-localized effect of endotoxin on hexose transport is not necessarily mediated by the insulin receptor and (4) the entry of 2-deoxyglucose and 3-methylglucose may involve two separate transport systems.     

Inhibition of glucose transport by benzoquinone and the addition product of benzoquinone and dithiothreitol

Hydroxylated benzene derivatives inhibited transport of d-glucose into calf-thymocyte plasma-membrane vesicles. The relative effectiveness of these was pyrogallol >hydroquinone catechol phloroglucinol. The most thoroughly studied of these agents, hydroquinone, produced weak, immediate inhibition when first added to membranes (K_i > 10 mM). This was followed by a gradual, time-dependent inhibition of the residual transport activity. The instantaneous inhibition could not be prevented by any agent tested, whereas the time-dependent phase was affected by reducing agents and superoxide dismutase. Several reducing agents (dithiothreitol, glutathione, NADH, ascorbate, bisulfite but not cysteine) prevented, while superoxide dismutase and cysteine potentiated time-dependent inhibition when added to the membrane suspension simultaneously with hydroquinone. NADH and ascorbate also prevented, whereas dithiothreitol potentiated, further time-dependent inhibition when added to membranes 2 h after hydroquinone. In contrast, all three reducing agents arrested time-dependent inhibition when added 2 h after pyrogallol. Numerous agents had no effect on time-dependent hydroquinone inhibition: oxidants (H₂O₂), metal chelators (EDTA, bathophenanthroline disulfonate, Desferral), radical scavengers (benzoate, ethanol), anti-oxidants (butylated hydroxytoluene) and catalase. Benzoquinone, an oxidation product of hydroquinone, was a much more potent inhibitor (K_i 1 mM) than hydroquinone. Several reducing agents (ascorbate, NADH, bisulfite) prevented this effect, while cysteine and dithiothreitol potentiated it. Below 300 μM, benzoquinone had little or no effect on sugar transport with or without glutathione or cysteine. Addition of dithiothreitol to benzoquinone (10–300 μM) resulted in potent inhibition of sugar transport (K_i 50 μM). Maximal inhibition occurred with a 1 : 1 mol ratio of these agents or with excess dithiothreitol. The inhibitory agent from benzoquinone and dithiothreitol lost potency in the presence of air and membranes, but was stable for hours in the presence of either of these alone. dl-threo-1,4-bis(2,5-dihydroxyphenylthio)-2,3-butanediol was obtained from the reaction of equimolar quantities of dithiothreitol and benzoquinone in ethanol. The structure of this adduct was established by spectroscopic and chemical methods. This compound exhibited all of the properties of the inhibitor which had been formed from benzoquinone and dithiothreitol in aqueous solution.     

13C-NMR study of the glucose synthesis pathways in the bacterium Chlorobium thiosulfatophilum

Photoassimilation of ¹³CO₂ and acetate by the photosynthetic bacterium Chlorobium thiosulfatophilum was investigated using ¹³C-NMR as the method for determination of the labelling pattern of the glucose synthesized by the bacterium. Metabolic pathways functioning in the bacterium were identified by analysis of the multiplet structure of the spectra of tightly coupled systems. The labelling pattern showed that glucose was synthesized in C. thiosulfatophilum mainly by the gluconeogenesis pathway. In agreement with previous investigations, the reserve polysaccharide of C. thiosulfatophilum was shown to be polyglucose, with the glucose units linked predominantly by (1→4)α-glucosidic linkages.     

Glucose starvation-induced turnover of the yeast glucose transporter Hxt1

Background

The budding yeast Saccharomyces cerevisiae possesses multiple glucose transporters with different affinities for glucose that enable it to respond to a wide range of glucose concentrations. The steady-state levels of glucose transporters are regulated in response to changes in the availability of glucose. This study investigates the glucose regulation of the low affinity, high capacity glucose transporter Hxt1.

Methods and results

Western blotting and confocal microscopy were performed to evaluate glucose regulation of the stability of Hxt1. Our results show that glucose starvation induces endocytosis and degradation of Hxt1 and that this event requires End3, a protein required for endocytosis, and the Doa4 deubiquitination enzyme. Mutational analysis of the lysine residues in the Hxt1 N-terminal domain demonstrates that the two lysine residues, K12 and K39, serve as the putative ubiquitin-acceptor sites by the Rsp5 ubiquitin ligase. We also demonstrate that inactivation of PKA (cAMP-dependent protein kinase A) is needed for Hxt1 turnover, implicating the role of the Ras/cAMP-PKA glucose signaling pathway in the stability of Hxt1.

Conclusion and general significance

Hxt1, most useful when glucose is abundant, is internalized and degraded when glucose becomes depleted. Of note, the stability of Hxt1 is regulated by PKA, known as a positive regulator for glucose induction of HXT1 gene expression, demonstrating a dual role of PKA in regulation of Hxt1.     

Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae

We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift.     

Membrane tension regulates water transport in yeast

Evidence that membrane surface tension regulates water fluxes in intact cells of a Saccharomyces cerevisiae strain overexpressing aquaporin AQY1 was obtained by assessing the osmotic water transport parameters in cells equilibrated in different osmolarities. The osmotic water permeability coefficients (P_f) obtained for yeast cells overexpressing AQY1 incubated in low osmolarity buffers were similar to those obtained for a double mutant aqy1aqy2 and approximately three times lower (with higher activation energy, E_a) than values obtained for cells incubated in higher osmolarities (with lower E_a). Moreover, the initial inner volumes attained a maximum value for cells equilibrated in lower osmolarities (below 0.75 M) suggesting a pre-swollen state with the membrane under tension, independent of aquaporin expression. In this situation, the impairment of water channel activity suggested by lower P_f and higher E_a could probably be the first available volume regulatory tool that, in cooperation with other osmosensitive solute transporters, aims to maintain cell volume. The results presented point to the regulation of yeast water channels by membrane tension, as previously described in other cell systems.     

Sugar transport in chick embryo cardiac cells in culture. Analysis by countertransport,relationship to phosphorylation and effect of glucose starvation

Embryonic chick heart cells in culture transport 2-deoxy-D-glucose and 3-O-methyl-D-glucose very rapidly. By direct measurements of uptake, it was not possible to estimate accurately transport rates, nor, with 2-deoxyglucose, to discriminate clearly between its transport and phosphorylation. In contrast, the technique of countertransport made it possible to determine precisely initial transport velocity and to make the following observations: (1) phosphorylation, and not transport, is rate-limiting in 2-deoxyglucose uptake; (2) hexose transport is stimulated 5-fold by removal of glucose from culture medium; and (3) this stimulation is followed by an increase in phosphorylation, but the effect is much less pronounced (2-fold stimulation only). In conclusion, the adaptative regulation of glucose transport described in many fibroblast cell lines exists also in cardiac cells.     

Cloning,characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica

Background

The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians.

Methods

GLUT2 was sequenced from northern and southern phenotypes of R. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques.

Results

GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150 mM urea. Proteins from all study subjects had similar affinity constants (~ 12 mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens.

Conclusion

Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival.

General significance

This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.     

Derepression of the high-affinity phosphate uptake in the yeast Saccharomyces cerevisiae

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb⁺ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the low-affinity system has decreased for about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.