Facilitated Diffusion Carrier Research Articles

Different Gene Expression Patterns of Hexose Transporter Genes Modulate Fermentation Performance of Four Saccharomyces cerevisiae Strains

In Saccharomyces cerevisiae, the fermentation rate and the ability to complete the sugar transformation process depend on the glucose and fructose transporter set-up. Hexose transport mainly occurs via facilitated diffusion carriers and these are encoded by the HXT gene family and GAL2. In addition, FSY1, coding a fructose/H+ symporter, was identified in some wine strains. This little-known transporter could be relevant in the last part of the fermentation process when fructose is the most abundant sugar. In this work, we investigated the gene expression of the hexose transporters during late fermentation phase, by means of qPCR. Four S. cerevisiae strains (P301.9, R31.3, R008, isolated from vineyard, and the commercial EC1118) were considered and the transporter gene expression levels were determined to evaluate how the strain gene expression pattern modulated the late fermentation process. The very low global gene expression and the poor fermentation performance of R008 suggested that the overall expression level is a determinant to obtain the total sugar consumption. Each strain showed a specific gene expression profile that was strongly variable. This led to rethinking the importance of the HXT3 gene that was previously considered to play a major role in sugar transport. In vineyard strains, other transporter genes, such as HXT6/7, HXT8, and FSY1, showed higher expression levels, and the resulting gene expression patterns properly supported the late fermentation process.

Caspofungin uptake is mediated by a high-affinity transporter in Candida albicans.

The uptake of the echinocandin drug caspofungin acetate in Candida albicans was evaluated at drug levels at or near the MIC for the organism. Maximal uptake was achieved in 10 min and was energy independent. A saturable transport system, consistent with a facilitated-diffusion carrier, was observed with the unlabeled drug competing with the labeled drug for uptake and efflux. More than 90% of the transported drug was observed in a single kinetic compartment that was available for efflux, indicating that the drug was free in the cytoplasm following uptake. Efflux was also energy independent but was sensitive to the presence of a fully loaded carrier on both faces of the bilayer. Overall, the data presented are consistent with the presence of a high-affinity facilitated-diffusion transporter that mediates caspofungin uptake and could be a potential source of transport-related reduced susceptibility.

Characterization of glycerol uptake in bloodstream and procyclic forms of Trypanosoma brucei.

Uptake of glycerol was studied in bloodstream and insect forms of the African parasite Trypanosoma brucei using [14C]glycerol in combination with the oil centrifugation technique. Our kinetic measurements revealed that in bloodstream forms glycerol appeared to be transported by two different mechanisms: firstly by a facilitated-diffusion carrier showing a Km of 0.17 mM and a Vmax of 44 nmol 10(-8) cells min(-1) that predominates at low glycerol concentrations, and secondly by simple diffusion. The effects induced by various inhibitors suggest that uptake is neither sodium dependent nor proton-motive-force driven. The saturable component of transport was phloretin and cytochalasin B sensitive and could also be inhibited by the substrate analogue glyceraldehyde, which led to a 74% decrease in glycerol uptake. In insect forms, however, glycerol is taken up by simple diffusion only. Uptake was insensitive to mercury ions and was not influenced by a variety of different channel inhibitors. Our data show that in T brucei glycerol transport across the plasma membrane occurs by simple diffusion. In addition, bloodstream forms express a carrier protein which promotes a rapid transport at low glycerol concentrations. Expression of this transport protein may account for a selective secretion of intracellular glycerol which otherwise could become toxic for the parasite due to its specific compartmentation of glycolysis.

The inhibition of pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei and its metabolic implications.

The pyruvate produced by glycolysis in the bloodstream form of the trypanosome is excreted into the host bloodstream by a facilitated diffusion carrier. The sensitivity of pyruvate transport for alpha-cyano-4-hydroxycinnamate and the compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate], which are known to be selective inhibitors of pyruvate (monocarboxylate) transporters present in mitochondria and the plasma membrane of eukaryotic cells, was examined. The trypanosomal pyruvate carrier was found to be rather insensitive to inhibition by alpha-cyano-4-hydroxycinnamate (Ki = 17 mM) but could be completely blocked by UK5099 (Ki = 49 microM). Inhibition of pyruvate transport resulted in the retention, and concomitant accumulation, of pyruvate within the trypanosomes, causing acidification of the cytosol and osmotic destabilization of the cells. Our results indicate that this physiological state has serious metabolic consequences and ultimately leads to cell death; thereby identifying the pyruvate carrier as a possible target for chemotherapeutic intervention.

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.

Membrane-related processes and overall energy metabolism in Trypanosoma brucei and other kinetoplastid species.

An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form of Trypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature. Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of the T. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane of T. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport in L. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.

The electrochemical proton gradient in the bloodstream form of Trypanosoma brucei is dependent on the temperature

The membrane potential and pH gradient over the plasma membrane of the protozoan parasite Trypanosoma brucei were measured with radioactive indicators in combination with the silicone oil centrifugation technique over a range of temperatures. At 37°C a small membrane potential and pH gradient of similar magnitude, but of opposite polarity, were measured. The resulting electrochemical proton gradient was almost zero. However, when the temperature was lowered from 37°C to 22°C, the internal pH was kept constant independent of the external pH and a membrane potential of between -- 100 and - 150 mV was measured, depending on the external pH. Measurements at various temperatures between 15°C and 37°C revealed that above 26°C the membrane potential collapsed and that this collapse correlated with a sudden increase in membrane fluidity. The uptake of 2-deoxy- d-glucose and of pyruvate, which are both mediated by facilitated diffusion carriers in the plasma membrane of the trypanosome, were also affected by this sudden increase in fluidity of the membrane. The overall rate of the conversion of glucose into its metabolites, which is independent of the plasma membrane, varied only gradually. We conclude (i) that major changes occur in the plasma membrane of T. brucei around 26°C, that affect all membrane related processes; (ii) that the electrochemical proton gradient plays a minor role in the energy metabolism of T. brucei when it resides in the bloodstream of the mammalian host at 37°C; and (iii) that below 26°C an electrochemical proton gradient is maintained over the plasma membrane.

Pyruvate transport across the plasma membrane of the bloodstream form of Trypanosoma brucei is mediated by a facilitated diffusion carrier

The characteristics of pyruvate transport across the plasma membrane in the bloodstream form of Trypanosoma brucei were studied using [ 14C]pyruvate in combination with the silicone-oil centrifugation technique. We present evidence for the existence of a facilitated diffusion carrier in the plasma membrane of T. brucei which specifically mediates the translocation of pyruvate. The uptake of pyruvate followed saturation kinetics (K m 1.96 ± 0.28 mM; C max 36.61 ± 1.15 nmol pyruvate/30 sec.mg protein), after correction of the data for a nonsaturable diffusion component. The uptake of pyruvate was competitively inhibited by a number of (oxo)monocarboxylic acids, including pyruvate analogs and metabolically related substances, but not by L-lactate. The transport exhibited the phenomenon of transacceleration, indicative for the involvement of a facilitated diffusion carrier. The carrier is highly specific for pyruvate and differs from other known monocarboxylate carriers present in the mitochondrial and/or plasma membrane of other eukaryotic cells in that it does not transport L-lactate.

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Glucose transport in crabtree-positive and crabtree-negative yeasts.

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.

Uniport carriers for metabolites

The analysis of membrane transport systems falls into two broad areas: those studies that investigate the kinetic aspects of the movement of solutes and ions, and more recently, investigations aimed at identifying and elucidating the structure of transporters. The former area has dominated the field of facilitated diffusion carriers for the past quarter century (see Stein, Transport and Diffusion Across Cell Membranes. Academic Press, 1986). Such investigations have concentrated on:

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na + concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na +/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na + concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.

Expression of transporters in Xenopus oocytes.

Xenopus oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to glucose and amino acids were measured electrophysiologically. Na+/glucose, amino acid cotransporters were expressed in the oocytes by injection of small intestine mRNA, while facilitated diffusion carrier protein(s) (uniporter) were mainly expressed by injection of kindney mRNA.

Synthetic Polymeric Membranes: Practical Applications - Past, Present and Future

Abstract