Molecular Mechanism of Nramp-Family Transition Metal Transport

The Natural resistance-associated macrophage protein (Nramp) family of transition metal transporters enables uptake and trafficking of essential micronutrients that all organisms must acquire to survive. Two decades after Nramps were identified as proton-driven, voltage-dependent secondary transporters, multiple Nramp crystal structures have begun to illustrate the fine details of the transport process and provide a new framework for understanding a wealth of preexisting biochemical data. Here we review the relevant literature pertaining to Nramps’ biological roles and especially their conserved molecular mechanism, including our updated understanding of conformational change, metal binding and transport, substrate selectivity, proton transport, proton-metal coupling, and voltage dependence. We ultimately describe how the Nramp family has adapted the LeuT fold common to many secondary transporters to provide selective transition-metal transport with a mechanism that deviates from the canonical model of symport.     

Auxin Transport in Suspension-Cultured Soybean Root Cells : II. Anion Effects on Carrier-Mediated Uptake

To test the hypothesis that the carrier-mediated component of the indoleacetic acid (IAA) influx involves an electrogenic proton/IAA anion symport, the effects on the IAA influx of salts expected to depolarize the membrane potential were examined in suspension-cultured soybean (Glycine max [L.] Merr.) root cells. Although KCl does inhibit carrier-mediated uptake, the effect is specific to the anion at low concentrations and not due to more general processes such as changes in ionic or osmotic strength. Other anions such as bromide, iodide, and fluoride inhibit the carrier more strongly. Because potassium iminodiacetate, which is also expected to depolarize the membrane potential, has no inhibitory effect on the IAA influx, there is no evidence for the involvement of the membrane potential in carrier-mediated uptake. It is therefore most likely that in soybean cells, if carrier-mediated uptake occurs via a proton symport, the H⁺:IAA— stoichiometry is 1:1. At concentrations greater than 70 millimolar, sorbitol, a nonionic osmoticum, inhibits carrier-mediated IAA uptake. The effects of specific anions and osmotic potential on the uptake carrier necessitates the reevaluation of other auxin transport studies in which KCl was routinely used as an agent with which to depolarize the membrane potential.     

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.     

FSY1, a novel gene encoding a specific fructose/H(+) symporter in the type strain of Saccharomyces carlsbergensis

A novel gene, FSY1, encoding a permease involved in active fructose uptake by a proton symport mechanism in the type strain of Saccharomyces carlsbergensis has been isolated. Fsy1p is only distantly related to the Hxt proteins that mediate facilitated diffusion of glucose and fructose in Saccharomyces cerevisiae and related species.     

A model for sucrose transport in the maize scutellum

A model originally developed for transport of neutral substrates in bacterial systems was tested for its suitability for depicting sucrose transport across the plasmalemma of the maize scutellum cell. The model contains a sucrose—proton symporter, a negatively-charged free carrier and a neutral sucrose—proton—carrier complex. Sucrose transport is driven by the sucrose gradient and by a proton electrochemical gradient set up by a proton-translocating ATPase. The results of experiments on sucrose uptake in scutellum slices are in accord with predictions based on the model. Evidence was obtained for an electrogenic proton pump in the plasmalemma, for sucrose—proton symport and for a sucrose transport mechanism driven by both electrical potential and pH gradients. It was found that treatments (dinitrophenol, N-ethylmaleimide or HCl) causing a net proton influx into the slices also caused an efflux of sucrose. Interpretations of these results compatible with the model are given.     

Significance of active fructose transport in the differentiation of the Saccharomyces sensu stricto group

Results of fructose proton symport and nDNA/nDNA reassociation measurements in 58 wine and beer yeast strains belonging to the Saccharomyces sensu stricto group are presented.
All strains were identified earlier using conventional physiological tests. Based on their fructose proton symport activity, four strains were found which did not correlate with their original classification, suggesting incorrect identification. The nDNA/nDNA reassociation measurements supported the results of the active fructose proton symport investigation.     

Catabolite interconversion of glucose transport systems in the yeast Candida wickerhamii

Candida wickerhamii IGC 3244 growing in glucose medium transported glucose by facilitated diffusion (at 25°C and pH 5, the K_s value was 1.7 mM and the V_max value was 1.6 mmol/h per g dry wt.), while cells grown under derepressed conditions produced a glucose proton symport (at 25°C and pH 5, the K_s value was 0.18 mM and the V_max value was 1.8–1.9 mmol/h per g dry wt.). In each case, the Lineweaver-Burk plot of initial uptake rates was linear, indicating the presence of a single system. In buffer with 2% glucose, the symport suffered catabolite inactivation while the facilitated diffusion system emerged concomitantly in such a way that the combined V_max remained nearly constant. During the conversion process, the Linewaver-Burk plots were biphasic, indicating the transitory co-existence of the two systems. A model is proposed that envisions the proton symport as composed of the facilitated diffusion system in association with (an)other transport protein(s), the latter being sensitive to carbon catabolite repression and inactivation.     

Anaerobic transport of serine and 2-aminoisobutyric acid by Staphylococcus epidermidis.

A membrane-bound ATPase detected in extracts of anaerobically grown Staphylococcus epidermidis was inhibited by a variety of compounds which inhibit ATPases in other organisms. Serine and 2-aminoisobutyric acid (AIB) were shown to enter the organism via the same transport system. The transport of AIB, the membrane potential and the transmembrane pH gradient were partially or completely abolished by the same inhibitors and also by uncoupling agents and lipid-soluble ions. It is proposed therefore that this ATPase generates and maintains an electrochemical gradient of protons across the cytoplasmic membrane of S. epidermidis capable of driving AIB uptake. Studies of AIB-induced proton movements suggested that AIB enters via a proton symport mechanism.     

Identification of an anaerobically induced phosphoenolpyruvate-dependent fructose-specific phosphotransferase system and evidence for the Embden-Meyerhof glycolytic pathway in the heterofermentative bacterium Lactobacillus brevis.

Heterofermentative gram-positive bacteria are believed to metabolize sugars exclusively via the pentose phosphoketolase pathway following uptake via sugar:cation symport. Here we show that anaerobic growth of one such bacterium, Lactobacillus brevis, in the presence of fructose induces the synthesis of a phosphotransferase system and glycolytic enzymes that allow fructose to be metabolized via the Embden-Meyerhof pathway.     

Transport kinetics of 6-deoxy-D-glucose inCandida parapsilosis

The strictly aerobic yeastCandida parapsilosis transports the nonmetabolizable monosaccharide 6-deoxy-D-glucose by an active process (inhibition by 2.4-dinitrophenol and other uncouplers but not by iodoacetamide), the accumulation ratio decreasing with increasing substrate concentration. Measured accumulation ratios are in agreement with those predicted from kinetic constants for influx and efflux. Energy for transport is probably required in the translocation step. The maximum rate is temperature-dependent with a transition point at 21 °C. the accumulation ratio is not, The uptake is most active at pH 4.5–8.5. It appears not to involve stoichiometric proton symport. The transport system is shared by D-glucose, D-mannose, D-galactose and possibly maltose but not by fructose, sucrose or pentoses. The apparent half-life of the transport system was 3.5–4 h.     

Coupling of Na<Superscript>+</Superscript>/α-ketoglutarate symport and PAH/α-ketoglutarate antiport in epithelial cells. Estimation of probability of the anion exchanger spontaneous re-orientation

In this work, dynamics was studied of uptake of p-aminohippurate by basolateral membrane vesicles isolated from rat kidney proximal tubules. The uphill PAH transport into the basolateral membrane vesicles was shown to occur in the presence of α-ketoglutarate and Na⁺-gradient. Based on mathematical model of symport and antiport cooperation, the mechanism of energy coupling of PAH transport via exchanger with Na⁺-dicarboxylate symport is discussed. Based on comparison of our own and literature data, the data analysis shows adequacy of the proposed mathematical model to describe the symport and antiport cooperation. This model has been shown to enable estimation of re-orientation probability of the empty anion exchanger (without substrate) from one membrane side to the other.     

Identification of Glucose Transporters in Aspergillus nidulans

To characterize the mechanisms involved in glucose transport, in the filamentous fungus Aspergillus nidulans, we have identified four glucose transporter encoding genes hxtB-E. We evaluated the ability of hxtB-E to functionally complement the Saccharomyces cerevisiae EBY.VW4000 strain that is unable to grow on glucose, fructose, mannose or galactose as single carbon source. In S. cerevisiae HxtB-E were targeted to the plasma membrane. The expression of HxtB, HxtC and HxtE was able to restore growth on glucose, fructose, mannose or galactose, indicating that these transporters accept multiple sugars as a substrate through an energy dependent process. A tenfold excess of unlabeled maltose, galactose, fructose, and mannose were able to inhibit glucose uptake to different levels (50 to 80 %) in these s. cerevisiae complemented strains. Moreover, experiments with cyanide-m-chlorophenylhydrazone (CCCP), strongly suggest that hxtB, -C, and –E mediate glucose transport via active proton symport. The A. nidulans ΔhxtB, ΔhxtC or ΔhxtE null mutants showed ~2.5-fold reduction in the affinity for glucose, while ΔhxtB and -C also showed a 2-fold reduction in the capacity for glucose uptake. The ΔhxtD mutant had a 7.8-fold reduction in affinity, but a 3-fold increase in the capacity for glucose uptake. However, only the ΔhxtB mutant strain showed a detectable decreased rate of glucose consumption at low concentrations and an increased resistance to 2-deoxyglucose.     

Sugar retrieval by coats of developing seeds of Phaseolus vulgaris L. and Vicia faba L

Influxes of glucose, fructose and sucrose were characterised for coat cells of developing seeds of Phaseolus vulgaris L. and Vicia faba L. by monitoring uptake of [(14)C]sugars into excised seed-coat halves and two different protoplast populations derived from seed coats. Sugar influxes by the two populations of protoplasts were similar for each sugar species [sucrose > (fructose approximately glucose)] and hexoses competed with sucrose. Concentration-dependent influxes of all three sugars by excised seed coats could be described by a simple directly proportional relationship between concentration ([S]) and uptake rate (v) in the physiological range of sugar concentrations (v approximately A.[S]). Alternatively, with the exception of fructose influx by Vicia, all could be fitted to a Michaelis-Menten relationship, as could sucrose uptake by Vicia protoplasts. Apparent K(m) values were high ( approximately 100-500 mM) compared with those reported for other systems. Sucrose transport was distinct from glucose and fructose transport in both species. Sugar influx was decreased by p-chloromercuribenzenesulfonic acid, carbonylcyanide m-chlorophenylhydrazone and erythrosin B. These responses are consistent with sugar/H(+) symport acting to retrieve photoassimilates leaked to the apoplasm during post-sieve element transport within seed coats.     

Mathematical modelling and assessment of the pH homeostasis mechanisms in Aspergillus niger while in citric acid producing conditions

In this work we introduce an extended model of the Aspergillus niger metabolism while in citrate production conditions. The model includes many recent findings related to various transport processes. It now considers a new information about the fructose uptake system and the proton and amino acids carriers between cytoplasm and the external medium. It also accounts for recent information about both the malate-citrate antiport between mitochondria and cytoplasm and the dihydrogen citrate ion excretion symport with protons. Finally, the model also accounts for new information about the glycerol-3-phosphate shuttle and pH buffering systems. Provided with this updated representation and after having assessed its quality and dynamic behaviour, we were able to explain the observed pH homoeostasis found in A. niger while in citrate producing conditions. The model also serves to enhance our comprehension of the molecular mechanisms operating in order to keep homoeostasis of pH in A. niger and other fungi, bacteria and yeast of biotechnological relevance.     

In Vitro Sugar Transport in Zea mays L. Kernels : II. Characteristics of Sugar Absorption and Metabolism by Isolated Developing Embryos

In vitro sugar transport into developing isolated maize embryos was studied. Embryo fresh and dry weight increased concomitantly with endogenous sucrose concentration and glucose uptake throughout development. However, endogenous glucose and fructose concentration and sucrose uptake remained constant. The uptake kinetics of radiolabeled sucrose, glucose, and fructose showed a biphasic dependence on exogenous substrate concentration. Hexose uptake was four to six times greater than sucrose uptake throughout development. Carbonylcyanide-m-chlorophenylhydrazone and dinitrophenol inhibited sucrose and glucose uptake significantly, but 3-O-methyl glucose uptake was less affected. The uptake of 1 millimolar sucrose was strongly pH dependent while glucose was not. Glucose and fructose were readily converted to sucrose and insoluble products soon after absorption into the embryo. Thus, sucrose accumulated, while glucose pools remained low. Based on the findings of this and other studies a model for sugar transport in the developing maize kernel is presented.     

GUP1 and its close homologue GUP2, encoding multimembrane-spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae

Many yeast species can utilize glycerol, both as a sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multimembrane-spanning protein, as being essential for proton symport of glycerol into S. cerevisiae. The gene is named GUP1 (glycerol uptake) and, for growth on glycerol, is important as a carbon and energy source. In addition, in strains deficient in glycerol production it also provides osmotic protection by the addition of glycerol. Another open reading frame (ORF), YPL189w, presenting a high degree of homology to YGL084c, similarly appears to be involved in active glycerol uptake in salt-containing glucose-based media in strains deficient in glycerol production. Analogously, this gene is named GUP2. To our knowledge, this is the first report on a gene product involved in active transport of glycerol in yeasts. Mutations with the same phenotypes occurred in two other ORFs of previously unknown function, YDL074c and YPL180w.     

Uptake of sucrose by Saccharomyces cerevisiae

Sucrose transport has been shown to occur in several Suc^? and Suc⁺Saccharomyces cerevisiae strains as an energy-dependent process. Assay conditions have been established to avoid both extra- and intracellular hydrolysis of the disaccharide thus allowing the identification of sucrose as such inside the cell immediately after the uptake; acid pH values (4.0–5.0) were optimal for transport although significant uptake was also detected at neutral pH. Transport of sucrose was not dependent on ATP and seemed to be driven by protonmotive force supplied by the electrochemical gradient of protons across the plasma membrane. The actual symport of protons along with sucrose was directly detected by continuous pH measurement of the reaction mixtures and the initial rate of proton movement in the symport process was determined. KC1 inhibited transport of sucrose suggesting that exit of K⁺ ions might well be involved in maintaining the electroneutrality of the process. On the other hand, NaCl stimulated transport by 50% in our experimental conditions. The specificity of sucrose transport was also tested using different disaccharides.     

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.