Symport Mechanism Research Articles

Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways

In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure-function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg(168) and Lys(492), and Asp(178), Asp(358) and Glu(473) as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp(358) (helix 7) and Lys(492) (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp(358) are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.

Experimental conditions can obscure the second high-affinity site in LeuT

Neurotransmitter:Na+ Symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na+-dependent symport mechanism. The crystal structure of the NSS homologue LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded centrally-located (S1) binding site next to two Na+. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a novel molecular mechanism of Na+/substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na+-coupled symport. This finding brings to light the caution needed in the selection of experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

Conserved Tyrosine in the First Transmembrane Segment of Solute:Sodium Symporters Is Involved in Na+-coupled Substrate Co-transport

Solute:sodium symporters (SSSs) transport vital molecules across the plasma membrane of all living organisms. vSGLT, the Na(+)/galactose transporter of Vibrio parahemeolyticus, is the only SSS for which high resolution structural information is available, revealing a LeuT-like fold and a Na(+)-binding site analogous to the Na2 site of LeuT. Whereas the core transmembrane segments (TMs) of SSSs share high structural similarity with other transporters of LeuT-like fold, TM1 does not correspond to any TM in those structural homologs and was only resolved for the backbone atoms in the initial vSGLT structure (Protein Data Bank code 3DH4). To assess the role of TM1 in Na(+)-coupled substrate symport by the SSSs, here we have studied the role of a conserved residue in TM1 by computational modeling in conjunction with radiotracer transport and binding studies. Based on our sequence alignment and much topological data for homologous PutP, the Na(+)/proline transporter, we have simulated a series of vSGLT models with shifted TM1 residue assignments. We show that in two converged vSGLT models that retained the original TM1 backbone conformation, a conserved residue, Tyr-19, is associated with the Na(+) binding interaction network. In silico and in vitro mutagenesis of homologous Tyr-14 in PutP revealed the involvement of this conserved residue in Na(+)-dependent substrate binding and transport. Thus, our combined computational and experimental data provide the first clues about the importance of a conserved residue in TM1, a unique TM in the proteins with LeuT-like fold, in the Na(+)-coupled symport mechanism of SSSs.

Impaired Function of the Tonoplast-Localized Sucrose Transporter in Rice, OsSUT2, Limits the Transport of Vacuolar Reserve Sucrose and Affects Plant Growth

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::β-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.

Helix Dynamics in LacY: Helices II and IV

Biochemical and biophysical studies based upon crystal structures of both a mutant and wild-type lactose permease from Escherichia coli (LacY) in an inward-facing conformation have led to a model for the symport mechanism in which both sugar and H+ binding sites are alternatively accessible to both sides of the membrane. Previous findings indicate that the face of helix II with Asp68 is important for the conformational changes that occur during turnover. As shown here, replacement of Asp68 at the cytoplasmic end of helix II, particularly with Glu, abolishes active transport but the mutants retain the ability to bind galactopyranoside. In the x-ray structure, Asp68 and Lys131 (helix IV) lie within ∼4.2 Å of each other. Although a double mutant with Cys replacements at both position 68 and position 131 cross-links efficiently, single replacements for Lys131 exhibit very significant transport activity. Site-directed alkylation studies show that sugar binding by the Asp68 mutants causes closure of the cytoplasmic cavity, similar to wild-type LacY; however, strikingly, the probability of opening the periplasmic pathway upon sugar binding is markedly reduced. Taken together with results from previous mutagenesis and cross-linking studies, these findings lead to a model in which replacement of Asp68 blocks a conformational transition involving helices II and IV that is important for opening the periplasmic cavity. Evidence suggesting that movements of helices II and IV are coupled functionally with movements in the pseudo-symmetrically paired helices VIII and X is also presented.

Leucine transport by the larval midgut of the parasitoid Aphidius ervi (Hymenoptera)

The larval midgut of the hymenopteran parasitoid Aphidius ervi accomplishes a large transport of nutrients from the lumen to the haemocoel, providing most of the organic molecules necessary for rapid insect development. l-amino acids in general, and leucine in particular, are efficiently accumulated in the larval body. We show here that the intact midgut of early 3rd instar larvae incubated in vitro can take up [(3)H]l-leucine from the basolateral side of the epithelium by transporters insensitive to the presence of monovalent cations. When the midgut is opened and the apical membrane of the absorbing epithelial cells is exposed to the medium containing radiolabelled leucine, a sodium-dependent uptake of the amino acid becomes apparent, disclosing the presence of a symport mechanism. Inhibition experiments of leucine uptake by a 100-fold excess of different amino acids, selected according to the properties of their side chain, revealed that this apical sodium-dependent mechanism is a broad spectrum transport system with a specialization for the absorption of aliphatic amino acids, that can also transfer glutamine and proline, but not phenylalanine, lysine and arginine. Altogether the experimental results obtained with intact- and open-gut preparations suggest that leucine transport across the basolateral membrane is mediated by both an uniporter and an obligatory amino acid exchange mechanism.

Evidence for a Proton–Protein Symport Mechanism in the Anthrax Toxin Channel

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titratable, is introduced at most positions in LFN, through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LFN translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA63)7 channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.

Mathematical model of cooperative work of ion pump, symport and antiport in epithelial cells

Transcellular transport in epithelial cells plays an important role in providing such physiological functions as excretion of cytotoxic substances or reabsorption of metabolites useful for the body life activity. These functions have been shown to be performed by the mechanisms - symport, antiport, ion pumps, and channels - that often function cooperatively. Kinetic models of the substrate transport with the aid of the above mechanisms are widely described in the literature. Much less attention is paid to modeling of cooperative activity of transporters that have different transport mechanisms. In this work we propose a mathematical model for flux coupling of three transporters - the ion pump, symporter, and antiporter as well as of two substrates, one of which (A) can be transported simultaneously by the symport and antiport mechanisms, while the other (B) - only by the latter mechanisms. Analysis of the model has shown that for the pair of substrates (A and B) the flux coupling becomes possible if the following conditions are met: (1) the substrate A flux into the internal cell volume using the symport mechanism is to exceed its antiporter-realized flux in the opposite direction; (2) probability of reorientation from one side of membrane to the other side for the antiporter loaded with the substrate is to be essentially higher than that for empty transporter. The proposed model can be used for comparing efficiency both of excretion and of reabsorption of cell metabolites in representatives of different taxa.

Substrate Selectivity of the Melibiose Permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β, d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)–C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.

Structurally reduced monosaccharide transporters in an evolutionarily conserved red alga.

The unicellular red alga Galdieria sulphuraria is a facultative heterotrophic member of the Cyanidiaceae, a group of evolutionary highly conserved extremophilic red algae. Uptake of various sugars and polyols is accomplished by a large number of distinct plasma membrane transporters. We have cloned three transporters [GsSPT1 (G. sulphuraria sugar and polyol transporter 1), GsSPT2 and GsSPT4], followed their transcriptional regulation and assayed their transport capacities in the heterologous yeast system. SPT1 is a conserved type of sugar/H(+) symporter with 12 predicted transmembrane-spanning domains, whereas SPT2 and SPT4 represent monosaccharide transporters, characterized by only nine hydrophobic domains. Surprisingly, all three proteins are functional plasma membrane transporters, as demonstrated by genetic complementation of a sugar uptake-deficient yeast mutant. Substrate specificities were broad and largely redundant, except for glucose, which was only taken up by SPT1. Comparison of SPT1 and truncated SPT1(Delta1-3) indicated that the N-terminus of the protein is not required for sugar transport or substrate recognition. However, its deletion affected substrate affinity as well as maximal transport velocity and released the pH dependency of sugar uptake. In line with these results, uptake by SPT2 and SPT4 was active but not pH-dependent, making a H(+) symport mechanism unlikely for the truncated proteins. We postulate SPT2 and SPT4 as functional plasma membrane transporters in G. sulphuraria. Most likely, they originated from genes encoding active monosaccharide/H(+) symporters with 12 transmembrane-spanning domains.

Thermodynamic stoichiometry of Na+-coupled glutathione transport

Ambiguity exists with respect to mechanisms of glutathione (GSH) transport and the molecular identity of GSH transporters. Empirical and theoretical limitations have hindered functional and molecular characterizations. Published literature referring to the isolation and molecular identification of Na+-coupled GSH transporters that mediate the cellular uptake of GSH is highly debated. Whereas a number of functional and kinetic reports of this putative symport mechanism exist, the hypothetical transmembrane Na+-coupled GSH transporter protein or the genetic message encoding it has not been isolated. Theoretical thermodynamic calculations to support the concept of secondary active GSH transport and to rationalize accounts of physical-kinetic measurements describing Na+-coupled cellular GSH uptake were performed. The adequacy of requisite energy and stoichiometric conservation of the separate electrical and chemical components of a Na+ gradient in maintaining a high cellular accumulation gradient for GSH was examined through a purely phenomenological perspective. Dependent on the biological context, the energetic coupling between Na+ and GSH cotransport may occur at ratios from 1:1 to 3:1. Molecular identification of specific transporters responsible for cellular Na+-coupled GSH uptake will facilitate determination of their relative contribution to the overall plasma membrane resting potential. In tissues with a high GSH concentration relative to their extracellular milieu, particularly in pathologies of cystic fibrosis and dry eye syndromes, large energy coupling ratios in cotransport of Na+ and GSH may be expected. Na+-coupled GSH transport may play an important role in disease onset and (or) progression, or treatment modalities thereof.

Leucine transport in brush border membrane vesicles from freshwater insect larvae

Leucine transport across brush border membrane vesicles prepared from four insect species common to European freshwater streams has been characterized. The species studied were: Ephemera danica (Ephemeroptera: Ephemeridae), Isoperla grammatica (Plecoptera: Perlodidae), Hydropsyche pellucidula (Trichoptera: Hydropsychidae), and Hybomitra bimaculata (Diptera: Tabanidae). The transport differed among the studied taxa for several features, including pH and sodium dependence, substrate affinity and specificity, and efficiency. In H. pellucidula and E. danica, leucine uptake was higher at pH 7.4 than at more alkaline or acidic pH values, whereas in I. grammatica and H. bimaculata, the uptake was rather constant when pH varied from 5.0 to 7.4, then strongly decreased at pH 8.8. All but E. danica displayed a transient intravescicular leucine accumulation in the presence of sodium, suggesting the existence of a cation-leucine symport mechanism. The sodium dependence ranged according to the following order: H. pellucidula > I. grammatica > H. bimaculata > E. danica. Moreover, in H. pellucidula and I. grammatica, the sodium-dependence was stronger at pH 8.8 than at pH 7.4. In E. danica, leucine uptake was sodium-independent at all pH values. The highest value of V(max) (45.3 pmol.s(-1).mg proteins(-1)) was in E. danica, which, however, displayed the lowest affinity (K(m) 137 muM) when compared to the kinetic parameters of other taxa. The V(max) and K(m) values were: 40 and 52.5, 32.1 and 12.5, and 4.5 and 230 for H. bimaculata, H. pellucidula, and I. grammatica, respectively. The obtained results are discussed within our current knowledge of amino acid transport systems in insects.

Jejunal Creatine Absorption: What is the Role of the Basolateral Membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.

(461) Endocytic Uptake of Quantum Dots and Endocytic Markers into `Sweet Lime' Juice Cells

To determine whether the mechanisms of sucrose accumulation into the low acid `Sweet Lime' (Citrus limmetioides Blanco) juice cells are consistent with those previously reported for the more acidic cultivars, we followed similar developmental changes in determinants of sink strength. In addition, we followed the incorporation and distribution of quantum dots and fluorescent endocytic probes into the cell with time of incubation. As in other citrus fruits, sucrose levels, sucrose synthase, sucrose phosphate synthase, and sucrose phosphate phosphatase increased throughout fruit development. The pH however, was much higher than in the more acidic cultivars. Sucrose uptake into energized plasmalemma vesicles was inhibited by gramicidin, in accordance with the presence of an active symport mechanism of sucrose from the apoplast into the cytosol. On the contrary, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. In conformity with recent findings showing the occurrence of an endocytic mechanism in `Murcott' mandarin, `Sweet Lime' juice cells were shown to incorporate membrane-impermeable dyes into their vacuoles in the presence of sucrose. High-definition confocal microscopy revealed the co-localization of membrane-impermeable markers in cytoplasmic vesicles, in membrane-bound intermediate structures such as the endosome and multi-vesicular body, and the eventual distribution of such fluorescent particles. The data provide strong evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole and for the visualization of intermediate distribution and cargo centers in the cell.

Sucrose Transport into Citrus Juice Cells: Evidence for an Endocytic Transport System

To investigate the mechanisms of sucrose transport and its accumulation into `Murcott' mandarin (Citrus reticulata Blanco) fruit, developmental changes in determinants of sink strength such as sucrose metabolizing enzymes, and sucrose transport across both plasmalemma and tonoplast were analyzed. Concurrently with sucrose levels, sucrose synthase, sucrose phosphate synthase and sucrose phosphate phosphatase increased throughout fruit development. Plasmalemma and tonoplast vesicles isolated from fruit collected at different developmental stages were analyzed for their transport capabilities. Sucrose uptake into energized plasmalemma vesicles was abolished by gramicidin, which is in accordance with the presence of an active symport mechanism of sucrose transport from the apoplast into the cytosol. Unexpectedly, tonoplast vesicles were shown to lack active transport mechanism of sucrose into the vacuole. More importantly, however, and in conformity with recent findings showing the occurrence of an endocytic mechanism of ion uptake in maize (Zea mays L.) root cells, citrus (Citrus L.) juice cells were shown to incorporate membrane impermeable dyes into their vacuoles in the presence of sucrose. High definition confocal microscopy revealed the co-localization of membrane impermeable markers in cytoplasmic vesicles and the formation of vesicles at the plasmalemma. The data provide evidence for an endocytic system of transport that allows direct incorporation of sucrose from the apoplast to the vacuole bypassing both the plasmalemma and tonoplast.

Colorimetric determination of active α-glucoside transport in Saccharomyces cerevisiae

Fermentation of α-glucosides (maltose, maltotriose) by Saccharomyces cerevisiae cells is a critical phase in the processes of brewing and breadmaking. Utilization of α-glucosides requires the active transport of the sugar across the cell membrane and, subsequently, its hydrolysis by cytoplasmic glucosidases. Although transport activities are usually assayed using radiolabeled substrates, we have developed a simple, cheap and reliable colorimetric assay for the determination of α-glucoside uptake using p-nitrophenyl-α- d-glucopyranoside ( pNPαG) as substrate. Our results show that pNPαG is actively transported by S. cerevisiae cells by a H +-symport mechanism, which depends on the electrochemical proton gradient across the plasma membrane. pNPαG uptake is mediated by the AGT1 α-glucoside permease, which has a high affinity ( K m=3 mM) for this chromogenic substrate. This simple colorimetric uptake assay can be used to analyze the expression and regulation of the AGT1 permease in S. cerevisiae cells.

The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae.

Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants. These haustoria have been linked to nutrient uptake ever since their discovery. We have for the first time to our knowledge shown that the flow of sugars from the host Vicia faba to the rust fungus Uromyces fabae seems to occur largely through the haustorial complex. One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter. Functional expression of the gene termed HXT1 in Saccharomyces cerevisiae and Xenopus laevis oocytes assigned a substrate specificity for D-glucose and D-fructose and indicated a proton symport mechanism. Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy. These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex. The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast. This organization might facilitate the competition of the parasite with natural sink organs of the host.

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.

Phosphate and sulfate uptake in the halotolerant alga Dunaliella are driven by Na +-symport mechanism

Summary Phosphate and sulfate uptake were measured in the halotolerant alga Dunaliella salina under anion-sufficient or anion-deficient conditions. Both phosphate and sulfate uptake were strictly and specifically dependent on Na+ ions. Pi accumulation was inhibited by Na+ ionophores and by inhibitors of Na+ extrusion. Pi uptake was associated with a stoichiometric influx of Na+ ions at a ratio of 1 Na+/1 Pi. Depletion of phosphate or sulfate from growth media induced a 20-fold or a 2.5-fold stimulation of the corresponding rate of uptake, respectively. The enhancements were associated with an increase in Vmax values, but not in the Km for anion substrates (Km(Pi)∼1 μmol/L, Km(SO4)∼15 μmol/L) or for Na+ (Km(Na+)∼35 mmol/L for Pi, ∼170 mmol/L for SO42-). These results suggest that uptake of phosphate and of sulfate in D. salina are mediated by Na+/anion symporters and are driven by δμNa+ across the plasma membrane. The implications of these results on the availability of phosphate and sulfate in hypersaline solutions are discussed.

Plant sucrose-H+ symporters mediate the transport of vitamin H.

A cDNA coding for a vitamin H (biotin) transport protein from Arabidopsis was identified by genetic complementation of a biotin uptake-deficient yeast mutant. Vitamin H transport by this protein was sensitive to the SH-group inhibitor p-chloromercuribenzene sulfonic acid (PCMBS) and to the uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP), suggesting an energy-dependent biotin-H+ symport mechanism. The transport activity could contribute to the so-far uncharacterized plant sucrose-H+ symporter AtSUC5 which mediates the energy-dependent transport of biotin and sucrose, and restores growth of the biotin transport-deficient yeast mutant on medium with low biotin concentrations. Functional comparison of the AtSUC5 transporter with previously characterized plant sucrose or monosaccharide transporters revealed that biotin transport may be a general and specific property of all plant sucrose transporters (sucrose/biotin-H+ symporters). This first report on a transporter with dual substrate specificity for two structurally unrelated molecules has a major impact on general thinking concerning the specificity of membrane transporters. The physiological relevance of this finding is discussed.