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Sample records for high-affinity sucrose transporter

  1. A Novel High-Affinity Sucrose Transporter Is Required for Virulence of the Plant Pathogen Ustilago maydis

    PubMed Central

    Goos, Sarah; Kämper, Jörg; Sauer, Norbert

    2010-01-01

    Plant pathogenic fungi cause massive yield losses and affect both quality and safety of food and feed produced from infected plants. The main objective of plant pathogenic fungi is to get access to the organic carbon sources of their carbon-autotrophic hosts. However, the chemical nature of the carbon source(s) and the mode of uptake are largely unknown. Here, we present a novel, plasma membrane-localized sucrose transporter (Srt1) from the corn smut fungus Ustilago maydis and its characterization as a fungal virulence factor. Srt1 has an unusually high substrate affinity, is absolutely sucrose specific, and allows the direct utilization of sucrose at the plant/fungal interface without extracellular hydrolysis and, thus, without the production of extracellular monosaccharides known to elicit plant immune responses. srt1 is expressed exclusively during infection, and its deletion strongly reduces fungal virulence. This emphasizes the central role of this protein both for efficient carbon supply and for avoidance of apoplastic signals potentially recognized by the host. PMID:20161717

  2. The High-Affinity Maltose/Trehalose ABC Transporter in the Extremely Thermophilic Bacterium Thermus thermophilus HB27 Also Recognizes Sucrose and Palatinose

    PubMed Central

    Silva, Zélia; Sampaio, Maria-Manuel; Henne, Anke; Böhm, Alex; Gutzat, Ruben; Boos, Winfried; da Costa, Milton S.; Santos, Helena

    2005-01-01

    examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose. PMID:15687184

  3. The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose.

    PubMed

    Silva, Zélia; Sampaio, Maria-Manuel; Henne, Anke; Böhm, Alex; Gutzat, Ruben; Boos, Winfried; da Costa, Milton S; Santos, Helena

    2005-02-01

    did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.

  4. Arabidopsis Sucrose Transporter AtSUC9. High-Affinity Transport Activity, Intragenic Control of Expression, and Early Flowering Mutant Phenotype1[OA

    PubMed Central

    Sivitz, Alicia B.; Reinders, Anke; Johnson, Meghan E.; Krentz, Anthony D.; Grof, Christopher P.L.; Perroux, Jai M.; Ward, John M.

    2007-01-01

    AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K0.5 = 0.066 ± 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and α-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to β-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition. PMID:17098854

  5. Arabidopsis sucrose transporter AtSUC9. High-affinity transport activity, intragenic control of expression, and early flowering mutant phenotype.

    PubMed

    Sivitz, Alicia B; Reinders, Anke; Johnson, Meghan E; Krentz, Anthony D; Grof, Christopher P L; Perroux, Jai M; Ward, John M

    2007-01-01

    AtSUC9 (At5g06170), a sucrose (Suc) transporter from Arabidopsis (Arabidopsis thaliana) L. Heynh., was expressed in Xenopus (Xenopus laevis) oocytes, and transport activity was analyzed. Compared to all other Suc transporters, AtSUC9 had an ultrahigh affinity for Suc (K(0.5) = 0.066 +/- 0.025 mm). AtSUC9 showed low substrate specificity, similar to AtSUC2 (At1g22710), and transported a wide range of glucosides, including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alpha-methyl-d-glucose. The ability of AtSUC9 to transport 10 glucosides was compared directly with that of AtSUC2, HvSUT1 (from barley [Hordeum vulgare]), and ShSUT1 (from sugarcane [Saccharum hybrid]), and results indicate that type I and type II Suc transporters have different substrate specificities. AtSUC9 protein was localized to the plasma membrane by transient expression in onion (Allium cepa) epidermis. Using a whole-gene translational fusion to beta-glucuronidase, AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression in Arabidopsis was dependent on intragenic sequence, and this was found to also be true for AtSUC1 (At1g71880) but not AtSUC2. Plants containing mutations in Suc transporter gene AtSUC9 were found to have an early flowering phenotype under short-day conditions. The transport properties of AtSUC9 indicate that it is uniquely suited to provide cellular uptake of Suc at very low extracellular Suc concentrations. The mutant phenotype of atsuc9 alleles indicates that AtSUC9 activity leads to a delay in floral transition.

  6. Jen1p: A High Affinity Selenite Transporter in Yeast

    PubMed Central

    McDermott, Joseph R.; Rosen, Barry P.

    2010-01-01

    Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p. PMID:20861301

  7. Jen1p: a high affinity selenite transporter in yeast.

    PubMed

    McDermott, Joseph R; Rosen, Barry P; Liu, Zijuan

    2010-11-15

    Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.

  8. High-affinity ammonium transporters and nitrogen sensing in mycorrhizas.

    PubMed

    Javelle, Arnaud; André, Bruno; Marini, Anne Marie; Chalot, Michel

    2003-02-01

    Most terrestrial plants live in mutualistic symbiosis with root-infecting mycorrhizal fungi. This association requires a molecular dialogue between the two partners. However, the nature of the chemical signals that induce hyphal differentiation are not well characterized and the mechanisms for signal reception are still unknown. In addition to its role in ammonium scavenging, the Mep2 protein from Saccharomyces cerevisiae has been proposed to act as an ammonium sensor that is essential for pseudohyphal differentiation in response to ammonium limitation. We propose that the high-affinity ammonium transporters from mycorrhizal fungi act in a similar manner to sense the environment and induce, via as-yet-unidentified signal transduction cascades, the switch in the mode of fungal growth observed during the formation of mycorrhiza.

  9. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed

    Xavier, K B; Martins, L O; Peist, R; Kossmann, M; Boos, W; Santos, H

    1996-08-01

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

  10. High-affinity maltose/trehalose transport system in the hyperthermophilic archaeon Thermococcus litoralis.

    PubMed Central

    Xavier, K B; Martins, L O; Peist, R; Kossmann, M; Boos, W; Santos, H

    1996-01-01

    The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85 degrees C. The K(m) for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the Vmax for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 microM concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, Ki, of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [14C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 microM maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M(r) of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon. PMID:8759837

  11. Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2004-10-01

    High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

  12. Expression Cloning of the High Affinity Choline Transporter

    DTIC Science & Technology

    1993-05-05

    clones. It encodes a GABA transporter that we found to be localized to the glial cells of the purely cholinergic electromotor nucleus of Torpedo. In a...expression cloning approach employing frog oocytes and mRNA from Torpedo C.B. Gundersen electromotor nucleus to isolate a choline transporter cDNA...The rationale for this is that the electromotor neurons should harbor one of the highest abundances of choline transporter mRNA in the animal kingdom

  13. Turgor regulation of sucrose transport in sugar beet taproot tissue.

    PubMed

    Wyse, R E; Zamski, E; Tomos, A D

    1986-06-01

    Sink tissues that store osmotically active compounds must osmoregulate to prevent excessively high turgor. The ability to regulate turgor may be related to membrane transport of solutes and thus sink strength. To study this possibility, the kinetics of sugar uptake were determined in sugar beet (Beta vulgaris L.) taproot tissue discs over a range of cell turgors. Sucrose uptake followed biphasic kinetics with a high affinity saturating component below 20 millimolar and a low affinity linear component at higher concentrations. Glucose uptake exhibited only simple saturation type kinetics. The high affinity saturating component of sucrose and glucose uptake was inhibited by increasing cell turgor (decreasing external mannitol concentrations). The inhibition was evident as a decrease in V(max) but no effect on K(m). Sucrose uptake by tissue equilibrated in dilute buffer exhibited no saturating component. Ethylene glycol, a permeant osmoticum, had no effect on uptake kinetics, suggesting that the effect was due to changes in cell turgor and not due to decreased water potential per se. p-(Chloromercuri)benzene sulfonic acid (PCMBS) inhibited sucrose uptake at low but not high cell turgor. High cell turgor caused the tissue to become generally leaky to potassium, sucrose, amino acids, and reducing sugars. PCMBS had no effect on sucrose leakage, an indication that the turgor-induced leakage of sucrose was not via back flow through the carrier. The ability of the tissue to acidify the external media was turgor dependent with an optimum at 300 kilopascals. Acidification was sharply reduced at cell turgors above or below the optimum. The results suggest that the secondary transport of sucrose is reduced at high turgor as a result of inhibition of the plasma membrane ATPase. This inhibition of ATPase activity would explain the reduced V(max) and leakiness to low molecular weight solutes. Cell turgor is an important regulator of sucrose uptake in this tissue and thus may be an

  14. Plant physiology: The importance of sucrose transporters.

    PubMed

    Truernit, E

    2001-03-06

    Sucrose transport is essential for the distribution of carbohydrates in plants. Recent studies have shown that a specific transporter protein plays an essential role in loading sucrose into the phloem component of the plant vasculature.

  15. The Saccharomyces cerevisiae high affinity phosphate transporter encoded by PHO84 also functions in manganese homeostasis.

    PubMed

    Jensen, Laran T; Ajua-Alemanji, Mispa; Culotta, Valeria Cizewski

    2003-10-24

    In the bakers' yeast Saccharomyces cerevisiae, high affinity manganese uptake and intracellular distribution involve two members of the Nramp family of genes, SMF1 and SMF2. In a search for other genes involved in manganese homeostasis, PHO84 was identified. The PHO84 gene encodes a high affinity inorganic phosphate transporter, and we find that its disruption results in a manganese-resistant phenotype. Resistance to zinc, cobalt, and copper ions was also demonstrated for pho84Delta yeast. When challenged with high concentrations of metals, pho84Delta yeast have reduced metal ion accumulation, suggesting that resistance is due to reduced uptake of metal ions. Pho84p accounted for virtually all the manganese accumulated under metal surplus conditions, demonstrating that this transporter is the major source of excess manganese accumulation. The manganese taken in via Pho84p is indeed biologically active and can not only cause toxicity but can also be incorporated into manganese-requiring enzymes. Pho84p is essential for activating manganese enzymes in smf2Delta mutants that rely on low affinity manganese transport systems. A role for Pho84p in manganese accumulation was also identified in a standard laboratory growth medium when high affinity manganese uptake is active. Under these conditions, cells lacking both Pho84p and the high affinity Smf1p transporter accumulated low levels of manganese, although there was no major effect on activity of manganese-requiring enzymes. We conclude that Pho84p plays a role in manganese homeostasis predominantly under manganese surplus conditions and appears to be functioning as a low affinity metal transporter.

  16. Functional transformations of bile acid transporters induced by high-affinity macromolecules

    PubMed Central

    Al-Hilal, Taslim A.; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-01-01

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery. PMID:24566561

  17. Functional transformations of bile acid transporters induced by high-affinity macromolecules.

    PubMed

    Al-Hilal, Taslim A; Chung, Seung Woo; Alam, Farzana; Park, Jooho; Lee, Kyung Eun; Jeon, Hyesung; Kim, Kwangmeyung; Kwon, Ick Chan; Kim, In-San; Kim, Sang Yoon; Byun, Youngro

    2014-02-25

    Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery.

  18. The HXT2 gene of Saccharomyces cerevisiae is required for high-affinity glucose transport.

    PubMed Central

    Kruckeberg, A L; Bisson, L F

    1990-01-01

    The HXT2 gene of the yeast Saccharomyces cerevisiae was identified on the basis of its ability to complement the defect in glucose transport of a snf3 mutant when present on the multicopy plasmid pSC2. Analysis of the DNA sequence of HXT2 revealed an open reading frame of 541 codons, capable of encoding a protein of Mr 59,840. The predicted protein displayed high sequence and structural homology to a large family of procaryotic and eucaryotic sugar transporters. These proteins have 12 highly hydrophobic regions that could form transmembrane domains; the spacing of these putative transmembrane domains is also highly conserved. Several amino acid motifs characteristic of this sugar transporter family are also present in the HXT2 protein. An hxt2 null mutant strain lacked a significant component of high-affinity glucose transport when under derepressing (low-glucose) conditions. However, the hxt2 null mutation did not incur a major growth defect on glucose-containing media. Genetic and biochemical analyses suggest that wild-type levels of high-affinity glucose transport require the products of both the HXT2 and SNF3 genes; these genes are not linked. Low-stringency Southern blot analysis revealed a number of other sequences that cross-hybridize with HXT2, suggesting that S. cerevisiae possesses a large family of sugar transporter genes. Images PMID:2233722

  19. Pharmacological characterization of a high-affinity p-tyramine transporter in rat brain synaptosomes

    PubMed Central

    Berry, Mark D.; Hart, Shannon; Pryor, Anthony R.; Hunter, Samantha; Gardiner, Danielle

    2016-01-01

    p-Tyramine is an archetypal member of the endogenous family of monoamines known as trace amines, and is one of the endogenous agonists for trace amine-associated receptor (TAAR)1. While much work has focused on the function of TAAR1, very little is known about the regulation of the endogenous agonists. We have previously reported that p-tyramine readily crosses lipid bilayers and that its release from synaptosomes is non-exocytotic. Such release, however, showed characteristics of modification by one or more transporters. Here we provide the first characterization of such a transporter. Using frontal cortical and striatal synaptosomes we show that p-tyramine passage across synaptosome membranes is not modified by selective inhibition of either the dopamine, noradrenaline or 5-HT transporters. In contrast, inhibition of uptake-2 transporters significantly slowed p-tyramine re-uptake. Using inhibitors of varying selectivity, we identify Organic Cation Transporter 2 (OCT2; SLC22A2) as mediating high affinity uptake of p-tyramine at physiologically relevant concentrations. Further, we confirm the presence of OCT2 protein in synaptosomes. These results provide the first identification of a high affinity neuronal transporter for p-tyramine, and also confirm the recently described localization of OCT2 in pre-synaptic terminals. PMID:27901065

  20. The Phn system of Mycobacterium smegmatis: a second high-affinity ABC-transporter for phosphate.

    PubMed

    Gebhard, Susanne; Tran, Sieu L; Cook, Gregory M

    2006-11-01

    Uptake of inorganic phosphate, an essential but often limiting nutrient, in bacteria is usually accomplished by the high-affinity ABC-transport system Pst. Pathogenic species of mycobacteria contain several copies of the genes encoding the Pst system (pstSCAB), and two of the encoded proteins, PstS1 and PstS2, have been shown to be virulence factors in Mycobacterium tuberculosis. The fast-growing Mycobacterium smegmatis contains only a single copy of the pst operon. This study reports the biochemical and molecular characterization of a second high-affinity phosphate transport system, designated Phn. The Phn system is encoded by a three-gene operon that constitutes the components of a putative ABC-type phosphonate/phosphate transport system. Expression studies using phnD- and pstS-lacZ transcriptional fusions showed that both operons were induced when the culture entered phosphate limitation, indicating a role for both systems in phosphate uptake at low extracellular concentrations. Deletion mutants in either phnD or pstS failed to grow in minimal medium with a 10 mM phosphate concentration, while the isogenic wild-type strain mc(2)155 grew at micromolar phosphate concentrations. Analysis of the kinetics of phosphate transport in the wild-type and mutant strains led to the proposal that the Phn and Pst systems are both high-affinity phosphate transporters with similar affinities for phosphate (i.e. apparent K(m) values between 40 and 90 muM P(i)). The Phn system of M. smegmatis appears to be unique in that, unlike previously identified Phn systems, it does not recognize phosphonates or phosphite as substrates.

  1. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity.

    PubMed Central

    Kim, E J; Kwak, J M; Uozumi, N; Schroeder, J I

    1998-01-01

    Because plants grow under many different types of soil and environmental conditions, we investigated the hypothesis that multiple pathways for K+ uptake exist in plants. We have identified a new family of potassium transporters from Arabidopsis by searching for homologous sequences among the expressed sequence tags of the GenBank database. The deduced amino acid sequences of AtKUP (for Arabidopsis thaliana K+ uptake transporter) cDNAs are highly homologous to the non-plant Kup and HAK1 potassium transporters from Escherichia coli and Schwanniomyces occidentalis, respectively. Interestingly, AtKUP1 and AtKUP2 are able to complement the potassium transport deficiency of an E. coli triple mutant. In addition, transgenic Arabidopsis suspension cells overexpressing AtKUP1 showed increased Rb+ uptake at micromolar concentrations with an apparent K(m) of approximately 22 microM, indicating that AtKUP1 encodes a high-affinity potassium uptake activity in vivo. A small, low-affinity Rb+ uptake component was also detected in AtKUP1-expressing cells. RNA gel blot analysis showed that the various members of the AtKUP family have distinct patterns of expression, with AtKUP3 transcript levels being strongly induced by K+ starvation. It is proposed that plants contain multiple potassium transporters for high-affinity uptake and that the AtKUP family may provide important components of high- and low-affinity K+ nutrition and uptake into various plant cell types. PMID:9477571

  2. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  3. Characterization of the Saccharomyces cerevisiae high affinity copper transporter Ctr3.

    PubMed

    Pena, M M; Puig, S; Thiele, D J

    2000-10-27

    Copper is an essential nutrient required for the activity of a number of enzymes with diverse biological roles. In the bakers' yeast Saccharomyces cerevisiae, copper is transported into cells by two high affinity copper transport proteins, Ctr1 and Ctr3. Although Ctr1 and Ctr3 are functionally redundant, they bear little homology at the amino acid sequence level. In this report, we characterize Ctr3 with respect to its localization, assembly, and post-transcriptional regulation. Ctr3 is an integral membrane protein that assembles as a trimer to form a competent copper uptake permease at the plasma membrane. Whereas the CTR1 and CTR3 genes are similarly regulated at the transcriptional level in response to copper, post-transcriptional regulation of these proteins is distinct. Unlike Ctr1, the Ctr3 transporter is neither regulated at the level of protein degradation nor endocytosis as a function of elevated copper levels. Our studies suggest that Ctr3 constitutes a fundamental module found in all eukaryotic high affinity copper transporters to date, which is sufficient for copper uptake but lacks elements for post-transcriptional regulation by copper.

  4. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

    PubMed Central

    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

  5. Mechanism of high affinity inhibition of the human urate transporter URAT1

    PubMed Central

    Tan, Philip K.; Ostertag, Traci M.; Miner, Jeffrey N.

    2016-01-01

    Gout is caused by elevated serum urate levels, which can be treated using inhibitors of the uric acid transporter, URAT1. We exploited affinity differences between the human and rat transporters to map inhibitor binding sites in URAT1. Human-rat transporter chimeras revealed that human URAT1 serine-35, phenylalanine-365 and isoleucine-481 are necessary and sufficient to provide up to a 100-fold increase in affinity for inhibitors. Moreover, serine-35 and phenylalanine-365 are important for high-affinity interaction with the substrate urate. A novel URAT1 binding assay provides support for direct interaction with these amino acids; thus, current clinically important URAT1 inhibitors likely bind the same site in URAT1. A structural model suggests that these three URAT1 residues are in close proximity potentially projecting within the channel. Our results indicate that amino acids from several transmembrane segments functionally cooperate to form a high-affinity URAT1 inhibitor binding site that, when occupied, prevents substrate interactions. PMID:27713539

  6. Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

    PubMed Central

    Cássio, F; Leáo, C

    1991-01-01

    Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664712

  7. Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

    PubMed Central

    Kranzler, Chana; Lis, Hagar; Finkel, Omri M; Schmetterer, Georg; Shaked, Yeala; Keren, Nir

    2014-01-01

    Iron bioavailability limits biological activity in many aquatic and terrestrial environments. Broad scale genomic meta-analyses indicated that within a single organism, multiple iron transporters may contribute to iron acquisition. Here, we present a functional characterization of a cyanobacterial iron transport pathway that utilizes concerted transporter activities. Cyanobacteria are significant contributors to global primary productivity with high iron demands. Certain cyanobacterial species employ a siderophore-mediated uptake strategy; however, many strains possess neither siderophore biosynthesis nor siderophore transport genes. The unicellular, planktonic, freshwater cyanobacterium, Synechocystis sp. PCC 6803, employs an alternative to siderophore-based uptake-reduction of Fe(III) species before transport through the plasma membrane. In this study, we combine short-term radioactive iron uptake and reduction assays with a range of disruption mutants to generate a working model for iron reduction and uptake in Synechocystis sp. PCC 6803. We found that the Fe(II) transporter, FeoB, is the major iron transporter in this organism. In addition, we uncovered a link between a respiratory terminal oxidase (Alternate Respiratory Terminal Oxidase) and iron reduction - suggesting a coupling between these two electron transfer reactions. Furthermore, quantitative RNA transcript analysis identified a function for subunits of the Fe(III) transporter, FutABC, in modulating reductive iron uptake. Collectively, our results provide a molecular basis for a tightly coordinated, high-affinity iron transport system. PMID:24088625

  8. Functional Analysis of Arabidopsis Sucrose Transporters

    SciTech Connect

    John M. Ward

    2009-03-31

    Sucrose is the main photosynthetic product that is transported in the vasculature of plants. The long-distance transport of carbohydrates is required to support the growth and development of net-importing (sink) tissues such as fruit, seeds and roots. This project is focused on understanding the transport mechanism sucrose transporters (SUTs). These are proton-coupled sucrose uptake transporters (membrane proteins) that are required for transport of sucrose in the vasculature and uptake into sink tissues. The accomplishments of this project included: 1) the first analysis of substrate specificity for any SUT. This was accomplished using electrophysiology to analyze AtSUC2, a sucrose transporter from companion cells in Arabidopsis. 2) the first analysis of the transport activity for a monocot SUT. The transport kinetics and substrate specificity of HvSUT1 from barley were studied. 3) the first analysis of a sucrose transporter from sugarcane. and 4) the first analysis of transport activity of a sugar alcohol transporter homolog from plants, AtPLT5. During this period four primary research papers, funded directly by the project, were published in refereed journals. The characterization of several sucrose transporters was essential for the current effort in the analysis of structure/function for this gene family. In particular, the demonstration of strong differences in substrate specificity between type I and II SUTs was important to identify targets for site-directed mutagenesis.

  9. High Affinity Transport of CO2 in the Cyanobacterium Synechococcus UTEX 625 1

    PubMed Central

    Espie, George S.; Miller, Anthony G.; Canvin, David T.

    1991-01-01

    The active transport of CO2 in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO3− transport. The substrate concentration required to give one half the maximum rate for whole cell CO2 transport was determined to be 0.4 ± 0.2 micromolar (mean ± standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO2 transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O2 evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO2 transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO2 transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO2 transport was caused by HCO3− or by Na+-dependent HCO3− transport. However, 12CO2 readily interfered with 13CO2 transport. CO2 transport and Na+-dependent HCO3− transport are separate, independent processes and the high affinity CO2 transporter is not only responsible for the initial transport of CO2 into the cell but also for scavenging any CO2 that may leak from the cell during ongoing photosynthesis. PMID:16668535

  10. Substrate-induced internalization of the high-affinity choline transporter.

    PubMed

    Okuda, Takashi; Konishi, Asami; Misawa, Hidemi; Haga, Tatsuya

    2011-10-19

    Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

  11. High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter.

    PubMed

    Ozvegy-Laczka, Csilla; Hegedus, Tamás; Várady, György; Ujhelly, Olga; Schuetz, John D; Váradi, András; Kéri, György; Orfi, László; Német, Katalin; Sarkadi, Balázs

    2004-06-01

    Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents. Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2. Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients. These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.

  12. The High-Affinity E. Coli Methionine ABC Transporter: Structure And Allosteric Regulation

    SciTech Connect

    Kadaba, N.S.; Kaiser, J.T.; Johnson, E.; Lee, A.; Rees, D.C.

    2009-05-18

    The crystal structure of the high-affinity Escherichia coli MetNI methionine uptake transporter, a member of the adenosine triphosphate (ATP)-binding cassette (ABC) family, has been solved to 3.7 angstrom resolution. The overall architecture of MetNI reveals two copies of the adenosine triphosphatase (ATPase) MetN in complex with two copies of the transmembrane domain MetI, with the transporter adopting an inward-facing conformation exhibiting widely separated nucleotide binding domains. Each MetI subunit is organized around a core of five transmembrane helices that correspond to a subset of the helices observed in the larger membrane-spanning subunits of the molybdate (ModBC) and maltose (MalFGK) ABC transporters. In addition to the conserved nucleotide binding domain of the ABC family, MetN contains a carboxyl-terminal extension with a ferredoxin-like fold previously assigned to a conserved family of regulatory ligand-binding domains. These domains separate the nucleotide binding domains and would interfere with their association required for ATP binding and hydrolysis. Methionine binds to the dimerized carboxyl-terminal domain and is shown to inhibit ATPase activity. These observations are consistent with an allosteric regulatory mechanism operating at the level of transport activity, where increased intracellular levels of the transported ligand stabilize an inward-facing, ATPase-inactive state of MetNI to inhibit further ligand translocation into the cell.

  13. Sucrose transport into stalk tissue of sugarcane

    SciTech Connect

    Thom, M.; Maretzki, A. )

    1990-05-01

    The productivity of higher plants is, in part, dependent on transport of photosynthate from source to sink (in sugarcane, stalk) and upon its assimilation in cells of the sink tissue. In sugarcane, sucrose has been reported to undergo hydrolysis in the apoplast before uptake into the storage parenchyma, whereas recently, sucrose was reported to be taken up intact. This work was based on lack of randomization of ({sup 14}C)fructosyl sucrose accumulated after feeding tissue slices with this sugar. In this report, we present evidence from slices of stalk tissue that sucrose is taken up intact via a carrier-mediated, energy-dependent process. The evidence includes: (1) uptake of fluorosucrose, an analog of sucrose not subject to hydrolysis by invertase; (2) little or no randomization of ({sup 14}C) fructosyl sucrose taken up; (3) the presence of a saturable as well as a linear component of sucrose uptake; and (4) inhibition of both the saturable and linear components of sucrose uptake by protonophore and sulhydryl agents. Hexoses can also be taken up, and at a greater efficiency than sucrose. It is probable that both hexose and sucrose can be transported across the plasma membrane, depending on the physiological status of the plant.

  14. Restricted spatial expression of a high-affinity phosphate transporter in potato roots.

    PubMed

    Gordon-Weeks, Ruth; Tong, Yiping; Davies, T G Emyr; Leggewie, Georg

    2003-08-01

    Phosphorus deficiency limits plant growth, and high-affinity phosphate transporters, of the Pht1 family, facilitate phosphate uptake and translocation. The family is subdivided into root specific, phosphate deprivation induced members and those also expressed in leaves. An antibody to StPT2, a potato root specific transporter, detected two bands (52 kDa and 30 kDa) on western blots of root plasma membrane extracts that were most intense in whole extracts from the root tip and slightly increased throughout the root in response to phosphate depletion. RT-PCR, using StPT2 specific primers, confirmed these findings. Low power confocal immunofluorescent images showed StPT2 expression mainly in the elongation zone at the root tip. By contrast, a vacuolar pyrophosphatase and a plasma membrane ATPase antibody labelled the whole root. High power images showed, by comparison with alpha-tubulin, cell wall and plasma membrane ATPase labelling, that StPT2 was in the epidermal plasma membrane and restricted to the apical surface. This is the first evidence of polar plasma membrane localisation of a plant nutrient transporter and is consistent with a role for StPT2 in phosphate capture and uptake.

  15. Molecular evolutionary analysis of the high-affinity K+ transporter gene family in angiosperms.

    PubMed

    Yang, P; Hua, C; Zhou, F; Zhang, B-J; Cai, X-N; Chen, Q-Z; Wang, R-L

    2016-07-15

    The high-affinity K(+) transporter (HKT) family comprises a group of multifunctional cation transporters widely distributed in organisms ranging from Bacteria to Eukarya. In angiosperms, the HKT family consists primarily of nine types, whose evolutionary relationships are not fully understood. The available sequences from 31 plant species were used to perform a comprehensive evolutionary analysis, including an examination of selection pressure and estimating phylogenetic tree and gene duplication events. Our results show that a gene duplication in the HKT1;5/HKT1;4 cluster might have led to the divergence of the HKT1;5 and HKT1;4 subfamilies. Additionally, maximum likelihood analysis revealed that the HKT family has undergone a strong purifying selection. An analysis of the amino acids provided strong statistical evidence for a functional divergence between subfamilies 1 and 2. Our study was the first to provide evidence of this functional divergence between these two subfamilies. Analysis of co-evolution in HKT identified 25 co-evolved groups. These findings expanded our understanding of the evolutionary mechanisms driving functional diversification of HKT proteins.

  16. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

    PubMed Central

    Cánovas, D; Vargas, C; Csonka, L N; Ventosa, A; Nieto, J J

    1996-01-01

    The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium. PMID:8955405

  17. Molecular physiology of higher plant sucrose transporters.

    PubMed

    Sauer, Norbert

    2007-05-25

    Sucrose is the primary product of photosynthetic CO(2) fixation that is used for the distribution of assimilated carbon within higher plants. Its partitioning from the site of synthesis to different sites of storage, conversion into other storage compounds or metabolic degradation involves various steps of cell-to-cell movement and transport. Many of these steps occur within symplastic domains, i.e. sucrose moves passively cell-to-cell through plasmodesmata. Some essential steps, however, occur between symplastically isolated cells or tissues. In these cases, sucrose is transiently released into the apoplast and its cell-to-cell transport depends on the activity of plasma membrane-localized, energy dependent, H(+)-symporting carrier proteins. This paper reviews the current knowledge of sucrose transporter physiology and molecular biology.

  18. Insulin Regulates the Activity of the High-Affinity Choline Transporter CHT

    PubMed Central

    Fishwick, Katherine J.; Rylett, R. Jane

    2015-01-01

    Studies in humans and animal models show that neuronal insulin resistance increases the risk of developing Alzheimer’s Disease (AD), and that insulin treatment may promote memory function. Cholinergic neurons play a critical role in cognitive and attentional processing and their dysfunction early in AD pathology may promote the progression of AD pathology. Synthesis and release of the neurotransmitter acetylcholine (ACh) is closely linked to the activity of the high-affinity choline transporter protein (CHT), but the impact of insulin receptor signaling and neuronal insulin resistance on these aspects of cholinergic function are unknown. In this study, we used differentiated SH-SY5Y cells stably-expressing CHT proteins to study the effect of insulin signaling on CHT activity and function. We find that choline uptake activity measured after acute addition of 20 nM insulin is significantly lower in cells that were grown for 24 h in media containing insulin compared to cells grown in the absence of insulin. This coincides with loss of ability to increase phospho-Protein Kinase B (PKB)/Akt levels in response to acute insulin stimulation in the chronic insulin-treated cells. Inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3-kinase) in cells significantly lowers phospho-PKB/Akt levels and decreases choline uptake activity. We show total internal reflection microscopy (TIRF) imaging of the dynamic movement of CHT proteins in live cells in response to depolarization and drug treatments. These data show that acute exposure of depolarized cells to insulin is coupled to transiently increased levels of CHT proteins at the cell surface, and that this is attenuated by chronic insulin exposure. Moreover, prolonged inhibition of PI3-kinase results in enhanced levels of CHT proteins at the cell surface by decreasing their rate of internalization. PMID:26161852

  19. In silico analysis of high affinity potassium transporter (HKT) isoforms in different plants.

    PubMed

    Zamani Babgohari, Mahbobeh; Ebrahimie, Esmaeil; Niazi, Ali

    2014-01-01

    High affinity potassium transporters (HKTs) are located in the plasma membrane of the vessels and have significant influence on salt tolerance in some plants. They exclude Na(+) from the parenchyma cells to reduce Na(+) concentration. Despite many studies, the underlying regulatory mechanisms and the exact functions of HKTs within different genomic backgrounds are relatively unknown. In this study, various bioinformatics techniques, including promoter analysis, identification of HKT-surrounding genes, and construction of gene networks, were applied to investigate the HKT regulatory mechanism. Promoter analysis showed that rice HKTs carry ABA response elements. Additionally, jasmonic acid response elements were detected on promoter region of TmHKT1;5. In silico synteny highlighted several unknown and new loci near rice, Arabidopsis thaliana and Physcomitrella patent HKTs, which may play a significant role in salt stress tolerance in concert with HKTs. Gene network prediction unravelled that crosstalk between jasmonate and ethylene reduces AtHKT1;1 expression. Furthermore, antiporter and transferase proteins were found in AtHKT1;1 gene network. Interestingly, regulatory elements on the promoter region of HKT in wild genotype (TmHKT1;5) were more frequent and variable than the ones in cultivated wheat (TaHKT1;5) which provides the possibility of rapid response and better understanding of environmental conditions for wild genotype. Detecting ABA and jasmonic acid response elements on promoter regions of HKTs provide valuable clues on underlying regulatory mechanisms of HKTs. In silico synteny and pathway discovery indicated several candidates which act in concert with HKTs in stress condition. We highlighted different arrangement of regulatory elements on promoter region of wild wheat (TmHKT1;5) compared to bread wheat (TaHKT1;5) in this study.

  20. Functional Analysis of an Arabidopsis T-DNA “Knockout” of the High-Affinity NH4+ Transporter AtAMT1;11

    PubMed Central

    Kaiser, Brent N.; Rawat, Suman R.; Siddiqi, M. Yaeesh; Masle, Josette; Glass, Anthony D.M.

    2002-01-01

    NH4+ acquisition by plant roots is thought to involve members of the NH4+ transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH4+. Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH4+ influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity 13NH4+ influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity 13NH4+ influx in the mutant by 30% compared with wild-type plants, whereas low-affinity 13NH4+ influx (250 μm–10 mm NH4+) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH4+ and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH4+ transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1. PMID:12427993

  1. Free radical modification of high-affinity GABA transport in the central nervous system

    SciTech Connect

    Debler, E.A.

    1985-01-01

    Presynaptic nerve terminals of DNS neurons can be characterized as dynamic self-contained subunits of the overall neuron. These neuron subunits together with the adjacent post-synaptic terminals constitute the functional unit of neurotransmission. One major function of the presynaptic nerve terminal in neurotransmission is its role in the release and subsequent reuptake of neurotransmitters. Neurochemical and morphological analysis of this high-affinity uptake process, for the amino acid neurotransmitter gamma-aminobutyric acid (GABA), revealed that free radical modification (lipid peroxidation) of presynaptic nerve terminal membrane structures resulted in a decrease in high-affinity (/sup 14/C)GABA uptake. Depolarized nerve terminals exposed to horseradish peroxidase (HRP)-generated superoxide radicals (.O/sub 2//sup -/) exhibited an alteration of synaptic vesicle membrane structure and a reduction of high-affinity (/sup 14/C)GABA uptake. In the kinetic analysis of high affinity (/sup 14/C)GABA uptake, hypoxanthine/xanthine oxidase (HPX/XOD)-treated synaptosomes exhibited a significant reduction in V/sub max/ but no significant alteration of K/sub m/. This HPX/XOD treatment also resulted in significant structural alterations of both the synaptic vesicle membrane and the synaptosomal plasma membrane. Structural and functional correlates appear to exist between synaptic vesicle and plasma membrane systems and high-affinity (/sup 14/C)GABA uptake. Together, the selectivity of the .O/sub 2//sup -/ toxicity and that of the high-K/sup +//Na/sup +/-free effect support the idea of two separate and different amino acid uptake systems in CNS tissue.

  2. Inhibition of the high affinity choline transporter enhances hyperalgesia in a rat model of chronic pancreatitis.

    PubMed

    Luo, Dan; Chen, Lei; Yu, Baoping

    2017-06-17

    The mechanisms underlying chronic and persistent pain associated with chronic pancreatitis (CP) are not completely understood. The cholinergic system is one of the major neural pathways of the pancreas. Meanwhile, this system plays an important role in chronic pain. We hypothesized that the high affinity choline transporter CHT1, which is a main determinant of cholinergic signaling capacity, is involved in regulating pain associated with CP. CP was induced by intraductal injection of 2% trinitrobenzene sulfonic acid (TNBS) in Sprague-Dawley rats. Pathological examination was used to evaluate the inflammation of pancreas and hyperalgesia was assessed by measuring the number of withdrawal events evoked by application of the von Frey filaments. CHT1 expression in pancreas-specific dorsal root ganglia (DRGs) was assessed through immunohistochemistry and western blotting. We also intraperitoneally injected the rats with hemicholinium-3 (HC-3, a specific inhibitor of CHT1). Then we observed its effects on the visceral hyperalgesia induced by CP, and on the acetylcholine (ACh) levels in the DRGs through using an acetylcholine/acetylcholinesterase assay kit. Signs of CP were observed 21 days after TNBS injection. Rats subjected to TNBS infusions had increased sensitivity to mechanical stimulation of the abdomen. CHT1-immunoreactive cells were increased in the DRGs from rats with CP compared to naive or sham rats. Western blots indicated that CHT1 expression was significantly up-regulated in TNBS-treated rats when compared to naive or sham-operated rats at all time points following surgery. In the TNBS group, CHT1 expression was higher on day 28 than on day 7 or day 14, but there was no statistical difference in CHT1 expression on day 28 vs. day 21. Treatment with HC-3 (60 μg/kg, 80 μg/kg, or 100 μg/kg) markedly enhanced the mechanical hyperalgesia and reduced ACh levels in a dose-dependent manner in rats with CP. We report for the first time that CHT1 may be involved

  3. Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding.

    PubMed

    Fischer, Marcus; Hopkins, Adam P; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G; Hubbard, Roderick E; Thomas, Gavin H

    2015-11-06

    Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. High Affinity S-Adenosylmethionine Plasma Membrane Transporter of Leishmania Is a Member of the Folate Biopterin Transporter (FBT) Family*

    PubMed Central

    Dridi, Larbi; Ahmed Ouameur, Amin; Ouellette, Marc

    2010-01-01

    S-Adenosylmethionine (AdoMet) is an important methyl group donor that plays a central role in many essential biochemical processes. The parasite Leishmania can both synthesize and transport AdoMet. Leishmania cells resistant to the antifolate methotrexate due to a rearrangement in folate biopterin transporter (FBT) genes were cross-resistant to sinefungin, an AdoMet analogue. FBT gene rearrangements were also observed in Leishmania major cells selected for sinefungin resistance. One of the rearranged FBT genes corresponded to the main AdoMet transporter (AdoMetT1) of Leishmania as determined by gene transfection and gene inactivation experiments. AdoMetT1 was determined to be a high affinity plasma membrane transporter expressed constitutively throughout the growth phases of the parasite. Leishmania cells selected for resistance or naturally insensitive to sinefungin had lower expression of AdoMetT1. A new function in one carbon metabolism, also a pathway of interest for chemotherapeutic interventions, is described for a novel class of membrane proteins found in diverse organisms. PMID:20406813

  5. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  6. Insights from the Fungus Fusarium oxysporum Point to High Affinity Glucose Transporters as Targets for Enhancing Ethanol Production from Lignocellulose

    PubMed Central

    Ali, Shahin S.; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M.

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km(glucose) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing. PMID:23382943

  7. Insights from the fungus Fusarium oxysporum point to high affinity glucose transporters as targets for enhancing ethanol production from lignocellulose.

    PubMed

    Ali, Shahin S; Nugent, Brian; Mullins, Ewen; Doohan, Fiona M

    2013-01-01

    Ethanol is the most-widely used biofuel in the world today. Lignocellulosic plant biomass derived from agricultural residue can be converted to ethanol via microbial bioprocessing. Fungi such as Fusarium oxysporum can simultaneously saccharify straw to sugars and ferment sugars to ethanol. But there are many bottlenecks that need to be overcome to increase the efficacy of microbial production of ethanol from straw, not least enhancement of the rate of fermentation of both hexose and pentose sugars. This research tested the hypothesis that the rate of sugar uptake by F. oxysporum would enhance the ethanol yields from lignocellulosic straw and that high affinity glucose transporters can enhance ethanol yields from this substrate. We characterized a novel hexose transporter (Hxt) from this fungus. The F. oxysporum Hxt represents a novel transporter with homology to yeast glucose signaling/transporter proteins Rgt2 and Snf3, but it lacks their C-terminal domain which is necessary for glucose signalling. Its expression level decreased with increasing glucose concentration in the medium and in a glucose uptake study the Km((glucose)) was 0.9 mM, which indicated that the protein is a high affinity glucose transporter. Post-translational gene silencing or over expression of the Hxt in F. oxysporum directly affected the glucose and xylose transport capacity and ethanol yielded by F. oxysporum from straw, glucose and xylose. Thus we conclude that this Hxt has the capacity to transport both C5 and C6 sugars and to enhance ethanol yields from lignocellulosic material. This study has confirmed that high affinity glucose transporters are ideal candidates for improving ethanol yields from lignocellulose because their activity and level of expression is high in low glucose concentrations, which is very common during the process of consolidated processing.

  8. Identification of an animal sucrose transporter.

    PubMed

    Meyer, Heiko; Vitavska, Olga; Wieczorek, Helmut

    2011-06-15

    According to a classic tenet, sugar transport across animal membranes is restricted to monosaccharides. Here, we present the first report of an animal sucrose transporter, SCRT, which we detected in Drosophila melanogaster at each developmental stage. We localized the protein in apical membranes of the late embryonic hindgut as well as in vesicular membranes of ovarian follicle cells. The fact that knockdown of SCRT expression results in significantly increased lethality demonstrates an essential function for the protein. Experiments with Saccharomyces cerevisiae as a heterologous expression system revealed that sucrose is a transported substrate. Because the knockout of SLC45A2, a highly similar protein belonging to the mammalian solute carrier family 45 (SLC45) causes oculocutaneous albinism and because the vesicular structures in which SCRT is located appear to contain melanin, we propose that these organelles are melanosome-like structures and that the transporter is necessary for balancing the osmotic equilibrium during the polymerization process of melanin by the import of a compatible osmolyte. In the hindgut epithelial cells, sucrose might also serve as a compatible osmolyte, but we cannot exclude the possibility that transport of this disaccharide also serves nutritional adequacy.

  9. Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae: a platform strain for engineering sucrose metabolism.

    PubMed

    Marques, Wesley Leoricy; Mans, Robert; Marella, Eko Roy; Cordeiro, Rosa Lorizolla; van den Broek, Marcel; Daran, Jean-Marc G; Pronk, Jack T; Gombert, Andreas K; van Maris, Antonius J A

    2017-01-01

    Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h-1 after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport. © FEMS 2017.

  10. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

    PubMed Central

    Gaber, R F; Styles, C A; Fink, G R

    1988-01-01

    We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion. Images PMID:3043197

  11. Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter.

    PubMed Central

    Kasahara, Toshiko; Kasahara, Michihiro

    2003-01-01

    Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares approximately 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports D-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The K (m) and V (max) values for D-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2. PMID:12603199

  12. Evolution of Plant Sucrose Uptake Transporters

    PubMed Central

    Reinders, Anke; Sivitz, Alicia B.; Ward, John M.

    2012-01-01

    In angiosperms, sucrose uptake transporters (SUTs) have important functions especially in vascular tissue. Here we explore the evolutionary origins of SUTs by analysis of angiosperm SUTs and homologous transporters in a vascular early land plant, Selaginella moellendorffii, and a non-vascular plant, the bryophyte Physcomitrella patens, the charophyte algae Chlorokybus atmosphyticus, several red algae and fission yeast, Schizosaccharomyces pombe. Plant SUTs cluster into three types by phylogenetic analysis. Previous studies using angiosperms had shown that types I and II are localized to the plasma membrane while type III SUTs are associated with vacuolar membrane. SUT homologs were not found in the chlorophyte algae Chlamydomonas reinhardtii and Volvox carterii. However, the characean algae Chlorokybus atmosphyticus contains a SUT homolog (CaSUT1) and phylogenetic analysis indicated that it is basal to all other streptophyte SUTs analyzed. SUTs are present in both red algae and S. pombe but they are less related to plant SUTs than CaSUT1. Both Selaginella and Physcomitrella encode type II and III SUTs suggesting that both plasma membrane and vacuolar sucrose transporter activities were present in early land plants. It is likely that SUT transporters are important for scavenging sucrose from the environment and intracellular compartments in charophyte and non-vascular plants. Type I SUTs were only found in eudicots and we conclude that they evolved from type III SUTs, possibly through loss of a vacuolar targeting sequence. Eudicots utilize type I SUTs for phloem (vascular tissue) loading while monocots use type II SUTs for phloem loading. We show that HvSUT1 from barley, a type II SUT, reverted the growth defect of the Arabidopsis atsuc2 (type I) mutant. This indicates that type I and II SUTs evolved similar (and interchangeable) phloem loading transporter capabilities independently. PMID:22639641

  13. A low K+ signal is required for functional high-affinity K+ uptake through HAK5 transporters.

    PubMed

    Rubio, Francisco; Fon, Mario; Ródenas, Reyes; Nieves-Cordones, Manuel; Alemán, Fernando; Rivero, Rosa M; Martínez, Vicente

    2014-11-01

    The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species.

  14. Functional assessment of the Medicago truncatula NIP/LATD protein demonstrates that it is a high-affinity nitrate transporter.

    PubMed

    Bagchi, Rammyani; Salehin, Mohammad; Adeyemo, O Sarah; Salazar, Carolina; Shulaev, Vladimir; Sherrier, D Janine; Dickstein, Rebecca

    2012-10-01

    The Medicago truncatula NIP/LATD (for Numerous Infections and Polyphenolics/Lateral root-organ Defective) gene encodes a protein found in a clade of nitrate transporters within the large NRT1(PTR) family that also encodes transporters of dipeptides and tripeptides, dicarboxylates, auxin, and abscisic acid. Of the NRT1(PTR) members known to transport nitrate, most are low-affinity transporters. Here, we show that M. truncatula nip/latd mutants are more defective in their lateral root responses to nitrate provided at low (250 μm) concentrations than at higher (5 mm) concentrations; however, nitrate uptake experiments showed no discernible differences in uptake in the mutants. Heterologous expression experiments showed that MtNIP/LATD encodes a nitrate transporter: expression in Xenopus laevis oocytes conferred upon the oocytes the ability to take up nitrate from the medium with high affinity, and expression of MtNIP/LATD in an Arabidopsis chl1(nrt1.1) mutant rescued the chlorate susceptibility phenotype. X. laevis oocytes expressing mutant Mtnip-1 and Mtlatd were unable to take up nitrate from the medium, but oocytes expressing the less severe Mtnip-3 allele were proficient in nitrate transport. M. truncatula nip/latd mutants have pleiotropic defects in nodulation and root architecture. Expression of the Arabidopsis NRT1.1 gene in mutant Mtnip-1 roots partially rescued Mtnip-1 for root architecture defects but not for nodulation defects. This suggests that the spectrum of activities inherent in AtNRT1.1 is different from that possessed by MtNIP/LATD, but it could also reflect stability differences of each protein in M. truncatula. Collectively, the data show that MtNIP/LATD is a high-affinity nitrate transporter and suggest that it could have another function.

  15. Defective high-affinity thiamine transporter leads to cell death in thiamine-responsive megaloblastic anemia syndrome fibroblasts

    PubMed Central

    Stagg, Amy R.; Fleming, Judith C.; Baker, Meghan A.; Sakamoto, Massayuki; Cohen, Nadine; Neufeld, Ellis J.

    1999-01-01

    We have investigated the cellular pathology of the syndrome called thiamine-responsive megaloblastic anemia (TRMA) with diabetes and deafness. Cultured diploid fibroblasts were grown in thiamine-free medium and dialyzed serum. Normal fibroblasts survived indefinitely without supplemental thiamine, whereas patient cells died in 5–14 days (mean 9.5 days), and heterozygous cells survived for more than 30 days. TRMA fibroblasts were rescued from death with 10–30 nM thiamine (in the range of normal plasma thiamine concentrations). Positive terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) staining suggested that cell death was due to apoptosis. We assessed cellular uptake of [3H]thiamine at submicromolar concentrations. Normal fibroblasts exhibited saturable, high-affinity thiamine uptake (Km 400–550 nM; Vmax 11 pmol/min/106 cells) in addition to a low-affinity unsaturable component. Mutant cells lacked detectable high-affinity uptake. At 30 nM thiamine, the rate of uptake of thiamine by TRMA fibroblasts was 10-fold less than that of wild-type, and cells from obligate heterozygotes had an intermediate phenotype. Transfection of TRMA fibroblasts with the yeast thiamine transporter gene THI10 prevented cell death when cells were grown in the absence of supplemental thiamine. We therefore propose that the primary abnormality in TRMA is absence of a high-affinity thiamine transporter and that low intracellular thiamine concentrations in the mutant cells cause biochemical abnormalities that lead to apoptotic cell death. J. Clin. Invest. 103:723–729 (1999). PMID:10074490

  16. Paracetamol and cytarabine binding competition in high affinity binding sites of transporting protein

    NASA Astrophysics Data System (ADS)

    Sułkowska, A.; Bojko, B.; Równicka, J.; Sułkowski, W. W.

    2006-07-01

    Paracetamol (acetaminophen, AA) the most popular analgesic drug is commonly used in the treatment of pain in patients suffering from cancer. In our studies, we evaluated the competition in binding with serum albumin between paracetamol (AA) and cytarabine, antyleukemic drug (araC). The presence of one drug can alter the binding affinity of albumin towards the second one. Such interaction can result in changing of the free fraction of the one of these drugs in blood. Two spectroscopic methods were used to determine high affinity binding sites and the competition of the drugs. Basing on the change of the serum albumin fluorescence in the presence of either of the drugs the quenching ( KQ) constants for the araC-BSA and AA-BSA systems were calculated. Analysis of UV difference spectra allowed us to describe the changes in drug-protein complexes (araC-albumin and AA-albumin) induced by the presence of the second drug (AA and araC, respectively). The mechanism of competition between araC and AA has been proposed.

  17. High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

    PubMed Central

    Nau-Wagner, Gabriele; Boch, Jens; Le Good, J. Ann; Bremer, Erhard

    1999-01-01

    We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a Ki of approximately 4 μM. Uptake studies with [1,2-dimethyl-14C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a Km value of 4 ± 1 μM and a maximum rate of transport (Vmax) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline

  18. Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

    PubMed

    Erickson, Jeffrey D

    2017-07-01

    Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K(+) -stimulated MeAIB transport displays an affinity (Km ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca(2+) , and is blocked by inhibition of voltage-gated Ca(2+) channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca(2+) and voltage-gated calcium channels, but is also blocked by the Na(+) channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca(2+) , but on Na(+) ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K(+) -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017

  19. Regulation of the high-affinity copper transporter (hCtr1) expression by cisplatin and heavy metals.

    PubMed

    Liang, Zheng Dong; Long, Yan; Chen, Helen H W; Savaraj, Niramol; Kuo, Macus Tien

    2014-01-01

    Platinum-based antitumor agents have been the mainstay in cancer chemotherapy for many human malignancies. Drug resistance is an important obstacle to achieving the maximal therapeutic efficacy of these drugs. Understanding how platinum drugs enter cells is of great importance in improving therapeutic efficacy. It has been demonstrated that human high-affinity copper transporter 1 (hCtr1) is involved in transporting cisplatin into cells to elicit cytotoxic effects, although other mechanisms may exist. In this communication, we demonstrate that cisplatin transcriptionally induces the expression of hCtr1 in time- and concentration-dependent manners. Cisplatin functions as a competitor for hCtr1-mediated copper transport, resulting in reduced cellular copper levels and leading to upregulated expression of Sp1, which is a positive regulator for hCtr1 expression. Thus, regulation of hCtr1 expression by cisplatin is an integral part of the copper homeostasis regulation system. We also demonstrate that Ag(I) and Zn(II), which are known to suppress hCtr1-mediated copper transport, can also induce hCtr1/Sp1 expression. In contrast, Cd(II), another inhibitor of copper transport, downregulates hCtr1 expression by suppressing Sp1 expression. Collectively, our results demonstrate diverse mechanisms of regulating copper metabolism by these heavy metals.

  20. Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter

    PubMed Central

    Rannversson, Hafsteinn; Andersen, Jacob; Sørensen, Lena; Bang-Andersen, Benny; Park, Minyoung; Huber, Thomas; Sakmar, Thomas P.; Strømgaard, Kristian

    2016-01-01

    Despite the well-established role of the human serotonin transporter (hSERT) in the treatment of depression, the molecular details of antidepressant drug binding are still not fully understood. Here we utilize amber codon suppression in a membrane-bound transporter protein to encode photocrosslinking unnatural amino acids (UAAs) into 75 different positions in hSERT. UAAs are incorporated with high specificity, and functionally active transporters have similar transport properties and pharmacological profiles compared with wild-type transporters. We employ ultraviolet-induced crosslinking with p-azido-L-phenylalanine (azF) at selected positions in hSERT to map the binding site of imipramine, a prototypical tricyclic antidepressant, and vortioxetine, a novel multimodal antidepressant. We find that the two antidepressants crosslink with azF incorporated at different positions within the central substrate-binding site of hSERT, while no crosslinking is observed at the vestibular-binding site. Taken together, our data provide direct evidence for defining the high-affinity antidepressant binding site in hSERT. PMID:27089947

  1. The fungal UmSrt1 and maize ZmSUT1 sucrose transporters battle for plant sugar resources.

    PubMed

    Wittek, Anke; Dreyer, Ingo; Al-Rasheid, Khaled A S; Sauer, Norbert; Hedrich, Rainer; Geiger, Dietmar

    2017-03-15

    The biotrophic fungus Ustilago maydis causes the corn smut disease inducing tumor formation in its host Zea mays. Upon infection, the fungal hyphae invaginate the plasma membrane of infected maize cells establishing an interface where pathogen and host are separated only by their plasma membranes. At this interface the fungal and maize sucrose transporters, UmSrt1 and ZmSUT1, compete for extracellular sucrose in the corn smut/maize pathosystem. Here we biophysically characterized ZmSUT1 and UmSrt1 in Xenopus oocytes with respect to their voltage-, pH- and substrate-dependence and determined affinities towards protons and sucrose. In contrast to ZmSUT1, UmSrt1 has a high-affinity for sucrose and is relatively pH- and voltage-independent. Using these quantitative parameters, we developed a mathematical model to simulate the competition for extracellular sucrose at the contact zone between the fungus and the host plant. This approach revealed that UmSrt1 exploits the apoplastic sucrose resource, which forces the plant transporter into a sucrose export mode providing the fungus with sugar from the phloem. Importantly, the high sucrose concentration in the phloem appeared disadvantageous for the ZmSUT1, preventing sucrose recovery from the apoplastic space in the fungus/plant interface.

  2. Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity.

    PubMed

    Schulze, W; Weise, A; Frommer, W B; Ward, J M

    2000-11-24

    AtSUT2 was found to be a low-affinity sucrose transporter (K(M)=11.7 mM at pH 4). Chimeric proteins between AtSUT2 and the high-affinity StSUT1 were constructed in which the extended N-terminus and central loop of AtSUT2 were exchanged with those domains of StSUT1 and vice versa. Chimeras containing the N-terminus of AtSUT2 showed significantly lower affinity for sucrose compared to chimeras containing the N-terminus of StSUT1. The results indicate a significant function of the N-terminus but not the central cytoplasmic loop in determining substrate affinity. Expression of AtSUT2 in major veins of source leaves and in flowers is compatible with a role as a second low-affinity sucrose transporter or as a sucrose sensor.

  3. Fructose Uptake in Sinorhizobium meliloti Is Mediated by a High-Affinity ATP-Binding Cassette Transport System

    PubMed Central

    Lambert, Annie; Østerås, Magne; Mandon, Karine; Poggi, Marie-Christine; Le Rudulier, Daniel

    2001-01-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (Km of 6 μM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  4. Fructose uptake in Sinorhizobium meliloti is mediated by a high-affinity ATP-binding cassette transport system.

    PubMed

    Lambert, A; Østerås, M; Mandon, K; Poggi, M C; Le Rudulier, D

    2001-08-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  5. Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1.

    PubMed

    Belenky, Peter A; Moga, Tiberiu G; Brenner, Charles

    2008-03-28

    NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.

  6. Sucrose transport into vacuoles isolated from barley mesophyll protoplasts.

    PubMed

    Kaiser, G; Heber, U

    1984-11-01

    Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent Km of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg(2+) present, nor by protonophores and valinomycin (with K(+) present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.

  7. Mutational analysis of the high-affinity zinc binding site validates a refined human dopamine transporter homology model.

    PubMed

    Stockner, Thomas; Montgomery, Therese R; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F; Freissmuth, Michael; Sitte, Harald H

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter's movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle.

  8. A Dualistic Conformational Response to Substrate Binding in the Human Serotonin Transporter Reveals a High Affinity State for Serotonin*

    PubMed Central

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-01-01

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT. PMID:25614630

  9. A dualistic conformational response to substrate binding in the human serotonin transporter reveals a high affinity state for serotonin.

    PubMed

    Bjerregaard, Henriette; Severinsen, Kasper; Said, Saida; Wiborg, Ove; Sinning, Steffen

    2015-03-20

    Serotonergic neurotransmission is modulated by the membrane-embedded serotonin transporter (SERT). SERT mediates the reuptake of serotonin into the presynaptic neurons. Conformational changes in SERT occur upon binding of ions and substrate and are crucial for translocation of serotonin across the membrane. Our understanding of these conformational changes is mainly based on crystal structures of a bacterial homolog in various conformations, derived homology models of eukaryotic neurotransmitter transporters, and substituted cysteine accessibility method of SERT. However, the dynamic changes that occur in the human SERT upon binding of ions, the translocation of substrate, and the role of cholesterol in this interplay are not fully elucidated. Here we show that serotonin induces a dualistic conformational response in SERT. We exploited the substituted cysteine scanning method under conditions that were sensitized to detect a more outward-facing conformation of SERT. We found a novel high affinity outward-facing conformational state of the human SERT induced by serotonin. The ionic requirements for this new conformational response to serotonin mirror the ionic requirements for translocation. Furthermore, we found that membrane cholesterol plays a role in the dualistic conformational response in SERT induced by serotonin. Our results indicate the existence of a subpopulation of SERT responding differently to serotonin binding than hitherto believed and that membrane cholesterol plays a role in this subpopulation of SERT.

  10. Constitutive expression of high-affinity sulfate transporter (HAST) gene in Indian mustard showed enhanced sulfur uptake and assimilation.

    PubMed

    Abdin, M Z; Akmal, M; Ram, M; Nafis, T; Alam, P; Nadeem, M; Khan, M A; Ahmad, A

    2011-07-01

    Lycopersicon esculantum sulfate transporter gene (LeST 1.1) encodes a high-affinity sulfate transporter (HAST) located in root epidermis. In this study, the LeST 1.1 gene was constitutively expressed in Indian mustard (Brassica juncea cv. Pusa Jai Kisan). Transgenic as well as untransformed plants were grown in sulfur-insufficient (25 and 50 μM) and sulfur-sufficient (1,000 μM) conditions for 30 days. Two-fold increase was noticed in the sulfate uptake rate of transgenic plants grown in both sulfur-insufficient and -sufficient conditions as compared to untransformed plants. The transgenic B. juncea plants were able to accumulate higher biomass and showed improved sulfur status even in sulfur-insufficient conditions when compared with untransformed plants. Chlorophyll content, ATP sulfurylase activity and protein content were also higher in transgenic plants than untranformed plants under sulfur-insufficient conditions. Our results, thus, clearly indicate that constitutive expression of LeST 1.1 gene in B. juncea had led to enhanced capacity of sulfur uptake and assimilation even in sulfur-insufficient conditions. This approach can also be used in other crops to enhance their sulfate uptake and assimilation potential under S-insufficient conditions.

  11. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis.

    PubMed

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Rajulu, Charukesi; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier; Rubio, Vicente

    2015-09-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life.

  12. ESCRT-III-Associated Protein ALIX Mediates High-Affinity Phosphate Transporter Trafficking to Maintain Phosphate Homeostasis in Arabidopsis

    PubMed Central

    Cardona-López, Ximena; Cuyas, Laura; Marín, Elena; Irigoyen, María Luisa; Gil, Erica; Puga, María Isabel; Bligny, Richard; Nussaume, Laurent; Geldner, Niko; Paz-Ares, Javier

    2015-01-01

    Prior to the release of their cargoes into the vacuolar lumen, sorting endosomes mature into multivesicular bodies (MVBs) through the action of ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT (ESCRT) protein complexes. MVB-mediated sorting of high-affinity phosphate transporters (PHT1) to the vacuole limits their plasma membrane levels under phosphate-sufficient conditions, a process that allows plants to maintain phosphate homeostasis. Here, we describe ALIX, a cytosolic protein that associates with MVB by interacting with ESCRT-III subunit SNF7 and mediates PHT1;1 trafficking to the vacuole in Arabidopsis thaliana. We show that the partial loss-of-function mutant alix-1 displays reduced vacuolar degradation of PHT1;1. ALIX derivatives containing the alix-1 mutation showed reduced interaction with SNF7, providing a simple molecular explanation for impaired cargo trafficking in alix-1 mutants. In fact, the alix-1 mutation also hampered vacuolar sorting of the brassinosteroid receptor BRI1. We also show that alix-1 displays altered vacuole morphogenesis, implying a new role for ALIX proteins in vacuolar biogenesis, likely acting as part of ESCRT-III complexes. In line with a presumed broad target spectrum, the alix-1 mutation is pleiotropic, leading to reduced plant growth and late flowering, with stronger alix mutations being lethal, indicating that ALIX participates in diverse processes in plants essential for their life. PMID:26342016

  13. Plant High-Affinity Potassium (HKT) Transporters involved in salinity tolerance: structural insights to probe differences in ion selectivity.

    PubMed

    Waters, Shane; Gilliham, Matthew; Hrmova, Maria

    2013-04-09

    High-affinity Potassium Transporters (HKTs) belong to an important class of integral membrane proteins (IMPs) that facilitate cation transport across the plasma membranes of plant cells. Some members of the HKT protein family have been shown to be critical for salinity tolerance in commercially important crop species, particularly in grains, through exclusion of Na+ ions from sensitive shoot tissues in plants. However, given the number of different HKT proteins expressed in plants, it is likely that different members of this protein family perform in a range of functions. Plant breeders and biotechnologists have attempted to manipulate HKT gene expression through genetic engineering and more conventional plant breeding methods to improve the salinity tolerance of commercially important crop plants. Successful manipulation of a biological trait is more likely to be effective after a thorough understanding of how the trait, genes and proteins are interconnected at the whole plant level. This article examines the current structural and functional knowledge relating to plant HKTs and how their structural features may explain their transport selectivity. We also highlight specific areas where new knowledge of plant HKT transporters is needed. Our goal is to present how knowledge of the structure of HKT proteins is helpful in understanding their function and how this understanding can be an invaluable experimental tool. As such, we assert that accurate structural information of plant IMPs will greatly inform functional studies and will lead to a deeper understanding of plant nutrition, signalling and stress tolerance, all of which represent factors that can be manipulated to improve agricultural productivity.

  14. Regulation of the high-affinity choline transporter activity and trafficking by its association with cholesterol-rich lipid rafts.

    PubMed

    Cuddy, Leah K; Winick-Ng, Warren; Rylett, Rebecca Jane

    2014-03-01

    The sodium-coupled, hemicholinium-3-sensitive, high-affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts in both SH-SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH-SY5Y cells expressing rat CHT with filipin, methyl-β-cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol-saturated MβC. Kinetic analysis of binding of [(3)H]hemicholinium-3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (KD) and maximum number of binding sites (Bmax ); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol-rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis. The sodium-coupled choline transporter CHT moves choline into cholinergic nerve terminals to serve as substrate for acetylcholine synthesis. We show for the first time that CHT is concentrated in cholesterol-rich lipid rafts, and decreasing membrane cholesterol significantly reduces both choline uptake activity and cell surface CHT protein levels. CHT association with cholesterol-rich rafts is critical for its function, and alterations in plasma membrane cholesterol could diminish cholinergic

  15. Mutational Analysis of the High-Affinity Zinc Binding Site Validates a Refined Human Dopamine Transporter Homology Model

    PubMed Central

    Stockner, Thomas; Montgomery, Therese R.; Kudlacek, Oliver; Weissensteiner, Rene; Ecker, Gerhard F.; Freissmuth, Michael; Sitte, Harald H.

    2013-01-01

    The high-resolution crystal structure of the leucine transporter (LeuT) is frequently used as a template for homology models of the dopamine transporter (DAT). Although similar in structure, DAT differs considerably from LeuT in a number of ways: (i) when compared to LeuT, DAT has very long intracellular amino and carboxyl termini; (ii) LeuT and DAT share a rather low overall sequence identity (22%) and (iii) the extracellular loop 2 (EL2) of DAT is substantially longer than that of LeuT. Extracellular zinc binds to DAT and restricts the transporter‚s movement through the conformational cycle, thereby resulting in a decrease in substrate uptake. Residue H293 in EL2 praticipates in zinc binding and must be modelled correctly to allow for a full understanding of its effects. We exploited the high-affinity zinc binding site endogenously present in DAT to create a model of the complete transmemberane domain of DAT. The zinc binding site provided a DAT-specific molecular ruler for calibration of the model. Our DAT model places EL2 at the transporter lipid interface in the vicinity of the zinc binding site. Based on the model, D206 was predicted to represent a fourth co-ordinating residue, in addition to the three previously described zinc binding residues H193, H375 and E396. This prediction was confirmed by mutagenesis: substitution of D206 by lysine and cysteine affected the inhibitory potency of zinc and the maximum inhibition exerted by zinc, respectively. Conversely, the structural changes observed in the model allowed for rationalizing the zinc-dependent regulation of DAT: upon binding, zinc stabilizes the outward-facing state, because its first coordination shell can only be completed in this conformation. Thus, the model provides a validated solution to the long extracellular loop and may be useful to address other aspects of the transport cycle. PMID:23436987

  16. Sucrose transport by the alkaliphilic, thermophilic Bacillus sp. strain TA2.A1 is dependent on a sodium gradient.

    PubMed

    Peddie, C J; Cook, G M; Morgan, H W

    2000-10-01

    An alkaliphilic Bacillus designated strain TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C. Sodium chloride (>5mM) was an obligate requirement for the growth of strain TA2.A1 on sucrose, and growth on sucrose was inhibited by monensin, an ionophore that collapses the sodium gradient (ApNa+) across the cell membrane. Sucrose transport by strain TA2.A1 was sodium dependent and was inhibited by monensin. The Kt for sucrose transport was 33 microM and the Eadie-Hofstee plot was linear, suggesting one high-affinity uptake system for sucrose. The affinity for sodium was low (0.5 mM), and the Hill plot had a slope of 1.6, suggesting that sodium binding was noncooperative and that the sucrose transporter had more than one binding site for sodium. Based on these results, Bacillus strain TA2.A1 uses a sodium gradient for sucrose uptake, in addition to the sodium-dependent glutamate uptake system reported previously.

  17. The High-Affinity Phosphate Transporter GmPT5 Regulates Phosphate Transport to Nodules and Nodulation in Soybean1[W][OA

    PubMed Central

    Qin, Lu; Zhao, Jing; Tian, Jiang; Chen, Liyu; Sun, Zhaoan; Guo, Yongxiang; Lu, Xing; Gu, Mian; Xu, Guohua; Liao, Hong

    2012-01-01

    Legume biological nitrogen (N) fixation is the most important N source in agroecosystems, but it is also a process requiring a considerable amount of phosphorus (P). Therefore, developing legume varieties with effective N2 fixation under P-limited conditions could have profound significance for improving agricultural sustainability. We show here that inoculation with effective rhizobial strains enhanced soybean (Glycine max) N2 fixation and P nutrition in the field as well as in hydroponics. Furthermore, we identified and characterized a nodule high-affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterologous expression verified that GmPT5 was indeed a high-affinity Pi transporter. Localization of GmPT5 expression based on β-glucuronidase staining in soybean composite plants with transgenic roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from the root vascular system into nodules. Overexpression or knockdown of GmPT5 in transgenic composite soybean plants altered nodulation and plant growth performance, which was partially dependent on P supply. Through both in situ and in vitro 33P uptake assays using transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functions in transporting Pi from roots to nodules, especially under P-limited conditions. We conclude that the high-affinity Pi transporter, GmPT5, controls Pi entry from roots to nodules, is critical for maintaining Pi homeostasis in nodules, and subsequently regulates soybean nodulation and growth performance. PMID:22740613

  18. Cloning, expression pattern and essentiality of the high-affinity copper transporter 1 (ctr1) gene in zebrafish.

    PubMed

    Mackenzie, Natalia C; Brito, Mónica; Reyes, Ariel E; Allende, Miguel L

    2004-03-17

    The high-affinity copper transporter 1 (Ctr1) is a highly conserved transmembrane protein that mediates the internalization of copper ions from the extracellular medium. In this study, we have isolated the zebrafish ctr1 gene. The zebrafish ctr1 cDNA encodes a protein with 69% identity to the human orthologue and shows conservation of specific amino acid residues involved in copper transport. We find only a single ctr1 gene in the zebrafish genome which maps to linkage group 5. The genomic structure of the zebrafish gene shows that it consists of five exons and that exon-intron boundaries are absolutely conserved with the mammalian ctr1 genes. Expression in embryos was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Zebrafish ctr1 is maternally loaded, and transcripts can be detected throughout development and in adult fish. Distribution of ctr1 message appears ubiquitous during early stages becoming restricted to the brain and ventral tissues by 24 h post fertilization (hpf). Beginning at 3 days post fertilization (dpf), expression is found mainly in the developing intestine. Specific knockdown of ctr1 by antisense morpholino oligonucleotides (MOs) causes early larval lethality. Defects include cell death in tissues where ctr1 is most heavily expressed, a finding similar to that described for a mouse knockout of mCtr1. Despite the existence of at least one other copper transport mechanism in the fish, our studies show that zebrafish ctr1 is an essential gene for development.

  19. Phosphate-dependent regulation of the low- and high-affinity transport systems in the model actinomycete Streptomyces coelicolor.

    PubMed

    Santos-Beneit, Fernando; Rodríguez-García, Antonio; Franco-Domínguez, Etelvina; Martín, Juan F

    2008-08-01

    The transport of inorganic phosphate (P(i)) is essential for the growth of all organisms. The metabolism of soil-dwelling Streptomyces species, and their ability to produce antibiotics and other secondary metabolites, are strongly influenced by the availability of phosphate. The transcriptional regulation of the SCO4138 and SCO1845 genes of Streptomyces coelicolor was studied. These genes encode the two putative low-affinity P(i) transporters PitH1 and PitH2, respectively. Expression of these genes and that of the high-affinity transport system pstSCAB follows a sequential pattern in response to phosphate deprivation, as shown by coupling their promoters to a luciferase reporter gene. Expression of pitH2, but not that of pap-pitH1 (a bicistronic transcript), is dependent upon the response regulator PhoP. PhoP binds to specific sequences consisting of direct repeats of 11 nt in the promoter of pitH2, but does not bind to the pap-pitH1 promoter, which lacks these direct repeats for PhoP recognition. The transcription start point of the pitH2 promoter was identified by primer extension analyses, and the structure of the regulatory sequences in the PhoP-protected DNA region was established. It consists of four central direct repeats flanked by two other less conserved repeats. A model for PhoP regulation of this promoter is proposed based on the four promoter DNA-PhoP complexes detected by electrophoretic mobility shift assays and footprinting studies.

  20. A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5.

    PubMed

    Nieves-Cordones, Manuel; Miller, Anthony J; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

    2008-12-01

    A chimeric CaHAK1-LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K(+) uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K(+) uptake shown by K(+)-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K(+) uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K(+) uptake was not correlated with the root K(+) content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K(+) starvation or growth in the presence of NH(4) (+), but which do not decrease the K(+) content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K(+) starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K(+) transporter.

  1. The Bacillus subtilis EfeUOB transporter is essential for high-affinity acquisition of ferrous and ferric iron.

    PubMed

    Miethke, Marcus; Monteferrante, Carmine G; Marahiel, Mohamed A; van Dijl, Jan Maarten

    2013-10-01

    Efficient uptake of iron is of critical importance for growth and viability of microbial cells. Nevertheless, several mechanisms for iron uptake are not yet clearly defined. Here we report that the widely conserved transporter EfeUOB employs an unprecedented dual-mode mechanism for acquisition of ferrous (Fe[II]) and ferric (Fe[III]) iron in the bacterium Bacillus subtilis. We show that the binding protein EfeO and the permease EfeU form a minimal complex for ferric iron uptake. The third component EfeB is a hemoprotein that oxidizes ferrous iron to ferric iron for uptake by EfeUO. Accordingly, EfeB promotes growth under microaerobic conditions where ferrous iron is more abundant. Notably, EfeB also fulfills a vital role in cell envelope stress protection by eliminating reactive oxygen species that accumulate in the presence of ferrous iron. In conclusion, the EfeUOB system contributes to the high-affinity uptake of iron that is available in two different oxidation states. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. A high affinity kidney targeting by chitobionic acid-conjugated polysorbitol gene transporter alleviates unilateral ureteral obstruction in rats.

    PubMed

    Islam, Mohammad Ariful; Kim, Sanghwa; Firdous, Jannatul; Lee, Ah-Young; Hong, Seong-Ho; Seo, Min Kyeong; Park, Tae-Eun; Yun, Cheol-Heui; Choi, Yun-Jaie; Chae, Chanhee; Cho, Chong-Su; Cho, Myung-Haing

    2016-09-01

    Aside from kidney transplantation - a procedure which is exceedingly dependent on donor-match and availability leading to excessive costs - there are currently no permanent treatments available which reverse kidney injury and failure. However, kidney-specific targeted gene therapy has outstanding potential to treat kidney-related dysfunction. Herein we report a novel kidney-specific targeted gene delivery system developed through the conjugation of chitobionic acid (CBA) to a polysorbitol gene transporter (PSGT) synthesized from sorbitol diacrylate and low molecular weight polyethylenimine (PEI) carrying hepatocyte growth factor (HGF) gene to alleviate unilateral ureteral obstruction (UUO) in rats. CBA-PSGT performed exceptionally well for targeted delivery of HGF to kidney tissues compared to its non-targeted counterparts (P < 0.001) after systemic tail-vein injection and significantly reduced the UUO symptoms, returning the UUO rats to a normal health status. The kidney-targeted CBA-PSGT-delivered HGF also strikingly reduced various pathologic and molecular markers in vivo such as the level of collagens (type I and II), blood urea nitrogen (BUN), creatinine, and the expressions of ICAM-1, TIMP-1 and α-SMA which play a critical role in obstructive kidney functions. Therefore, CBA-PSGT should be further investigated because of its potential to alleviate UUO and kidney-related diseases using high affinity kidney targeting.

  3. Sucrose transporter1 functions in phloem loading in maize leaves

    PubMed Central

    Slewinski, Thomas L.; Meeley, Robert; Braun, David M.

    2009-01-01

    In most plants, sucrose is exported from source leaves to carbon-importing sink tissues to sustain their growth and metabolism. Apoplastic phloem-loading species require sucrose transporters (SUTs) to transport sucrose into the phloem. In many dicot plants, genetic and biochemical evidence has established that SUT1-type proteins function in phloem loading. However, the role of SUT1 in phloem loading in monocot plants is not clear since the rice (Oryza sativa) and sugarcane (Saccharum hybrid) SUT1 orthologues do not appear to function in phloem loading of sucrose. A SUT1 gene was previously cloned from maize (Zea mays) and shown to have expression and biochemical activity consistent with a hypothesized role in phloem loading. To determine the biological function of SUT1 in maize, a sut1 mutant was isolated and characterized. sut1 mutant plants hyperaccumulate carbohydrates in mature leaves and display leaf chlorosis with premature senescence. In addition, sut1 mutants have greatly reduced stature, altered biomass partitioning, delayed flowering, and stunted tassel development. Cold-girdling wild-type leaves to block phloem transport phenocopied the sut1 mutants, supporting a role for maize SUT1 in sucrose export. Furthermore, application of 14C-sucrose to abraded sut1 mutant and wild-type leaves showed that sucrose export was greatly diminished in sut1 mutants compared with wild type. Collectively, these data demonstrate that SUT1 is crucial for efficient phloem loading of sucrose in maize leaves. PMID:19181865

  4. Characterization of Three Functional High-Affinity Ammonium Transporters in Lotus japonicus with Differential Transcriptional Regulation and Spatial Expression1

    PubMed Central

    D'Apuzzo, Enrica; Rogato, Alessandra; Simon-Rosin, Ulrike; El Alaoui, Hicham; Barbulova, Ani; Betti, Marco; Dimou, Maria; Katinakis, Panagiotis; Marquez, Antonio; Marini, Anne-Marie; Udvardi, Michael K.; Chiurazzi, Maurizio

    2004-01-01

    Ammonium is a primary source of nitrogen for plants. In legume plants ammonium can also be obtained by symbiotic nitrogen fixation, and \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document} is also a regulator of early and late symbiotic interaction steps. Ammonium transporters are likely to play important roles in the control of nodule formation as well as in nitrogen assimilation. Two new genes, LjAMT1;2 and LjAMT1;3, were cloned from Lotus japonicus. Both were able to complement the growth defect of a yeast (Saccharomyces cerevisiae) ammonium transport mutant. Measurement of [14C]methylammonium uptake rates and competition experiments revealed that each transporter had a high affinity for \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{NH}}_{4}^{+}\\end{equation*}\\end{document}. The Ki for ammonium was 1.7, 3, and 15 μm for LjAMT1;1, 1;2, and 1;3, respectively. Real-time PCR revealed higher expression of LjAMT1;1, 1;2, and 1;3 genes in leaves than in roots and nodule, with expression levels decreasing in the order LjAMT1;1 > 1;2 > 1;3 except in flowers, in which LjAMT1;3 was expressed at higher level than in leaves, and LjAMT1;1 showed the lowest level of expression. Expression of LjAMT1;1 and 1;2 in roots was induced by nitrogen deprivation. Expression of LjAMT1;1 was repressed in leaves exposed to elevated CO2 concentrations, which also suppress photorespiration. Tissue and cellular localization of LjAMT1 genes expression, using promoter-β-glucuronidase and in situ RNA hybridization approaches, revealed distinct cellular spatial

  5. Molecular identification and functional analysis of a maize (Zea mays) DUR3 homolog that transports urea with high affinity.

    PubMed

    Liu, Guo-Wei; Sun, Ai-Li; Li, Di-Qin; Athman, Asmini; Gilliham, Matthew; Liu, Lai-Hua

    2015-04-01

    Successful molecular cloning and functional characterization of a high-affinity urea permease ZmDUR3 provide convincing evidence of ZmDUR3 roles in root urea acquisition and internal urea-N-remobilization of maize plants. Urea occurs ubiquitously in both soils and plants. Being a major form of nitrogen fertilizer, large applications of urea assist cereals in approaching their genetic yield potential, but due to the low nitrogen-use efficiency of crops, this practice poses a severe threat to the environment through their hypertrophication. To date, except for paddy rice, little is known about the biological basis for urea movement in dryland crops. Here, we report the molecular and physiological characterization of a maize urea transporter, ZmDUR3. We show using gene prediction, PCR-based cloning and yeast complementation, that a functional full-length cDNA encoding a 731 amino acids-containing protein with putative 15 transmembrane α-helixes for ZmDUR3 was successfully cloned. Root-influx studies using (15)N-urea demonstrated ZmDUR3 catalyzes urea transport with a K m at ~9 µM when expressed in the Arabidopsis dur3-mutant. qPCR analysis revealed that ZmDUR3 mRNA in roots was significantly upregulated by nitrogen depletion and repressed by reprovision of nitrogen after nitrogen starvation, indicating that ZmDUR3 is a nitrogen-responsive gene and relevant to plant nitrogen nutrition. Moreover, detection of higher urea levels in senescent leaves and obvious occurrence of ZmDUR3 transcripts in phloem-cells of mature/aged leaves strongly implies a role for ZmDUR3 in urea vascular loading. Significantly, expression of ZmDUR3 complemented atdur3-mutant of Arabidopsis, improving plant growth on low urea and increasing urea acquisition. As it also targets to the plasma membrane, our data suggest that ZmDUR3 functions as an active urea permease playing physiological roles in effective urea uptake and nitrogen remobilization in maize.

  6. Monomeric TonB and the Ton box are required for the Formation of a High-Affinity Transporter-TonB Complex†

    PubMed Central

    Freed, Daniel M.; Lukasik, Stephen M.; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S.

    2013-01-01

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In the present study, a soluble construct of Escherichia coli TonB (residues 33–239) was used to determine the affinity of TonB to the outer membrane transporters BtuB, FecA and FhuA. Using fluorescence anisotropy, TonB(33–239) was found to bind with high-affinity (tens of nM) to both BtuB and FhuA; however, no high-affinity binding was observed to FecA. In BtuB, the high affinity binding of TonB(33–239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33–239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33–239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When bound to the outer membrane transporter, DEER shows that the TonB(33–239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle. PMID:23517233

  7. Monomeric TonB and the Ton box are required for the formation of a high-affinity transporter-TonB complex.

    PubMed

    Freed, Daniel M; Lukasik, Stephen M; Sikora, Arthur; Mokdad, Audrey; Cafiso, David S

    2013-04-16

    The energy-dependent uptake of trace nutrients by Gram-negative bacteria involves the coupling of an outer membrane transport protein to the transperiplasmic protein TonB. In this study, a soluble construct of Escherichia coli TonB (residues 33-239) was used to determine the affinity of TonB for outer membrane transporters BtuB, FecA, and FhuA. Using fluorescence anisotropy, TonB(33-239) was found to bind with high affinity (tens of nanomolar) to both BtuB and FhuA; however, no high-affinity binding to FecA was observed. In BtuB, the high-affinity binding of TonB(33-239) was eliminated by mutations in the Ton box, which yield transport-defective protein, or by the addition of a Colicin E3 fragment, which stabilizes the Ton box in a folded state. These results indicate that transport requires a high-affinity transporter-TonB interaction that is mediated by the Ton box. Characterization of TonB(33-239) using double electron-electron resonance (DEER) demonstrates that a significant population of TonB(33-239) exists as a dimer; moreover, interspin distances are in approximate agreement with interlocked dimers observed previously by crystallography for shorter TonB fragments. When the TonB(33-239) dimer is bound to the outer membrane transporter, DEER shows that the TonB(33-239) dimer is converted to a monomeric form, suggesting that a dimer-monomer conversion takes place at the outer membrane during the TonB-dependent transport cycle.

  8. Sucrose transporters in plants: update on function and structure.

    PubMed

    Lemoine, R

    2000-05-01

    In plants, sucrose is the major transport form for photoassimilated carbon and is both a source of carbon skeletons and energy for plant organs unable to perform photosynthesis (sink organs). As a molecule translocated over distance, sucrose has to pass through a number of membranes. Membrane transport of sucrose has therefore been considered for a long time as a major determinant of plant productivity. After several decades of physiological and biochemical experiments measuring the activity of sucrose carriers, unequivocal evidence came from the first identification of a cDNA coding a sucrose carrier (SoSUT1, Riesmeier et al. (1992) EMBO J. 11, 4705-4713). At present 20 different cDNAs encoding sucrose carriers have been identified in different plant species, in both dicots and monocots (one case). The total number is increasing rapidly and most importantly, it can be guessed from the results obtained for Arabidopsis, that in each species, sucrose transporters represent a gene family. The sequences are highly conserved and those carriers display the typical 12 transmembrane alpha-helices of members of the Major Facilitator superfamily. Yeast expression of those carriers indicate that they are all influx carriers, all cotransport sucrose and proton and that their affinity for sucrose is surprisingly similar (0.2-2 mM). All their characteristics are in agreement with those demonstrated at the physiological level in plants. These characteristics are discussed in relation to the function in plants and the few data available on the structure of those transporters in relation to their function are presented.

  9. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1

    PubMed Central

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  10. Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake.

    PubMed

    Winterberg, Markus; Rajendran, Esther; Baumeister, Stefan; Bietz, Sven; Kirk, Kiaran; Lingelbach, Klaus

    2012-04-12

    Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.

  11. Zinc Deficiency Up-Regulates Expression of High-Affinity Phosphate Transporter Genes in Both Phosphate-Sufficient and -Deficient Barley Roots1

    PubMed Central

    Huang, Chunyuan; Barker, Susan Jane; Langridge, Peter; Smith, Frank W.; Graham, Robin David

    2000-01-01

    Phosphate (P) is taken up by plants through high-affinity P transporter proteins embedded in the plasma membrane of certain cell types in plant roots. Expression of the genes that encode these transporters responds to the P status of the plants, and their transcription is normally tightly controlled. However, this tight control of P uptake is lost under Zn deficiency, leading to very high accumulation of P in plants. We examined the effect of plant Zn status on the expression of the genes encoding the HVPT1 and HVPT2 high-affinity P transporters in barley (Hordeum vulgare L. cv Weeah) roots. The results show that the expression of these genes is intimately linked to the Zn status of the plants. Zn deficiency induced the expression of genes encoding these P transporters in plants grown in either P-sufficient or -deficient conditions. Moreover, the role of Zn in the regulation of these genes is specific in that it cannot be replaced by manganese (a divalent cation similar to Zn). It appears that Zn plays a specific role in the signal transduction pathway responsible for the regulation of genes encoding high-affinity P transporters in plant roots. The significance of Zn involvement in the regulation of genes involved in P uptake is discussed. PMID:10982454

  12. Platelets as potential peripheral markers to study functioning of the high-affinity sodium-dependent glutamate transporters in the nerve terminals of the brain

    NASA Astrophysics Data System (ADS)

    Borisova, T. A.; Kasatkina, L. A.

    Activity of the high-affinity sodium-dependent glutamate transporters in the brain nerve terminals is demonstrated to alter under artificial gravity conditions. A comparison analysis is made for L-[14C] glutamate transport in platelets and isolated nerve terminals. The kinetic characteristics of the transporters, [Na+]-dependence and influence of the transpoter inhibitor DL-threo-beta-benzyloxyaspartate on the L-[14C] glutamate uptake process are determined. It is shown that glutamate uptake process is very similar for platelets and nerve terminals. Thus it is reasonable to use platelets as a potential peripheral model for glutamate transport.

  13. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision.

    PubMed

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-11-16

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants.

  14. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision

    PubMed Central

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-01-01

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants. PMID:27849020

  15. Sucrose transport into developing seeds of Pisum sativum L.

    PubMed

    Tegeder, M; Wang, X D; Frommer, W B; Offler, C E; Patrick, J W

    1999-04-01

    The anatomy of developing pea seeds is characterized by transfer cells present in both coats and cotyledons at the maternal/filial interface. To determine the nature and cellular localization of sucrose transporters in pea seeds, a full-length clone of a sucrose/H+ symporter (PsSUT1) was isolated from a cotyledon cDNA library. Northern blot analyses of different organs showed that PsSUT1 is expressed in non-seed tissues, including sucrose sinks and sources. Within developing seeds, transcripts of PsSUT1 and PsAHA1 genes were detected in all tissues, while transcripts of a sucrose binding protein (GmSBP) were confined to cotyledon epidermal transfer cells. Signal intensities of PsSUT1 and PsAHA1 transcripts and protein products were most pronounced in the thin-walled parenchyma cells of seed coats and epidermal transfer cells of cotyledons. For cotyledons, the highest transporter densities were localized to those portions of plasma membranes lining the wall ingrowth regions of epidermal transfer cells. Responses of [14C]sucrose influx to metabolic inhibitors indicated that proton-coupled sucrose transport was operative in both seed coats and cotyledons. Cotyledon epidermal transfer cells were shown to support the highest sucrose flux. Maximal transport activity was found to account for the sucrose flux differences between seed tissues. Intercellular movement of the symplasmic tracer, 5-(6)-carboxyfluorescein (CF), demonstrated that symplasmic pathways interconnect the vascular tissues to thin-walled parenchyma transfer cells of seed coats and, for cotyledons, epidermal transfer cells to storage parenchyma cells.

  16. Expression of high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae is both repressed and induced by glucose and appears to be regulated posttranslationally.

    PubMed Central

    Wendell, D L; Bisson, L F

    1994-01-01

    Expression of putative high-affinity glucose transport protein Hxt2p of Saccharomyces cerevisiae was repressed 15- to 20-fold in high concentrations of glucose or fructose. S. cerevisiae with either the ssn6-delta 9 or the hxk2-delta 1::URA3 mutation, each of which relieves glucose repression, exhibited high Hxt2p expression in both 2.0% glucose (normally repressing) and 0.05% glucose (normally derepressing) while S. cerevisiae with the snf1-delta 10 mutation, which causes constitutive repression, did not detectably express Hxt2p in either glucose concentration. In addition to repressing at high concentrations, glucose or fructose is required for induction of Hxt2p expression. Hxt2p was not expressed by wild-type S. cerevisiae in media containing only ethanol or galactose as carbon and energy source but was expressed if glucose was added. An hxk2-delta 1::URA3 mutant did not detectably express Hxt2p in ethanol or galactose, but an ssn6-delta9 mutant did highly express Hxt2p in both carbon sources. Thus, simple relief of glucose repression as occurs with hxk2 null mutants is insufficient for high-level Hxt2p expression. Mutation of ssn6, a general transcriptional repressor, does lead to Hxt2p expression in the absence of glucose induction, suggesting relief of an additional negative regulatory system. High expression of Hxt2p does not always result in HXT2-dependent high-affinity transport, implying that Hxt2p activity is regulated posttranslationally. In the high glucose condition for the ssn6 mutant, high-affinity glucose transport is derepressed. Deletion of the HXT2 locus does not diminish this level of transport. However, high-affinity glucose transport is diminished in the ssn6-delta9 hxt2 delta1 double mutant compared with ssn6-delta9 alone in low glucose. Thus, while constitutively expressed in ssn6 mutants, Hxt2p only appears to be active as a transporter under low-glucose conditions. Similarly, Hxt2p was found to be expressed under low-glucose conditions

  17. Stem sugar accumulation in sweet sorghum - activity and expression of sucrose metabolizing enzymes and sucrose transporters.

    PubMed

    Qazi, Hilal Ahmad; Paranjpe, Sharayu; Bhargava, Sujata

    2012-04-15

    Sugar metabolism was studied in sweet sorghum (SSV74) that is known to store sugars in the mature internodes and which is reported to give grain yields twice that of a grain sorghum variety (SPV1616). Comparison of sugar accumulation in these two varieties was carried out at three stages of growth and in the upper and lower internodes. In spite of large differences in the level of sugar accumulation, osmolarity of the sap did not vary as significantly in the two varieties. Significant contribution of variety, stage and internode position was seen for the variation observed in sugar content. Though the activities of sugar metabolizing enzymes namely sucrose synthase (in the synthesis and cleavage directions), sucrose phosphate synthase and invertase (cytoplasmic and vacuolar) also varied in a stage- and internode-specific manner in the two varieties, these enzymes did not contribute significantly to the variation observed in sugar content. Transcriptional expression of one sucrose synthase (SUC1), two sucrose phosphate synthase (SPS2 and SPS3) and a vacuolar invertase (INV3) gene were lower in sweet sorghum as compared to grain sorghum. Sweet sorghum also showed lower expression of two sucrose transporters (SUT1 and SUT4), which correlated to higher sugar accumulation in this variety. Differential expression of the sugar metabolizing enzymes and sucrose transporters in sweet and grain sorghum suggest a role for signaling molecules and transcription factors in regulating sugar accumulation observed in the mature internodes of sweet sorghum, which needs to be investigated. Copyright © 2012 Elsevier GmbH. All rights reserved.

  18. Maltose and Maltodextrin Transport in the Thermoacidophilic Gram-Positive Bacterium Alicyclobacillus acidocaldarius Is Mediated by a High-Affinity Transport System That Includes a Maltose Binding Protein Tolerant to Low pH

    PubMed Central

    Hülsmann, Anja; Lurz, Rudi; Scheffel, Frank; Schneider, Erwin

    2000-01-01

    We have studied the uptake of maltose in the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius, which grows best at 57°C and pH 3.5. Under these conditions, accumulation of [14C]maltose was observed in cells grown with maltose but not in those grown with glucose. At lower temperatures or higher pH values, the transport rates substantially decreased. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The kinetic parameters (Km of 0.91 ± 0.06 μM and Vmax ranging from 0.6 to 3.7 nmol/min/mg of protein) are consistent with a binding protein-dependent ATP binding cassette (ABC) transporter. A corresponding binding protein (MalE) that interacts with maltose with high affinity (Kd of 1.5 μM) was purified from the culture supernatant of maltose-grown cells. Immunoelectron microscopy revealed distribution of the protein throughout the cell wall. The malE gene was cloned and sequenced. Five additional open reading frames, encoding components of a maltose transport system (MalF and MalG), a putative transcriptional regulator (MalR), a cyclodextrinase (CdaA), and an α-glucosidase (GlcA), were identified downstream of malE. The malE gene lacking the DNA sequence that encodes the signal sequence was expressed in Escherichia coli. The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80°C. Recombinant MalE cross-reacted with an antiserum raised against the wild-type protein, thereby indicating that the latter is the product of the malE gene. The MalE protein might be well suited as a model to study tolerance of proteins to low pH. PMID:11053372

  19. Identification of the High-affinity Substrate-binding Site of the Multidrug and Toxic Compound Extrusion (MATE) Family Transporter from Pseudomonas stutzeri*

    PubMed Central

    Nie, Laiyin; Grell, Ernst; Malviya, Viveka Nand; Xie, Hao; Wang, Jingkang; Michel, Hartmut

    2016-01-01

    Multidrug and toxic compound extrusion (MATE) transporters exist in all three domains of life. They confer multidrug resistance by utilizing H+ or Na+ electrochemical gradients to extrude various drugs across the cell membranes. The substrate binding and the transport mechanism of MATE transporters is a fundamental process but so far not fully understood. Here we report a detailed substrate binding study of NorM_PS, a representative MATE transporter from Pseudomonas stutzeri. Our results indicate that NorM_PS is a proton-dependent multidrug efflux transporter. Detailed binding studies between NorM_PS and 4′,6-diamidino-2-phenylindole (DAPI) were performed by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and spectrofluorometry. Two exothermic binding events were observed from ITC data, and the high-affinity event was directly correlated with the extrusion of DAPI. The affinities are about 1 μm and 0.1 mm for the high and low affinity binding, respectively. Based on our homology model of NorM_PS, variants with mutations of amino acids that are potentially involved in substrate binding, were constructed. By carrying out the functional characterization of these variants, the critical amino acid residues (Glu-257 and Asp-373) for high-affinity DAPI binding were determined. Taken together, our results suggest a new substrate-binding site for MATE transporters. PMID:27235402

  20. The Organization of High-Affinity Ammonium Uptake in Arabidopsis Roots Depends on the Spatial Arrangement and Biochemical Properties of AMT1-Type Transporters[W

    PubMed Central

    Yuan, Lixing; Loqué, Dominique; Kojima, Soichi; Rauch, Sabine; Ishiyama, Keiki; Inoue, Eri; Takahashi, Hideki; von Wirén, Nicolaus

    2007-01-01

    The AMMONIUM TRANSPORTER (AMT) family comprises six isoforms in Arabidopsis thaliana. Here, we describe the complete functional organization of root-expressed AMTs for high-affinity ammonium uptake. High-affinity influx of 15N-labeled ammonium in two transposon-tagged amt1;2 lines was reduced by 18 to 26% compared with wild-type plants. Enrichment of the AMT1;2 protein in the plasma membrane and localization of AMT1;2 promoter activity in the endodermis and root cortex indicated that AMT1;2 mediates the uptake of ammonium entering the root via the apoplasmic transport route. An amt1;1 amt1;2 amt1;3 amt2;1 quadruple mutant (qko) showed severe growth depression under ammonium supply and maintained only 5 to 10% of wild-type high-affinity ammonium uptake capacity. Transcriptional upregulation of AMT1;5 in nitrogen-deficient rhizodermal and root hair cells and the ability of AMT1;5 to transport ammonium in yeast suggested that AMT1;5 accounts for the remaining uptake capacity in qko. Triple and quadruple amt insertion lines revealed in vivo ammonium substrate affinities of 50, 234, 61, and 4.5 μM for AMT1;1, AMT1;2, AMT1;3, and AMT1;5, respectively, but no ammonium influx activity for AMT2;1. These data suggest that two principle means of achieving effective ammonium uptake in Arabidopsis roots are the spatial arrangement of AMT1-type ammonium transporters and the distribution of their transport capacities at different substrate affinities. PMID:17693533

  1. Assignment of the gene coding for the human high-affinity glutamate transporter EAAC1 to 9p24: Potential role in dicarboxylic aminoaciduria and neurodegenerative disorders

    SciTech Connect

    Smith, C.P.; Kanai, Y.; Stelzner, M.; Hediger, M.A.; Weremowicz, S.; Morton, C.C. )

    1994-03-15

    Functional defects of high-affinity glutamate transporters have been implicated in the pathophysiology of neurodegenerative diseases such as amyotrophic lateral sclerosis. In small intestine and kidney, in which the high-affinity glutamate transporter mediates net absorption of glutamate and aspartate across epithelial cells, an inborn error of glutamate transport is thought to cause dicarboxylic aminoaciduria. This disorder is characterized by increased urinary excretion of glutamate and aspartate and is, in general, associated with neurologic and developmental abnormalities. Recently, the authors isolated a cDNA encoding a high-affinity glutamate transporter (EAAC1) that also transports aspartate but not other amino acids. EAAC1 is ubiquitously expressed throughout the body, particularly in brain (neurons), intestine, and kidney. Here, the authors present mapping of the chromosome location of EAAC1 using Southern analysis of a panel of human/rodent somatic cell hybrids and fluorescence in situ hybridization (FISH). Southern analysis of EcoRI-digested DNA gave bands at 6.5, 5.6, 5.1, and 1.2 kb for human genomic DNA; 7.5 kb for mouse genomic DNA; and 7.3, 3.2, and 1 kb for hamster genomic DNA. All four human EAAC1-specific bands were observed in the lane corresponding to the human/Chinese hamster hybrid containing chromosome 9 but not in lanes corresponding to any other hybrid. Because the human/Chinese hamster hybrid is the only one retaining chromosome 9, this result unambiguously assigns human EAAC1 to chromosome 9. For precise chromosome assignment of the human EAAC1 gene, they employed FISH. Map position of the EAAC1 probe was assigned by visual inspection of the fluorescent signal on the DAPI-stained metaphase chromosomes. The human EAAC1 gene was assigned to 9p24.

  2. Interleukin-2 and concanavalin A upregulate a cat2 isoform encoding a high affinity L-arginine transporter in feline lymphocytes.

    PubMed Central

    Stevens, B R; Tellier, M; Harvey, W; Feldman, D H; Bosworth, J

    2000-01-01

    The immunological responses of activated lymphocytes are associated with increased nitric oxide (NO) biosynthesis. Studies in the literature have primarily approached control of NO by focusing on the regulation of the nitric oxide synthase (NOS) isoforms. However, the present study approaches the control of NO synthesis by addressing the regulation of L-arginine availability to lymphocytes via regulation of membrane transport. The guanidino nitrogen of L-arginine is the sole biosynthetic precursor of NO. We investigated cytokine and mitogen regulation of membrane L-arginine transporters for the first time in feline cells. Feline peripheral blood mononuclear cells were treated with interleukin-2 and concanavalin A, then alternatively spliced isoforms of L-arginine transporters known in other species were probed by RT-PCR, using various oligonucleotide primers that hybridized to several regions in common with the isoforms. Both high affinity and low affinity isoforms are encoded by mRNAs arising from mutually exclusive alternative splicing of the primary transcript. A region of 123 bp was obtained that encoded an extracellular polypeptide loop of 41 amino acids. The sequence of this region represented the high affinity L-arginine substrate binding site of a CAT2 transporter polypeptide isoform, but not the CAT2a isoform low affinity binding site. Neither of the inducible isoforms were constitutively expressed in unstimulated feline cells. This is the first report demonstrating that domestic cats possess the cat2 gene encoding an inducible L-arginine transporter, and, furthermore, that the high affinity isoform transcript is activated by interleukin-2 and concanavalin A in feline lymphocytes. Images Figure 1. Figure 3. PMID:10935886

  3. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    PubMed Central

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  4. Rapid Up-Regulation of HKT1, a High-Affinity Potassium Transporter Gene, in Roots of Barley and Wheat following Withdrawal of Potassium1

    PubMed Central

    Wang, Tie-Bang; Gassmann, Walter; Rubio, Francisco; Schroeder, Julian I.; Glass, Anthony D.M.

    1998-01-01

    High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied. These processes make important contributions to plant K+ homeostasis. A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized. We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media. This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels. Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+. Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected. Resupply of 1 mm K+ was sufficient to strongly reduce HKT1 transcript levels. In wheat root cortical cells, both membrane depolarizations in response to 100 μm K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation. Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression. Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed. PMID:9765551

  5. Physiological and Biochemical Characterization of AnNitA, the Aspergillus nidulans High-Affinity Nitrite Transporter

    PubMed Central

    Unkles, Shiela E.; Symington, Vicki F.; Kotur, Zorica; Wang, Ye; Siddiqi, M. Yaeesh; Kinghorn, James R.; Glass, Anthony D. M.

    2011-01-01

    High-affinity nitrite influx into mycelia of Aspergillus nidulans has been characterized by use of 13NO2−, giving average Km and Vmax values of 48 ± 8 μM and 228 ± 49 nmol mg−1 dry weight (DW) h−1, respectively. Kinetic analysis of a plot that included an additional large number of low-concentration fluxes gave an excellent monophasic fit (r2 = 0.96), with no indication of sigmoidal kinetics. Two-dimensional (2D) and three-dimensional (3D) models of AnNitA are presented, and the possible roles of conserved asparagine residues N122 (transmembrane domain 3 ]Tm 3]), N173 (Tm 4), N214 (Tm 5), and N246 (Tm 6) are discussed. PMID:22021238

  6. Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

    NASA Astrophysics Data System (ADS)

    Borisova, T.; Himmelreich, N.

    Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na+-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na+ elucidate further the mechanism underlying Ca2+-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca2+-free Na+-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na+ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the

  7. Pharmacological characterization of N,N-dimethyl-2-(2-amino-4-methylphenyl thio)benzylamine as a ligand of the serotonin transporter with high affinity and selectivity.

    PubMed

    Chalon, Sylvie; Tarkiainen, Jari; Garreau, Lucette; Hall, Hakan; Emond, Patrick; Vercouillie, Johnny; Farde, Lars; Dasse, Philippe; Varnas, Katarina; Besnard, Jean-Claude; Halldin, Christer; Guilloteau, Denis

    2003-01-01

    Serotonin transporter has a key-role in regulation of serotoninergic function, and is involved in numerous neurodegenerative and psychiatric disorders. To obtain an efficient radioactive ligand allowing the study of this transporter in vitro and in vivo, we synthesized a new diphenyl sulfide derivative, N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine or MADAM. We present here extensive pharmacological characterization of this compound. [3H]MADAM bound to serotonin transporters with a very high affinity in vitro on rat cortical membranes, at least 2 times better than the most commonly used radioactive probes (Kd, 60 pM; Bmax, 543 fmol/mg of protein). Competition studies showed few inhibitory effect of nisoxetine (Ki = 270 nM), no inhibitory effect of desipramine or 1-[2-(diphenylmethoxy) ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) (Ki >1000 nM), and strong effect of paroxetine (Ki = 0.32 nM) and citalopram (Ki = 1.57 nM). Therefore, MADAM has around 1000-fold better selectivity for the serotonin transporter than for other transporters. Autoradiographic studies both on rat and postmortem human brain slices demonstrated that the distribution of [3H]MADAM parallels the localization of serotonin transporters and is prevented by known inhibitors of them. The high affinity and selectivity of [3H]MADAM for the serotonin transporter show that it is very valuable for studies using in vitro approaches. The high selectivity and low nonspecific binding of [3H]MADAM on the postmortem human brain, together with preliminary in vivo results with [11C]MADAM, is a new argument for future use of this ligand in in vivo studies of the distribution, pharmacology, and pathophysiology of the serotonin transporter in the human brain with positron emission tomography.

  8. The sea urchin embryo as a model for mammalian developmental neurotoxicity: ontogenesis of the high-affinity choline transporter and its role in cholinergic trophic activity.

    PubMed

    Qiao, Dan; Nikitina, Lyudmila A; Buznikov, Gennady A; Lauder, Jean M; Seidler, Frederic J; Slotkin, Theodore A

    2003-11-01

    Embryonic development in the sea urchin requires trophic actions of the same neurotransmitters that participate in mammalian brain assembly. We evaluated the development of the high-affinity choline transporter, which controls acetylcholine synthesis. A variety of developmental neurotoxicants affect this transporter in mammalian brain. [3H]Hemicholinium-3 binding to the transporter was found in the cell membrane fraction at stages from the unfertilized egg to pluteus, with a binding affinity comparable with that seen in mammalian brain. Over the course of development, the concentration of transporter sites rose more than 3-fold, achieving concentrations comparable with those of cholinergically enriched mammalian brain regions. Dimethylaminoethanol (DMAE), a competitive inhibitor of choline transport, elicited dysmorphology beginning at the mid-blastula stage, with anomalies beginning progressively later as the concentration of DMAE was lowered. Pretreatment, cotreatment, or delayed treatment with acetylcholine or choline prevented the adverse effects of DMAE. Because acetylcholine was protective at a lower threshold, the DMAE-induced defects were most likely mediated by its effects on acetylcholine synthesis. Transient removal of the hyaline layer enabled a charged transport inhibitor, hemicholinium-3, to penetrate sufficiently to elicit similar anomalies, which were again prevented by acetylcholine or choline. These results indicate that the developing sea urchin possesses a high-affinity choline transporter analogous to that found in the mammalian brain, and, as in mammals, the functioning of this transporter plays a key role in the developmental, trophic activity of acetylcholine. The sea urchin model may thus be useful in high-throughput screening of suspected developmental neurotoxicants.

  9. Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

    PubMed Central

    Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

    1995-01-01

    The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

  10. Sucrose transporters in two members of the Scrophulariaceae with different types of transport sugar.

    PubMed

    Knop, C; Voitsekhovskaja, O; Lohaus, G

    2001-05-01

    In order to study differences between sugar transport in oligosaccharide-translocating and sucrose-translocating species, two members of the Scrophulariaceae, Asarina barclaiana Pennell and Alonsoa meridionalis O. Kuntze, were analysed regarding minor-vein anatomy, sugar concentrations in leaves and phloem sap, and expression of sucrose transporters. The minor veins of Asarina barclaiana possess mainly transfer cells and modified intermediary cells and those of Alonsoa meridionalis have intermediary cells and ordinary companion cells. Phloem sap from these plants was collected by the laser-aphid-stylet technique. The main carbon transport forms in Asarina were sucrose and in Alonsoa raffinose and stachyose. The sum of the carbohydrate concentrations in the phloem sap of both species was as high as that in apoplastic phloem loaders. In Asarina the ratio of the sucrose concentration in the phloem to that in the cytosol of source cells was about 35 and the corresponding ratio in Alonsoa was about two. Sucrose transporter cDNAs were isolated from leaves of both species. By means of semi-quantitative reverse transcription-polymerase chain reaction, sucrose transporter mRNA was detected in different organs and also in the phloem sap. This is the first time that sucrose transporters have been found in oligosaccharide-translocating species and that the mRNA of these sucrose transporters has been localized directly in the phloem sap. Taken together, our observations indicate that Asarina is an apoplastic phloem loader, while the results for Alonsoa are ambiguous: some properties are typical of the symplastic phloem-loading mechanism, but probably a sucrose transporter is involved in loading and/or retrieval of sucrose into the phloem.

  11. Sucrose- and H+-Dependent Charge Movements Associated with the Gating of Sucrose Transporter ZmSUT1

    PubMed Central

    Carpaneto, Armando; Koepsell, Hermann; Bamberg, Ernst; Hedrich, Rainer; Geiger, Dietmar

    2010-01-01

    Background In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly proton-driven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features. Methodology/Principal Findings To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependent-potassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I = Q/τ) was sufficient to predict ZmSUT1 transport-associated currents. Conclusions Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to

  12. Capacity and Plasticity of Potassium Channels and High-Affinity Transporters in Roots of Barley and Arabidopsis1[C][W

    PubMed Central

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Oh, Saehong; Kronzucker, Herbert J.

    2013-01-01

    The role of potassium (K+) transporters in high- and low-affinity K+ uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of 42K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K+ influx at low external K+ concentration ([K+]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K+ channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K+ influx in barley, indicating a shift from active to passive K+ uptake at low [K+]ext and yielding fluxes as high as 36 µmol g (root fresh weight)−1 h−1 at 5 mm [K+]ext, among the highest transporter-mediated K+ fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K+]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K+ efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K+ accumulation. We discuss key differences and similarities in K+ acquisition between two important model systems and reveal novel aspects of K+ transport in planta. PMID:23553635

  13. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

    SciTech Connect

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.

  14. Sucrose accumulation in sweet sorghum stems occurs by apoplasmic phloem unloading and does not involve differential Sucrose transporter expression.

    PubMed

    Bihmidine, Saadia; Baker, R Frank; Hoffner, Cassandra; Braun, David M

    2015-07-30

    Sorghum (Sorghum bicolor L. Moench) cultivars store non-structural carbohydrates predominantly as either starch in seeds (grain sorghums) or sugars in stems (sweet sorghums). Previous research determined that sucrose accumulation in sweet sorghum stems was not correlated with the activities of enzymes functioning in sucrose metabolism, and that an apoplasmic transport step may be involved in stem sucrose accumulation. However, the sucrose unloading pathway from stem phloem to storage parenchyma cells remains unelucidated. Sucrose transporters (SUTs) transport sucrose across membranes, and have been proposed to function in sucrose partitioning differences between sweet and grain sorghums. The purpose of this study was to characterize the key differences in carbohydrate accumulation between a sweet and a grain sorghum, to define the path sucrose may follow for accumulation in sorghum stems, and to determine the roles played by sorghum SUTs in stem sucrose accumulation. Dye tracer studies to determine the sucrose transport route revealed that, for both the sweet sorghum cultivar Wray and grain sorghum cultivar Macia, the phloem in the stem veins was symplasmically isolated from surrounding cells, suggesting sucrose was apoplasmically unloaded. Once in the phloem apoplasm, a soluble tracer diffused from the vein to stem parenchyma cell walls, indicating the lignified mestome sheath encompassing the vein did not prevent apoplasmic flux outside of the vein. To characterize carbohydrate partitioning differences between Wray and Macia, we compared the growth, stem juice volume, solute contents, SbSUTs gene expression, and additional traits. Contrary to previous findings, we detected no significant differences in SbSUTs gene expression within stem tissues. Phloem sieve tubes within sweet and grain sorghum stems are symplasmically isolated from surrounding cells; hence, unloading from the phloem likely occurs apoplasmically, thereby defining the location of the previously

  15. Rice DUR3 mediates high-affinity urea transport and plays an effective role in improvement of urea acquisition and utilization when expressed in Arabidopsis.

    PubMed

    Wang, Wei-Hong; Köhler, Barbara; Cao, Feng-Qiu; Liu, Guo-Wei; Gong, Yuan-Yong; Sheng, Song; Song, Qi-Chao; Cheng, Xiao-Yuan; Garnett, Trevor; Okamoto, Mamoru; Qin, Rui; Mueller-Roeber, Bernd; Tester, Mark; Liu, Lai-Hua

    2012-01-01

    • Despite the great agricultural and ecological importance of efficient use of urea-containing nitrogen fertilizers by crops, molecular and physiological identities of urea transport in higher plants have been investigated only in Arabidopsis. • We performed short-time urea-influx assays which have identified a low-affinity and high-affinity (K(m) of 7.55 μM) transport system for urea-uptake by rice roots (Oryza sativa). • A high-affinity urea transporter OsDUR3 from rice was functionally characterized here for the first time among crops. OsDUR3 encodes an integral membrane-protein with 721 amino acid residues and 15 predicted transmembrane domains. Heterologous expression demonstrated that OsDUR3 restored yeast dur3-mutant growth on urea and facilitated urea import with a K(m) of c. 10 μM in Xenopus oocytes. • Quantitative reverse-transcription polymerase chain reaction (qPCR) analysis revealed upregulation of OsDUR3 in rice roots under nitrogen-deficiency and urea-resupply after nitrogen-starvation. Importantly, overexpression of OsDUR3 complemented the Arabidopsis atdur3-1 mutant, improving growth on low urea and increasing root urea-uptake markedly. Together with its plasma membrane localization detected by green fluorescent protein (GFP)-tagging and with findings that disruption of OsDUR3 by T-DNA reduces rice growth on urea and urea uptake, we suggest that OsDUR3 is an active urea transporter that plays a significant role in effective urea acquisition and utilisation in rice. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  16. Blockade of the high-affinity noradrenaline transporter (NET) by the selective 5-HT reuptake inhibitor escitalopram: an in vivo microdialysis study in mice

    PubMed Central

    Nguyen, Hai T; Guiard, Bruno P; Bacq, Alexandre; David, Denis J; David, Indira; Quesseveur, Gaël; Gautron, Sophie; Sanchez, Connie; Gardier, Alain M

    2013-01-01

    BACKGROUND AND PURPOSE Escitalopram, the S(+)-enantiomer of citalopram is the most selective 5-HT reuptake inhibitor approved. Although all 5-HT selective reuptake inhibitors (SSRIs) increase extracellular levels of 5-HT ([5-HT]ext). some also enhance, to a lesser extent, extracellular levels of noradrenaline ([NA]ext). However, the mechanisms by which SSRIs activate noradrenergic transmission in the brain remain to be determined. EXPERIMENTAL APPROACH This study examined the effects of escitalopram, on both [5-HT]ext and [NA]ext in the frontal cortex (FCx) of freely moving wild-type (WT) and mutant mice lacking the 5-HT transporter (SERT−/−) by using intracerebral microdialysis. We explored the possibilities that escitalopram enhances [NA]ext, either by a direct mechanism involving the inhibition of the low- or high-affinity noradrenaline transporters, or by an indirect mechanism promoted by [5-HT]ext elevation. The forced swim test (FST) was used to investigate whether enhancing cortical [5-HT]ext and/or [NA]ext affected the antidepressant-like activity of escitalopram. KEY RESULTS In WT mice, a single systemic administration of escitalopram produced a significant increase in cortical [5-HT]ext and [NA]ext. As expected, escitalopram failed to increase cortical [5-HT]ext in SERT−/− mice, whereas its neurochemical effects on [NA]ext persisted in these mutants. In WT mice subjected to the FST, escitalopram increased swimming parameters without affecting climbing behaviour. Finally, escitalopram, at relevant concentrations, failed to inhibit cortical noradrenaline and 5-HT uptake mediated by low-affinity monoamine transporters. CONCLUSIONS AND IMPLICATIONS These experiments suggest that escitalopram enhances, although moderately, cortical [NA]extin vivo by a direct mechanism involving the inhibition of the high-affinity noradrenaline transporter (NET). PMID:22233336

  17. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  18. FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter*

    PubMed Central

    Boswell-Casteel, Rebba C.; Johnson, Jennifer M.; Duggan, Kelli D.; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A.

    2014-01-01

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  19. Arabidopsis INOSITOL TRANSPORTER4 Mediates High-Affinity H+ Symport of Myoinositol across the Plasma Membrane1

    PubMed Central

    Schneider, Sabine; Schneidereit, Alexander; Konrad, Kai R.; Hajirezaei, Mohammad-Reza; Gramann, Monika; Hedrich, Rainer; Sauer, Norbert

    2006-01-01

    Four genes of the Arabidopsis (Arabidopsis thaliana) monosaccharide transporter-like superfamily share significant homology with transporter genes previously identified in the common ice plant (Mesembryanthemum crystallinum), a model system for studies on salt tolerance of higher plants. These ice plant transporters had been discussed as tonoplast proteins catalyzing the inositol-dependent efflux of Na+ ions from vacuoles. The subcellular localization and the physiological role of the homologous proteins in the glycophyte Arabidopsis were unclear. Here we describe Arabidopsis INOSITOL TRANSPORTER4 (AtINT4), the first member of this subgroup of Arabidopsis monosaccharide transporter-like transporters. Functional analyses of the protein in yeast (Saccharomyces cerevisiae) and Xenopus laevis oocytes characterize this protein as a highly specific H+ symporter for myoinositol. These activities and analyses of the subcellular localization of an AtINT4 fusion protein in Arabidopsis and tobacco (Nicotiana tabacum) reveal that AtINT4 is located in the plasma membrane. AtINT4 promoter-reporter gene plants demonstrate that AtINT4 is strongly expressed in Arabidopsis pollen and phloem companion cells. The potential physiological role of AtINT4 is discussed. PMID:16603666

  20. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    PubMed Central

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence. PMID:23691177

  1. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis.

    PubMed

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-04-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway.

  2. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis

    PubMed Central

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-01-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose–H+ symporter. Analyses of STP10 mRNA and STP10 promoter–reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  3. ATP binding and hydrolysis disrupt the high-affinity interaction between the heme ABC transporter HmuUV and its cognate substrate-binding protein.

    PubMed

    Qasem-Abdullah, Hiba; Perach, Michal; Livnat-Levanon, Nurit; Lewinson, Oded

    2017-09-01

    Using the energy of ATP hydrolysis, ABC transporters catalyze the trans-membrane transport of molecules. In bacteria, these transporters partner with a high-affinity substrate-binding protein (SBP) to import essential micronutrients. ATP binding by Type I ABC transporters (importers of amino acids, sugars, peptides, and small ions) stabilizes the interaction between the transporter and the SBP, thus allowing transfer of the substrate from the latter to the former. In Type II ABC transporters (importers of trace elements, e.g. vitamin B12, heme, and iron-siderophores) the role of ATP remains debatable. Here we studied the interaction between the Yersinia pestis ABC heme importer (HmuUV) and its partner substrate-binding protein (HmuT). Using real-time surface plasmon resonance experiments and interaction studies in membrane vesicles, we find that in the absence of ATP the transporter and the SBP tightly bind. Substrate in excess inhibits this interaction, and ATP binding by the transporter completely abolishes it. To release the stable docked SBP from the transporter hydrolysis of ATP is required. Based on these results we propose a mechanism for heme acquisition by HmuUV-T where the substrate-loaded SBP docks to the nucleotide-free outward-facing conformation of the transporter. ATP binding leads to formation of an occluded state with the substrate trapped in the trans-membrane translocation cavity. Subsequent ATP hydrolysis leads to substrate delivery to the cytoplasm, release of the SBP, and resetting of the system. We propose that other Type II ABC transporters likely share the fundamentals of this mechanism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Modulation of high affinity ATP-dependent cyclic nucleotide transporters by specific and non-specific cyclic nucleotide phosphodiesterase inhibitors.

    PubMed

    Aronsen, Lena; Orvoll, Elin; Lysaa, Roy; Ravna, Aina W; Sager, Georg

    2014-12-15

    Intracellular cyclic nucleotides are eliminated by phosphodiesterases (PDEs) and by ATP Binding cassette transporters such as ABCC4 and ABCC5. PDE5 and ABCC5 have similar affinity for cGMP whereas ABCC5 has much higher affinity for cGMP compared with cAMP. Since the substrate (cGMP) is identical for these two eliminatory processes it is conceivable that various PDE inhibitors also modulate ABCC5-transport. Cyclic GMP is also transported by ABBC4 but the affinity is much lower with a Km 50-100 times higher than for that of ABBCC5. The present study aimed to determine Ki-values for specific or relative specific PDE5 inhibitors (vardenafil, tadalafil, zaprinast and dipyridamole) and the non-specific PDE inhibitors (IBMX, caffeine and theophylline) for ABCC5 and ABCC4 transport. The transport of [(3)H]-cGMP (2 µM) was concentration-dependently inhibited with the following Ki-values: vardenafil (0.62 µM), tadalafil (14.1 µM), zaprinast (0.68 µM) and dipyridamole (1.2 µM), IBMX (10 µM), caffeine (48 µM) and theophylline (69 µM). The Ki-values for the inhibition of the [(3)H]-cAMP (2 µM) transport were: vardenafil (3.4 µM), tadalafil (194 µM), zaprinast (2.8 µM), dipyridamole (5.5 µM), IBMX (16 µM), caffeine (41 µM) and theophylline (85 µM). The specificity for ABCC5 we defined as ratio between Ki-values for inhibition of [(3)H]-cGMP and [(3)H]-cAMP transport. Tadalafil showed the highest specificity (Ki-ratio: 0.073) and caffeine the lowest (Ki-ratio: 1.2). Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Genetic and biochemical characterization of a high-affinity betaine uptake system (BusA) in Lactococcus lactis reveals a new functional organization within bacterial ABC transporters.

    PubMed

    Obis, D; Guillot, A; Gripon, J C; Renault, P; Bolotin, A; Mistou, M Y

    1999-10-01

    The cytoplasmic accumulation of exogenous betaine stimulates the growth of Lactococcus lactis cultivated under hyperosmotic conditions. We report that L. lactis possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters. Through transposon mutagenesis, a mutant deficient in betaine transport was isolated. We identified two genes, busAA and busAB, grouped in an operon, busA (betaine uptake system). The transcription of busA is strongly regulated by the external osmolality of the medium. The busAA gene codes for the ATP-binding protein. busAB encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the Bacillus subtilis betaine-binding protein, OpuAC. BusA of L. lactis displays a high affinity towards betaine (K(m) = 1.7 microM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of L. lactis to be under dual control at the biochemical and genetic levels. A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of L. lactis. The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.

  6. Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions

    PubMed Central

    Kaler, Gregory; Truong, David M.; Sweeney, Derina E.; Logan, Darren W.; Nagle, Megha; Eraly, Satish A.; Nigam, Sanjay K.

    2007-01-01

    We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss. PMID:17094945

  7. The Diamidine Diminazene Aceturate Is a Substrate for the High-Affinity Pentamidine Transporter: Implications for the Development of High Resistance Levels in TrypanosomesS⃞

    PubMed Central

    Teka, Ibrahim A.; Kazibwe, Anne J. N.; El-Sabbagh, Nasser; Al-Salabi, Mohammed I.; Ward, Christopher P.; Eze, Anthonius A.; Munday, Jane C.; Mäser, Pascal; Matovu, Enock; Barrett, Michael P.

    2011-01-01

    African trypanosomiasis is a disease of humans and livestock in many areas south of the Sahara. Resistance to the few existing drugs is a major impediment to the control of these diseases, and we investigated how resistance to the main veterinary drug diminazene aceturate correlates with changes in drug transport in resistant strains. The strain tbat1(−/−), lacking the TbAT1/P2 aminopurine transporter implicated previously in diminazene transport, was adapted to higher levels of diminazene resistance. The resulting cell line was designated ABR and was highly cross-resistant to other diamidines and moderately resistant to cymelarsan. Procyclic trypanosomes were shown to be a convenient model to study diamidine uptake in Trypanosoma brucei brucei given the lack of TbAT1/P2 and a 10-fold higher activity of the high-affinity pentamidine transporter (HAPT1). Diminazene could be transported by HAPT1 in procyclic trypanosomes. This drug transport activity was lacking in the ABR line, as reported previously for the pentamidine-adapted line B48. The Km for diminazene transport in bloodstream tbat1(−/−) trypanosomes was consistent with uptake by HAPT1. Diminazene transport in ABR and B48 cells was reduced compared with tbat1(−/−), but their resistance phenotype was different: B48 displayed higher levels of resistance to pentamidine and the melaminophenyl arsenicals, whereas ABR displayed higher resistance to diminazene. These results establish a loss of HAPT1 function as a contributing factor to diminazene resistance but equally demonstrate for the first time that adaptations other than those determining the initial rates of drug uptake contribute to diamidine and arsenical resistance in African trypanosomes. PMID:21436312

  8. Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

    PubMed

    Chan, Helen; Zwingmann, Claudia; Pannunzio, Marc; Butterworth, Roger F

    2003-07-01

    Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at

  9. pH, Lactate, and Hypoxia: Reciprocity in Regulating High-Affinity Monocarboxylate Transporter Expression in Glioblastoma.

    PubMed

    Caruso, James P; Koch, Brandon J; Benson, Philip D; Varughese, Elsa; Monterey, Michael D; Lee, Amy E; Dave, Ajal M; Kiousis, Sam; Sloan, Andrew E; Mathupala, Saroj P

    2017-02-01

    Highly malignant brain tumors harbor the aberrant propensity for aerobic glycolysis, the excessive conversion of glucose to lactic acid even in the presence of ample tissue oxygen. Lactic acid is rapidly effluxed to the tumor microenvironment via a group of plasma-membrane transporters denoted monocarboxylate transporters (MCTs) to prevent "self-poisoning." One isoform, MCT2, has the highest affinity for lactate and thus should have the ability to respond to microenvironment conditions such as hypoxia, lactate, and pH to help maintain high glycolytic flux in the tumor. Yet, MCT2 is considered to not respond to hypoxia, which is counterintuitive. Its response to tumor lactate has not been reported. In this report, we experimentally identify the transcription initiation site/s for MCT2 in astrocytes (normal) and glioma (tumor). We then use a BACmid library to isolate a 4.2-kbp MCT2 promoter-exon I region and examine promoter response to glycolysis-mediated stimuli in glioma cells. Reporter analysis of nested-promoter constructs indicated response of MCT2 to hypoxia, pH, lactate, and glucose, the major physiological "players" that facilitate a tumor's growth and proliferation. Immunoblot analysis of native MCT2 expression under altered pH and hypoxia reflected the reporter data. The pH-mediated gene-regulation studies we describe are the first to record H(+)-based reporter studies for any mammalian system and demonstrate the exquisite response of the MCT2 gene to minute changes in tumor pH. Identical promoter usage also provides the first evidence of astrocytes harnessing the same gene regulatory regions to facilitate astrocyte-neuron lactate shuttling, a metabolic feature of normal brain. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2.

    PubMed

    He, Xiao; Garza, Denisse; Nigam, Sanjay K; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates--in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1-prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M(-1) s(-1) and an even slower dissociation rate (kd) in the range of 10-4 s(-1) for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1--which is normally expressed in liver and other tissues--interacts with prostaglandin analogs. While it is not

  11. Multispecific Organic Cation Transporter 1 (OCT1) from Bos taurus Has High Affinity and Slow Binding Kinetics towards Prostaglandin E2

    PubMed Central

    He, Xiao; Garza, Denisse; Nigam, Sanjay K.; Chang, Geoffrey

    2016-01-01

    Organic cation transporter 1 (OCT1, SLC22A1), like many solute carrier 22 (SLC22) family members, is important for the disposition of clinically important drugs, metabolites and signaling molecules. Several studies suggest that SLC22 family (eg. organic anion transporters or OATs and OCTs) bind and possibly transport prostaglandins with relatively high affinity (submicromolar). The affinities of OCT1 and OATs toward PGE2 and PGF2a reported in these cell-based transport studies are considerably greater than for xenobiotics and natural metabolite substrates—in many cases over 100-fold higher. This raises the possibility that prostaglandins are key endogenous substrates and/or that they act on the transporter in a manner different from other substrates such as xenobiotics and lower affinity metabolites. To further investigate OCT1—prostaglandin interactions, we designed biophysical studies using purified bovine OCT1 (Bos taurus, btOCT1/SLC22A1) with PGE2 analogs, in fluorescently labeled and label-free formats. Using fluorescence polarization (FP), we detected a binding of btOCT1 to the PGE2-Rhodamine conjugate at submicromolar affinity, consistent with affinity data for PGE2 from cells over-expressing the related human OCT1. Using purified native btOCT1 as analyte and biotinylated PGE2 analog as ligand, our data from surface plasmon resonance (SPR) revealed that btOCT1 specifically interacts to PGE2 with KD values in the hundred nanomolar range. BtOCT1 also demonstrated a slow association (ka) in the range of 103 M-1s-1 and an even slower dissociation rate (kd) in the range of 10−4 s-1 for PGE2, suggesting the possibility of a different mode of binding compared to other structurally unrelated transported substrates of low-affinity (eg. drugs, metabolites). Our results complement in vitro transport studies and provide direct evidence that OCT1—which is normally expressed in liver and other tissues—interacts with prostaglandin analogs. While it is not

  12. Components of the Vid30c are needed for the rapamycin-induced degradation of the high-affinity hexose transporter Hxt7p in Saccharomyces cerevisiae.

    PubMed

    Snowdon, Chris; Hlynialuk, Chris; van der Merwe, George

    2008-03-01

    Saccharomyces cerevisiae adapts to changing nutrient conditions by regulating its genome-wide transcription profile and cell-wide protein complement in correlation with the reigning nutrient conditions. The target of rapamycin (TOR) signalling pathway is one of the major control mechanisms within the cell that facilitates these changes. The transcription, intracellular trafficking, and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are regulated in response to nutrient conditions. The Vid and Gid proteins facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Three members of the VID and GID gene families, VID30/GID1, GID2, and VID28/GID5 are needed for the rapamycin or nitrogen starvation-induced degradation of the high-affinity hexose transporter Hxt7p is shown here. In addition, evidence that the functions of several Vid and Gid proteins are in close relation to the TOR signalling pathway is provided.

  13. [Structure-functional organization of eukaryotic high-affinity copper importer CTR1 determines its ability to transport copper, silver and cisplatin].

    PubMed

    Skvortsov, A N; Zatulovskiĭ, E A; Puchkova, L V

    2012-01-01

    It was shown recently, that high affinity Cu(I) importer eukaryotic protein CTR1 can also transport in vitro abiogenic Ag(I) ions and anticancer drug cisplatin. At present there is no rational explanation how CTR1 can transfer platinum group, which is different by coordination properties from highly similar Cu(I) and Ag(I). To understand this phenomenon we analyzed 25 sequences of chordate CTR1 proteins, and found out conserved patterns of organization of N-terminal extracellular part of CTR1 which correspond to initial metal binding. Extracellular copper-binding motifs were qualified by their coordination properties. It was shown that relative position of Met- and His-rich copper-binding motifs in CTR1 predisposes the extracellular CTR1 part to binding of copper, silver and cisplatin. Relation between tissue-specific expression of CTR1 gene, steady-state copper concentration, and silver and platinum accumulation in organs of mice in vivo was analyzed. Significant positive but incomplete correlation exists between these variables. Basing on structural and functional peculiarities of N-terminal part of CTR1 a hypothesis of coupled transport of copper and cisplatin has been suggested, which avoids the disagreement between CTR1-mediated cisplatin transport in vitro, and irreversible binding of platinum to Met-rich peptides.

  14. Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT).

    PubMed

    Hosford, Patrick S; Millar, Julian; Ramage, Andrew G

    2015-04-01

    The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12-50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg(-1) ). However, decynium-22 (600 μg kg(-1) ), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not noradrenaline, whereas desipramine had the reverse action. The OCT3 inhibitor corticosterone (10 mg kg(-1) ) had no effect. Blockade of glutamate receptors with topical kynurenate (10-50 nm) reduced the vagally evoked 5-HT signal by 50%, indicating that this release was from at least two sources. It is concluded that vagally evoked 5-HT release is under the regulation of the high-capacity, low-affinity transporter PMAT, not the low-capacity, high-affinity transporter SERT. This is the first demonstration that PMAT may be playing a physiological role in the regulation of 5-HT transmission and this could indicate that 5-HT is acting, in part, as a volume transmitter within the NTS. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  15. Cardiovascular afferents cause the release of 5-HT in the nucleus tractus solitarii; this release is regulated by the low- (PMAT) not the high-affinity transporter (SERT)

    PubMed Central

    Hosford, Patrick S; Millar, Julian; Ramage, Andrew G

    2015-01-01

    Key points The nucleus tractus solitarii (NTS) integrates visceral afferent information essential for cardiovascular haemostasis. Using fast-cyclic voltammetry in anaesthetized rats, 5-HT (serotonin) release was detected in NTS in response to activation of these afferents. Removal of 5-HT from the extracellular space is usually regulated by the low-capacity, high-affinity 5-HT transporter (5-HTT/SERT). The present data demonstrate that 5-HT removal in the NTS is regulated by the plasma membrane monoamine transporter (PMAT), a high-capacity, low-affinity transporter. The present data also demonstrate that the 5-HT released by afferent activation comes from at least two different sources. It is suggested that one of these sources is the afferents themselves. These results demonstrate a physiological role for the low-affinity uptake transporter in the regulation of 5-HT concentration in NTS. Abstract The nucleus tractus solitarii (NTS) integrates inputs from cardiovascular afferents and thus is crucial for cardiovascular homeostasis. These afferents primarily release glutamate, although 5-HT has also been shown to play a role in their actions. Using fast-cyclic voltammetry, an increase in 5-HT concentrations (range 12–50 nm) could be detected in the NTS in anaesthetized rats in response to electrical stimulation of the vagus and activation of cardiopulmonary, chemo- and baroreceptor reflexes. This 5-HT signal was not potentiated by the serotonin transporter (SERT) or the noradrenaline transporter (NET) inhibitors citalopram and desipramine (1 mg kg−1). However, decynium-22 (600 μg kg−1), an organic cation 3 transporter (OCT3)/plasma membrane monoamine transporter (PMAT) inhibitor, increased the 5-HT signal by 111 ± 21% from 29 ± 10 nm. The effectiveness of these inhibitors was tested against the removal time of 5-HT and noradrenaline applied by microinjection to the NTS. Citalopram and decynium-22 attenuated the removal of 5-HT but not

  16. The solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, rs3794087 variant and assessment risk for restless legs syndrome.

    PubMed

    Jiménez-Jiménez, Félix Javier; Alonso-Navarro, Hortensia; Martínez, Carmen; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Rojo-Sebastián, Ana; Rubio, Lluisa; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Martín, Elena; Agúndez, José A G

    2014-02-01

    A glutamatergic dysfunction has been postulated to play a role in restless legs syndrome (RLS) pathophysiology, as glutamate concentrations have been found to increase in the thalamus of RLS patients. The aim of our study was to investigate the possible association between the single nucleotide polymorphism (SNP) rs3794087 in the solute carrier family 1 (glial high affinity glutamate transporter), member 2 gene, SLC1A2, related with glutamate transport and the risk for RLS. We studied the allelic and genotype frequencies of the SNP rs3794087 in 205 patients with RLS and 328 healthy controls using TaqMan genotyping. The rs3794087 genotype and allelic frequencies did not significantly differ between patients with RLS and controls and were unrelated with the age at onset of RLS, gender, and family history of RLS. The results of our study suggest that the rs3794087 polymorphism is not related to the risk for RLS. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. The Rice High-Affinity Potassium Transporter1;1 Is Involved in Salt Tolerance and Regulated by an MYB-Type Transcription Factor.

    PubMed

    Wang, Rong; Jing, Wen; Xiao, Longyun; Jin, Yakang; Shen, Like; Zhang, Wenhua

    2015-07-01

    Sodium transporters play key roles in plant tolerance to salt stress. Here, we report that a member of the High-Affinity K(+) Transporter (HKT) family, OsHKT1;1, in rice (Oryza sativa 'Nipponbare') plays an important role in reducing Na(+) accumulation in shoots to cope with salt stress. The oshkt1;1 mutant plants displayed hypersensitivity to salt stress. They contained less Na(+) in the phloem sap and accumulated more Na(+) in the shoots compared with the wild type. OsHKT1;1 was expressed mainly in the phloem of leaf blades and up-regulated in response to salt stress. Using a yeast one-hybrid approach, a novel MYB coiled-coil type transcription factor, OsMYBc, was found to bind to the OsHKT1;1 promoter. In vivo chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that OsMYBc binds to AAANATNC(C/T) fragments within the OsHKT1;1 promoter. Mutation of the OsMYBc-binding nucleotides resulted in a decrease in promoter activity of OsHKT1;1. Knockout of OsMYBc resulted in a reduction in NaCl-induced expression of OsHKT1;1 and salt sensitivity. Taken together, these results suggest that OsHKT1;1 has a role in controlling Na(+) concentration and preventing sodium toxicity in leaf blades and is regulated by the OsMYBc transcription factor.

  18. Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner.

    PubMed

    Huang, Shuangjie; Chen, Si; Liang, Zhihao; Zhang, Chenming; Yan, Ming; Chen, Jingguang; Xu, Guohua; Fan, Xiaorong; Zhang, Yali

    2015-12-08

    The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3(-) transport protein OsNAR2.1 in NO3(-)-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3(-) concentrations, but not under low NH4(+) concentrations. (15)N-labelling NO3(-) supplies (provided at concentrations of 0-10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3(-) concentrations resulted from impaired NO3(-) uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3(-) supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3(-)-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3(-) uptake and NO3(-)-signalling.

  19. Deletion of SLC19A2, the high affinity thiamine transporter, causes selective inner hair cell loss and an auditory neuropathy phenotype.

    PubMed

    Liberman, M C; Tartaglini, E; Fleming, J C; Neufeld, E J

    2006-09-01

    Mutations in the gene coding for the high-affinity thiamine transporter Slc19a2 underlie the clinical syndrome known as thiamine-responsive megaloblastic anemia (TRMA) characterized by anemia, diabetes, and sensorineural hearing loss. To create a mouse model of this disease, a mutant line was created with targeted disruption of the gene. Cochlear function is normal in these mutants when maintained on a high-thiamine diet. When challenged with a low-thiamine diet, Slc19a2-null mice showed 40-60 dB threshold elevations by auditory brainstem response (ABR), but only 10-20 dB elevation by otoacoustic emission (OAE) measures. Wild-type mice retain normal hearing on either diet. Cochlear histological analysis showed a pattern uncommon for sensorineural hearing loss: selective loss of inner hair cells after 1-2 weeks on low thiamine and significantly greater inner than outer hair cell loss after longer low-thiamine challenges. Such a pattern is consistent with the observed discrepancy between ABR and OAE threshold shifts. The possible role of thiamine transport in other reported cases of selective inner hair cell loss is considered.

  20. Aspen SUCROSE TRANSPORTER3 allocates carbon into wood fibers.

    PubMed

    Mahboubi, Amir; Ratke, Christine; Gorzsás, András; Kumar, Manoj; Mellerowicz, Ewa J; Niittylä, Totte

    2013-12-01

    Wood formation in trees requires carbon import from the photosynthetic tissues. In several tree species, including Populus species, the majority of this carbon is derived from sucrose (Suc) transported in the phloem. The mechanism of radial Suc transport from phloem to developing wood is not well understood. We investigated the role of active Suc transport during secondary cell wall formation in hybrid aspen (Populus tremula × Populus tremuloides). We show that RNA interference-mediated reduction of PttSUT3 (for Suc/H(+) symporter) during secondary cell wall formation in developing wood caused thinner wood fiber walls accompanied by a reduction in cellulose and an increase in lignin. Suc content in the phloem and developing wood was not significantly changed. However, after (13)CO2 assimilation, the SUT3RNAi lines contained more (13)C than the wild type in the Suc-containing extract of developing wood. Hence, Suc was transported into developing wood, but the Suc-derived carbon was not efficiently incorporated to wood fiber walls. A yellow fluorescent protein:PttSUT3 fusion localized to plasma membrane, suggesting that reduced Suc import into developing wood fibers was the cause of the observed cell wall phenotype. The results show the importance of active Suc transport for wood formation in a symplasmically phloem-loading tree species and identify PttSUT3 as a principal transporter for carbon delivery into secondary cell wall-forming wood fibers.

  1. Physiological evidence for a sodium-dependent high-affinity phosphate and nitrate transport at the plasma membrane of leaf and root cells of Zostera marina L.

    PubMed

    Rubio, L; Linares-Rueda, A; García-Sánchez, M J; Fernández, J A

    2005-02-01

    Zostera marina L. is an angiosperm that grows in a medium in which inorganic phosphate (P(i)) and nitrate (NO(3)(-)) are present in micromolar concentrations and must be absorbed against a steep electrochemical potential gradient. The operation of a Na(+)-dependent NO(3)(-) transport was previously demonstrated in leaf cells of this plant, suggesting that other Na(+)-coupled systems could mediate the uptake of anions. To address this question, P(i) transport was studied in leaves and roots of Z. marina, as well as NO(3)(-) uptake in roots. Electrophysiological studies demonstrated that micromolar concentrations of P(i) induced depolarizations of the plasma membrane of root cells. However, this effect was not observed in leaf cells. P(i)-induced depolarizations showed Michaelis-Menten kinetics (K(m)=1.5+/-0.6 microM P(i); D(max)=7.8+/-0.8 mV), and were not observed in the absence of Na(+). However, depolarizations were restored when Na(+) was resupplied. NO(3)(-) additions also evoked depolarizations of the plasma membrane of root cells only in the presence of Na(+). Both NO(3)(-)- and P(i)-induced depolarizations were accompanied by an increase in cytoplasmic Na(+) activity, detected by Na(+)-sensitive microelectrodes. P(i) net uptake (measured in depletion experiments) was stimulated by Na(+). These results strongly suggest that P(i) uptake in roots of Z. marina is mediated by a high-affinity Na(+)-dependent transport system. Both NO(3)(-) and P(i) transport systems exploit the steep inwardly directed electrochemical potential gradient for Na(+), considering the low cytoplasmic Na(+) activity (10.7+/-3.3 mM Na(+)) and the high external Na(+) concentration (500 mM Na(+)).

  2. Dual regulation of root hydraulic conductivity and plasma membrane aquaporins by plant nitrate accumulation and high-affinity nitrate transporter NRT2.1.

    PubMed

    Li, Guowei; Tillard, Pascal; Gojon, Alain; Maurel, Christophe

    2016-04-01

    The water status and mineral nutrition of plants critically determine their growth and development. Nitrate (NO3(-)), the primary nitrogen source of higher plants, is known to impact the water transport capacity of roots (root hydraulic conductivity, Lpr). To explore the effects and mode of action of NO3(-) on Lpr, we used an extended set of NO3(-) transport (nrt1.1, nrt1.2, nrt1.5 and nrt2.1), signaling (nrt1.1 and nrt2.1) and metabolism (nia) mutants in Arabidopsis, grown under various NO3(-) conditions. First, a strong positive relationship between Lpr and NO3(-) accumulation, in shoots rather than in roots, was revealed. Secondly, a specific 30% reduction of Lpr in nrt2.1 plants unraveled a major role for the high-affinity NO3(-) transporter NRT2.1 in increasing Lpr These results indicate that NO3(-)signaling rather than nitrogen assimilation products governs Lpr in Arabidopsis. Quantitative real-time reverse transcription-PCR and enzyme-linked immunosorbent assays (ELISAs) were used to investigate the effects of NO3(-) availability on plasma membrane aquaporin (plasma membrane intrinsic protein; PIP) expression. Whereas PIP regulation mostly occurs at the post-translational level in wild-type plants, a regulation of PIPs at both the transcriptional and translational levels was uncovered in nrt2.1 plants. In conclusion, this work reveals that control of Arabidopsis Lpr and PIP functions by NO3(-) involves novel shoot to root signaling and NRT2.1-dependent functions.

  3. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status.

    PubMed

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-04-23

    AMT1-3 encodes the high affinity NH₄⁺ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

  4. Overexpressing of OsAMT1-3, a High Affinity Ammonium Transporter Gene, Modifies Rice Growth and Carbon-Nitrogen Metabolic Status

    PubMed Central

    Bao, Aili; Liang, Zhijun; Zhao, Zhuqing; Cai, Hongmei

    2015-01-01

    AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants. PMID:25915023

  5. Unequal functional redundancy between the two Arabidopsis thaliana high-affinity sulphate transporters SULTR1;1 and SULTR1;2.

    PubMed

    Barberon, Marie; Berthomieu, Pierre; Clairotte, Michael; Shibagaki, Nakako; Davidian, Jean-Claude; Gosti, Françoise

    2008-01-01

    * In Arabidopsis, SULTR1;1 and SULTR1;2 are two genes proposed to be involved in high-affinity sulphate uptake from the soil solution. We address here the specific issue of their functional redundancy for the uptake of sulphate and for the accumulation of its toxic analogue selenate with regard to plant growth and selenate tolerance. * Using the complete set of genotypes, including the wild-type, each one of the single sultr1;1 and sultr1;2 mutants and the resulting double sultr1;1-sultr1;2 mutant, we performed a detailed phenotypic analysis of root length, shoot biomass, sulphate uptake, sulphate and selenate accumulation and selenate tolerance. * The results all ordered the four different genotypes according to the same functional hierarchy. Wild-type and sultr1;1 mutant plants displayed similar phenotypes. By contrast, sultr1;1-sultr1;2 double-mutant plants showed the most extreme phenotype and the sultr1;2 mutant displayed intermediate performances. Additionally, the degree of selenate tolerance was directly related to the seedling selenate content according to a single sigmoid regression curve common to all the genotypes. * The SULTR1;1 and SULTR1;2 genes display unequal functional redundancy, which leaves open for SULTR1;1 the possibility of displaying an additional function besides its role in sulphate membrane transport.

  6. Aspects of sucrose transport in stem parenchyma of sweet sorghum. [Sorghum bicolor

    SciTech Connect

    Lingle, S.E.

    1987-08-01

    Sweet sorghum (Sorghum bicolor (L.) Moench) is a sucrose-storing crop with a storage tissue anatomically similar to that of sugarcane (Saccharum spp.). However, recent evidence suggests that sweet sorghum may be biochemically different from sugarcane. /sup 14/C-sucrose uptake was studied in excised tissue discs from fully-elongated internodes of Rio sweet sorghum. Washout studies gave results consistent with a 3 compartment system. After 3 hours of uptake, most of the /sup 14/C was found in the vacuole compartment, and was determined by HPLC to be sucrose. Total sucrose uptake consisted of a PCMBS-sensitive (active) and a PCMBS-insensitive (passive) component. Active sucrose uptake had a pH optimum of 4.5. Total sucrose uptake was negatively correlated with the internal sucrose content of the tissue. Fructosyl-labelled /sup 14/C-sucrose was not randomized during uptake, suggesting that sucrose cleavage is not a requirement for sucrose uptake in sweet sorghum. This data suggests that in sweet sorghum, sucrose is transported intact by a specific carrier, as opposed to the sucrose-cleavage-and-resynthesis transport system that apparently operates in sugarcane.

  7. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems.

    PubMed

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed.

  8. Tonoplast Sugar Transporters (SbTSTs) putatively control sucrose accumulation in sweet sorghum stems

    PubMed Central

    Bihmidine, Saadia; Julius, Benjamin T; Dweikat, Ismail; Braun, David M

    2016-01-01

    ABSTRACT Carbohydrates are differentially partitioned in sweet versus grain sorghums. While the latter preferentially accumulate starch in the grain, the former primarily store large amounts of sucrose in the stem. Previous work determined that neither sucrose metabolizing enzymes nor changes in Sucrose transporter (SUT) gene expression accounted for the carbohydrate partitioning differences. Recently, 2 additional classes of sucrose transport proteins, Tonoplast Sugar Transporters (TSTs) and SWEETs, were identified; thus, we examined whether their expression tracked sucrose accumulation in sweet sorghum stems. We determined 2 TSTs were differentially expressed in sweet vs. grain sorghum stems, likely underlying the massive difference in sucrose accumulation. A model illustrating potential roles for different classes of sugar transport proteins in sorghum sugar partitioning is discussed. PMID:26619184

  9. Role of the Human High-Affinity Copper Transporter (hCtr1) in Copper Homeostasis Regulation and Cisplatin Sensitivity in Cancer Chemotherapy

    PubMed Central

    Kuo, Macus Tien; Fu, Siqing; Savaraj, Niramol; Chen, Helen H. W.

    2012-01-01

    The high-affinity copper transporter (Ctr1) (SCLC31A1) plays an important role in regulating Cu homeostasis because copper (Cu) is an essential micronutrient and Cu deficiency is detrimental to many important cellular functions but excess Cu is toxic. Recent research has revealed that human Cu homeostasis is tightly controlled by interregulatory circuitry involving Cu, Sp1 and hCtr1. This circuitry uses Sp1 transcription factor as a Cu sensor in modulating hCtr1 expression which in turn controls cellular Cu and Sp1 levels in a three-way mutual regulatory loop. Posttranslational regulation of hCtr1 expression by Cu stresses has also been described in the literature. Because hCtr1 can also transport platinum (Pt) drugs, this finding underscores the important role of hCtr1 in Pt drug sensitivity in cancer chemotherapy. Consistent with this notion is the findings that elevated hCtr1 expression was associated with favorable treatment outcomes in cisplatin-based cancer chemotherapy. Moreover, cultured cell studies demonstrated that elevated hCtr1 expression can be induced by depleting cellular Cu levels, resulting in enhanced cisplatin uptake and its cell killing activity. A phase 1 clinical trial using a combination of trientine (a Cu chelator) and carboplatin has been performed with encouraging results. This review discusses new insights into the role of hCtr1 in regulating Cu homeostasis and explains how modulating cellular Cu availability could influence treatment efficacy in platinum-based cancer chemotherapy through hCtr1 regulation. PMID:22962276

  10. Genomic organization, promoter analysis, and chromosomal localization of the gene for the mouse glial high-affinity glutamate transporter Slc1a3

    SciTech Connect

    Hagiwara, Tatsuya; Tanaka, Kohichi; Maeno-Hikichi, Yuka

    1996-05-01

    The mouse gene encoding glial high-affinity, Na -dependent glutamate transporter Slcla3 (GluT-1/GLAST) was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the mouse genome and comprised 10 exons spanning more than 56 kilobases. The transcription initiation sites were mapped to positions 503, which is the first transcriptional point (defined as +1), 128 (+376), and 64 (+440) basepairs upstream of the 3{prime}-end of exon 1 by primer extension. The 5{prime}-flanking region of the mouse GluT-1 gene had a typical CCAAT box and a GC box but lacked at TATA box. These features of the promoter region were characteristic of housekeeping genes. The fusion plasmids containing approximately 4 kb of the 5{prime}-flanking region (-3830 to +450) and the firefly luciferase gene induced a significant luciferase activity when transfected into COS-1 cells. Distal deletion of the 5{prime}-flanking region, leaving 619 bp (-169 to +450), resulted in a marked decrease in luciferase activity in COS-1 cells, suggesting that a CCAAT box, which was positioned at -200, is necessary for the expression of this gene. In situ hybridization localized this gene. In situ hybridization localized this gene to mouse chromosome 15A2. These structural features will lead to a better understanding of the regulatory mechanism of the expression of the GluT-1 gene by ischemia and will also provide a basis for future evolutionary comparisons with other neurotransmitter transporters. 40 refs., 6 figs., 1 tab.

  11. Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

    PubMed

    Boscari, Alexandre; Van de Sype, Ghislaine; Le Rudulier, Daniel; Mandon, Karine

    2006-08-01

    Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

  12. Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner

    PubMed Central

    Huang, Shuangjie; Chen, Si; Liang, Zhihao; Zhang, Chenming; Yan, Ming; Chen, Jingguang; Xu, Guohua; Fan, Xiaorong; Zhang, Yali

    2015-01-01

    The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3− transport protein OsNAR2.1 in NO3−-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3− concentrations, but not under low NH4+ concentrations. 15N-labelling NO3− supplies (provided at concentrations of 0–10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3− concentrations resulted from impaired NO3− uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3− supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3−-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3− uptake and NO3−-signalling. PMID:26644084

  13. Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

    PubMed Central

    Cheng, Wang; Li, Qiong; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing

    2013-01-01

    Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome. PMID:23951167

  14. LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

    PubMed

    Duan, Guilan; Hakoyama, Tsuneo; Kamiya, Takehiro; Miwa, Hiroki; Lombardo, Fabien; Sato, Shusei; Tabata, Satoshi; Chen, Zheng; Watanabe, Toshihiro; Shinano, Takuro; Fujiwara, Toru

    2017-03-09

    Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp(292) to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nM. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not Mo delivery to the nodules. This article is protected by copyright. All rights reserved.

  15. Differential regulation of two sucrose transporters by defoliation and light conditions in perennial ryegrass.

    PubMed

    Furet, Pierre-Maxime; Berthier, Alexandre; Decau, Marie-Laure; Morvan-Bertrand, Annette; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie; Meuriot, Frédéric

    2012-12-01

    Sucrose transport between source and sink tissues is supposed to be a key-step for an efficient regrowth of perennial rye-grass after defoliation and might be altered by light conditions. We assessed the effect of different light regimes (high vs low light applied before or after defoliation) on growth, fructans and sucrose mobilization, as well as on sucrose transporter expression during 14 days of regrowth. Our results reported that defoliation led to a mobilization of C reserves (first sucrose and then fructans), which was parallel to an induction of LpSUT1 sucrose transporter expression in source and sink tissues (i.e. leaf sheaths and elongating leaf bases, respectively) irrespective to light conditions. Light regime (high or low light) had little effects on regrowth and on C reserves mobilization during the first 48 h of regrowth after defoliation. Thereafter, low light conditions, delaying the recovery of photosynthetic capacities, had a negative effect on C reserves re-accumulation (especially sucrose). Surprisingly, high light did not enhance sucrose transporter expression. Indeed, while light conditions had no effect on LpSUT1 expression, LpSUT2 transcripts levels were enhanced for low light grown plants. These results indicate that two sucrose transporter currently identified in Lolium perenne L. are differentially regulated by light and sucrose.

  16. Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3– High Affinity Transport System in Maize Roots

    PubMed Central

    Pii, Youry; Alessandrini, Massimiliano; Dall’Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms. PMID:27877183

  17. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    PubMed

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  18. Time-Resolved Investigation of Molecular Components Involved in the Induction of [Formula: see text] High Affinity Transport System in Maize Roots.

    PubMed

    Pii, Youry; Alessandrini, Massimiliano; Dall'Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H(+)-ATPase transcripts indicates that those mainly involved in the response to [Formula: see text] treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H(+)-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  19. Overexpression of sucrose transporter gene PbSUT2 from Pyrus bretschneideri, enhances sucrose content in Solanum lycopersicum fruit.

    PubMed

    Wang, Li-Fen; Qi, Xiao-Xiao; Huang, Xiao-San; Xu, Lin-Lin; Jin, Cong; Wu, Jun; Zhang, Shao-Ling

    2016-08-01

    Sucrose transporters (SUTs) belong to the major facilitator superfamily. The function of SUTs has been intensively investigated in some higher plants, whereas that in pear fruit is unknown. In this study, the cloning and functional characterization of a sucrose transporter, PbSUT2, in pear (Pyrus bretschneideri Rehd. cv. 'Yali') fruits are reported. PbSUT2 encoded a protein of 498 amino acid residues, and was localized in the plasma membrane of transformed onion epidermal cells and Arabidopsis protoplasts. Phylogenetic analysis revealed that PbSUT2 belonged to the SUT4 clade. The phenotype of overexpression of PbSUT2 tomato plants included early flowering, higher fruit quantity and lower plant height. Overexpression of PbSUT2 in transgenic tomato plants led to increases in the net photosynthetic rate in leaves and sucrose content in mature fruit compared with wild-type tomato plants, and a decrease in the contents of glucose, fructose and total soluble sugars in mature fruits. These results suggested that PbSUT2 affected sucrose content in sinks and the flowering phase during tomato plant growth and development. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Molecular cloning and functional characterization of the apple sucrose transporter gene MdSUT2.

    PubMed

    Ma, Qi-Jun; Sun, Mei-Hong; Liu, Ya-Jing; Lu, Jing; Hu, Da-Gang; Hao, Yu-Jin

    2016-12-01

    Sucrose is not only the primary photosynthetic product but also the major component translocated in the phloem of economically important plant species. Sucrose transporters or carriers (SUTs or SUCs), function as sucrose/H(+) symporters and play a crucial role in determining the cell-to-cell distribution of sucrose throughout the entire plant. However, whether such genes are involved in responses to abiotic stress and other biological processes is largely unknown. Here, we report that MdSUT2 in apple is a homolog of the Arabidopsis vacuolar sucrose transporter AtSUT2. Ectopic expression of MdSUT2 in Arabidopsis decreased sucrose sensitivity in germination and seeding stage and increased sucrose transport activity. In addition, our results showed that MdSUT2 impacted on plant growth by accelerating vegetative growth and promoting early flowering in Arabidopsis. Overexpression of MdSUT2 significantly improved abiotic stress tolerance including NaCl, ABA, and mannitol in apple calli and Arabidopsis. Together, these findings provide evidence that the apple sucrose transporter MdSUT2 is involved in abiotic stress resistance and the regulation of plant growth and development. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  1. High-affinity glutamate transporter and glutamine synthetase content in longissimus dorsi and adipose tissues of growing Angus steers differs among suckling, weanling, backgrounding, and finishing production stages.

    PubMed

    Matthews, J C; Huang, J; Rentfrow, G

    2016-03-01

    Skeletal muscle and adipose tissues play important roles in maintaining whole-body Glu and N homeostasis by the uptake of Glu and release of Gln. To test the hypothesis that expression of high-affinity Glu transporters (GLAST1, EAAT4, EAAC1, GLT-1) and glutamine synthetase (GS) would increase in longissimus dorsi and adipose tissue of newborn Angus steers randomly assigned ( = 6) to develop through suckling (S; 32 d) and/or weanling (W; 184 d), backgrounding (B; 248 d), and finishing (F; 423 d) production stages. Carcass quality was determined at slaughter to verify shifts in adipose and lean deposition with development. Expression of mRNA (RT-PCR/Southern) and relative protein abundance (Western analysis) were determined in tissue homogenates isolated from longissimus dorsi, and kidney and subcutaneous adipose. The effect of production stage or tissue type on carcass and protein abundance was assessed by 1-way ANOVA using the GLM procedure of SAS, and Fisher's protected LSD procedure was used to separate data means. Neither GLAST1 nor EAAT4 mRNA or protein was detected. EAAC1, GLT-1, and GS mRNA were identified in all tissues, but GLT-1 and GS protein were not detected in kidney or subcutaneous adipose, and GS protein was not detected in longissimus dorsi. The EAAC1 content of subcutaneous ( = 0.06) and kidney ( = 0.02) adipose was 2 times greater in B and F than W steers, whereas GS was 5 times greater ( < 0.07) in B than F steers (B = W > F). For longissimus dorsi, EAAC1 ( < 0.01) and GLT-1 ( < 0.04) content decreased with development (S > W > B = F, S = W > B = F, respectively). Within F steers, EAAC1 and GLT-1 mRNA was expressed by subcutaneous, kidney, omental, mesenchymal, and intramuscular adipose tissues, whereas GS mRNA was expressed by all except for intramuscular. Only EAAC1 protein was detected in any adipose tissue, with EAAC1 content being 104% and 112% greater ( < 0.01) in intramuscular than in kidney or subcutaneous adipose, respectively, and not

  2. Transport and sorting of the solanum tuberosum sucrose transporter SUT1 is affected by posttranslational modification.

    PubMed

    Krügel, Undine; Veenhoff, Liesbeth M; Langbein, Jennifer; Wiederhold, Elena; Liesche, Johannes; Friedrich, Thomas; Grimm, Bernhard; Martinoia, Enrico; Poolman, Bert; Kühn, Christina

    2008-09-01

    The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed.

  3. Sucrose-Metabolizing Enzymes in Transport Tissues and Adjacent Sink Structures in Developing Citrus Fruit 1

    PubMed Central

    Lowell, Cadance A.; Tomlinson, Patricia T.; Koch, Karen E.

    1989-01-01

    Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import

  4. Sucrose-metabolizing enzymes in transport tissues and adjacent sink structures in developing citrus fruit.

    PubMed

    Lowell, C A; Tomlinson, P T; Koch, K E

    1989-08-01

    Juice tissues of citrus lack phloem; therefore, photosynthates enroute to juice sacs exit the vascular system on the surface of each segment. Areas of extensive phloem unloading and transport (vascular bundles + segment epidermis) can thus be separated from those of assimilate storage (juice sacs) and adjacent tissues where both processes occur (peel). Sugar composition, dry weight accumulation, and activities of four sucrose-metabolizing enzymes (soluble and cell-wall-bound acid invertase, alkaline invertase, sucrose synthase, and sucrose phosphate synthase) were measured in these transport and sink tissues of grapefruit (Citrus paradisi Macf.) to determine more clearly whether a given enzyme appeared to be more directly associated with assimilate transport versus deposition or utilization. Results were compared at three developmental stages. Activity of sucrose (per gram fresh weight and per milligram protein) extracted from zones of extensive phloem unloading and transport was significantly greater than from adjacent sink tissues during the stages (II and III) when juice sacs grow most rapidly. In stage II fruit, activity of sucrose synthase also significantly surpassed that of all other sucrose-metabolizing enzymes in extracts from the transport tissues (vascular bundles + segment epidermis). In contrast, sucrose phosphate synthase and alkaline invertase at this stage of growth were the most active enzymes from adjacent, rapidly growing, phloem-free sink tissues (juice sacs). Activity of these two enzymes in extracts from juice sacs was significantly greater than that form the transport tissues (vascular bundles + segment epidermis). Soluble acid invertase was the most active enzyme in extracts from all tissues of very young fruit (stage I), including nonvascular regions, but nearly disappeared prior to the onset of juice sac sugar accumulation. The physiological function of high sucrose synthase activity in the transport tissues during rapid sucrose import

  5. Downregulating the sucrose transporter VpSUT1 in Verbascum phoeniceum does not inhibit phloem loading

    PubMed Central

    Zhang, Cankui; Turgeon, Robert

    2009-01-01

    Sucrose is loaded into the phloem in the minor veins of leaves before export. Two active, species-specific loading mechanisms have been proposed. One involves transporter-mediated sucrose transfer from the apoplast into the sieve element-companion cell complex, so-called apoplastic loading. In the putative second mechanism, sucrose follows an entirely symplastic pathway, and the solute concentration is elevated by the synthesis of raffinose and stachyose in the phloem, not by transporter activity. Several sucrose-transporting plants have been shown to be apoplastic loaders by downregulating sucrose transporter 1 (SUT1), leading to accumulation of sugars and leaf chlorosis. In this study we compared the effect of downregulating SUT1 in Nicotiana tabacum, a sucrose transporter, and Verbascum phoeniceum, a species that transports raffinose and stachyose. To test the effectiveness of RNAi downregulation, we measured SUT1 mRNA levels and sucrose-H+ symport in leaf discs. Mild NtSUT1 downregulation in N. tabacum resulted in the pronounced phenotype associated with loading inhibition. In contrast, no such phenotype developed when VpSUT1 was downregulated in V. phoeniceum, leaving minimal sucrose transport activity. Only those plants with the most severe VpSUT1 downregulation accumulated more carbohydrate than usual and these plants were normal by other criteria: growth rate, photosynthesis, and ability to clear starch during the night. The results provide direct evidence that the mechanism of phloem loading in V. phoeniceum does not require active sucrose uptake from the apoplast and strongly supports the conclusion that the loading pathway is symplastic in this species. PMID:19846784

  6. Evidence for the Transport of Maltose by the Sucrose Permease, CscB, of Escherichia coli

    PubMed Central

    Peng, Yang; Kumar, Sanath; Hernandez, Ricardo L.; Jones, Suzanna E.; Cadle, Kathleen M.; Smith, Kenneth P.; Varela, Manuel F.

    2009-01-01

    The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure “downhill” maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation (“uphill” transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport and permease structure should consider maltose, as well as sucrose. PMID:19294451

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

    PubMed

    Ludwig, A; Stolz, J; Sauer, N

    2000-11-01

    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.

  8. Post-translational regulation of sucrose transporters by direct protein–protein interactions

    PubMed Central

    Krügel, Undine; Kühn, Christina

    2013-01-01

    Sucrose transporters are essential membrane proteins for the allocation of carbon resources in higher plants and protein–protein interactions play a crucial role in the post-translational regulation of sucrose transporters affecting affinity, transport capacity, oligomerization, localization, and trafficking. Systematic screening for protein interactors using sucrose transporters as bait proteins helped identifying several proteins binding to sucrose transporters from apple, Arabidopsis, potato, or tomato using the split ubiquitin system. This mini-review summarizes known sucrose transporter-interacting proteins and their potential function in plants. Not all of the identified interaction partners are postulated to be located at the plasma membrane, but some are predicted to be endoplasmic reticulum-residing proteins such as a protein disulfide isomerase and members of the cytochrome b5 family. Many of the SUT1-interacting proteins are secretory proteins or involved in metabolism. Identification of actin and actin-related proteins as SUT1-interacting proteins confirmed the observation that movement of SUT1-containing intracellular vesicles can be blocked by inhibition of actin polymerization using specific inhibitors. Manipulation of expression of these interacting proteins represents one possible way to modify resource allocation by post-translational regulation of sucrose transporters. PMID:23847641

  9. Are sucrose transporter expression profiles linked with patterns of biomass partitioning in Sorghum phenotypes?

    PubMed Central

    Milne, Ricky J.; Byrt, Caitlin S.; Patrick, John W.; Grof, Christopher P. L.

    2013-01-01

    Sorghum bicolor is a genetically diverse C4 monocotyledonous species, encompassing varieties capable of producing high grain yields as well as sweet types which accumulate soluble sugars (predominantly sucrose) within their stems to high concentrations. Sucrose produced in leaves (sources) enters the phloem and is transported to regions of growth and storage (sinks). It is likely that sucrose transporter (SUT) proteins play pivotal roles in phloem loading and the delivery of sucrose to growth and storage sinks in all Sorghum ecotypes. Six SUTs are present in the published Sorghum genome, based on the BTx623 grain cultivar. Homologues of these SUTs were cloned and sequenced from the sweet cultivar Rio, and compared with the publically available genome information. SbSUT5 possessed nine amino acid sequence differences between the two varieties. Two of the remaining five SUTs exhibited single variations in their amino acid sequences (SbSUT1 and SbSUT2) whilst the rest shared identical sequences. Complementation of a mutant Saccharomyces yeast strain (SEY6210), unable to grow upon sucrose as the sole carbon source, demonstrated that the Sorghum SUTs were capable of transporting sucrose. SbSUT1, SbSUT4, and SbSUT6 were highly expressed in mature leaf tissues and hence may contribute to phloem loading. In contrast, SbSUT2 and SbSUT5 were expressed most strongly in sinks consistent with a possible role of facilitating sucrose import into stem storage pools and developing inflorescences. PMID:23805151

  10. Are sucrose transporter expression profiles linked with patterns of biomass partitioning in Sorghum phenotypes?

    PubMed

    Milne, Ricky J; Byrt, Caitlin S; Patrick, John W; Grof, Christopher P L

    2013-01-01

    Sorghum bicolor is a genetically diverse C4 monocotyledonous species, encompassing varieties capable of producing high grain yields as well as sweet types which accumulate soluble sugars (predominantly sucrose) within their stems to high concentrations. Sucrose produced in leaves (sources) enters the phloem and is transported to regions of growth and storage (sinks). It is likely that sucrose transporter (SUT) proteins play pivotal roles in phloem loading and the delivery of sucrose to growth and storage sinks in all Sorghum ecotypes. Six SUTs are present in the published Sorghum genome, based on the BTx623 grain cultivar. Homologues of these SUTs were cloned and sequenced from the sweet cultivar Rio, and compared with the publically available genome information. SbSUT5 possessed nine amino acid sequence differences between the two varieties. Two of the remaining five SUTs exhibited single variations in their amino acid sequences (SbSUT1 and SbSUT2) whilst the rest shared identical sequences. Complementation of a mutant Saccharomyces yeast strain (SEY6210), unable to grow upon sucrose as the sole carbon source, demonstrated that the Sorghum SUTs were capable of transporting sucrose. SbSUT1, SbSUT4, and SbSUT6 were highly expressed in mature leaf tissues and hence may contribute to phloem loading. In contrast, SbSUT2 and SbSUT5 were expressed most strongly in sinks consistent with a possible role of facilitating sucrose import into stem storage pools and developing inflorescences.

  11. Sugarcane ShSUT1: analysis of sucrose transport activity and inhibition by sucralose.

    PubMed

    Reinders, Anke; Sivitz, Alicia B; Hsi, Alex; Grof, Christopher P L; Perroux, Jai M; Ward, John M

    2006-10-01

    Plant sucrose transporters (SUTs) are members of the glycoside-pentoside-hexuronide (GPH) cation symporter family (TC2.A.2) that is part of the major facilitator superfamily (MFS). All plant SUTs characterized to date function as proton-coupled symporters and catalyze the cellular uptake of sucrose. SUTs are involved in loading sucrose into the phloem and sink tissues, such as seeds, roots and flowers. Because monocots are agriculturally important, SUTs from cereals have been the focus of recent research. Here we present a functional analysis of the SUT ShSUT1 from sugarcane, an important crop species grown for its ability to accumulate high amounts of sucrose in the stem. ShSUT1 was previously shown to be expressed in maturing stems and plays an important role in the accumulation of sucrose in this tissue. Using two-electrode voltage clamping in Xenopus oocytes expressing ShSUT1, we found that ShSUT1 is highly selective for sucrose, but has a relatively low affinity for sucrose (K(0.5) = 8.26 mM at pH 5.6 and a membrane potential of -137 mV). We also found that the sucrose analog sucralose (4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose) is a competitive inhibitor of ShSUT1 with an inhibition coefficient (K(i)) of 16.5 mM. The presented data contribute to our understanding of sucrose transport in plants in general and in monocots in particular.

  12. The Trehalose Phosphotransferase System (PTS) in E. coli W Can Transport Low Levels of Sucrose that Are Sufficient to Facilitate Induction of the csc Sucrose Catabolism Operon

    PubMed Central

    Steen, Jennifer A.; Bohlke, Nina; Vickers, Claudia E.; Nielsen, Lars K.

    2014-01-01

    Plasticity in substrate acceptance is a well-characterised phenomenon for disaccharide transporters. Sucrose, a non-reducing disaccharide, is usually metabolised via either the permease-mediated chromosomally-encoded sucrose catabolism (csc) regulon or the sucrose phosphotransferase system (PTS). E. coli W is a fast-growing strain which efficiently utilises sucrose at concentrations above 1% via the csc regulon. To examine if sucrose could be metabolised via other routes, a library of transposon mutants was generated and screened on 0.2% sucrose. One mutant identified from this library had an insertion in the repressor for the regulon controlling catabolism of the disaccharide trehalose (treR). A series of mutants was constructed to elucidate the mechanism of sucrose utilization in the treR insertion strain. Analysis of these mutants provided evidence that deletion of TreR enables uptake of sucrose via TreB, an enzyme II protein required for PTS-mediated uptake of trehalose. Once inside the cell, this sucrose is not processed by the TreC hydrolase, nor is it sufficient for growth of the strain. QRT-PCR analysis showed that levels of cscA (invertase) transcript increased in the WΔtreR mutant relative to the wild-type strain when grown under low sucrose conditions. This result suggests that the intracellular sucrose provided by TreB can facilitate de-repression of the csc regulon, leading to increased gene expression, sucrose uptake and sucrose utilization in the treR mutant. PMID:24586369

  13. Carbon partitioning and export in transgenic Arabidopsis thaliana with altered capacity for sucrose synthesis grown at low temperature: a role for metabolite transporters.

    PubMed

    Lundmark, Maria; Cavaco, Ana M; Trevanion, Stephen; Hurry, Vaughan

    2006-09-01

    We investigated the role of metabolite transporters in cold acclimation by comparing the responses of wild-type (WT) Arabidopsis thaliana (Heynh.) with that of transgenic plants over-expressing sucrose-phosphate synthase (SPSox) or with that of antisense repression of cytosolic fructose-1,6-bisphosphatase (FBPas). Plants were grown at 23 degrees C and then shifted to 5 degrees C. We compared the leaves shifted to 5 degrees C for 3 and 10 d with new leaves that developed at 5 degrees C with control leaves on plants at 23 degrees C. At 23 degrees C, ectopic expression of SPS resulted in 30% more carbon being fixed per day and an increase in sucrose export from source leaves. This increase in fixation and export was supported by increased expression of the plastidic triose-phosphate transporter AtTPT and, to a lesser extent, the high-affinity Suc transporter AtSUC1. The improved photosynthetic performance of the SPSox plants was maintained after they were shifted to 5 degrees C and this was associated with further increases in AtSUC1 expression but with a strong repression of AtTPT mRNA abundance. Similar responses were shown by WT plants during acclimation to low temperature and this response was attenuated in the low sucrose producing FBPas plants. These data suggest that a key element in recovering flux through carbohydrate metabolism in the cold is to control the partitioning of metabolites between the chloroplast and the cytosol, and Arabidopsis modulates the expression of AtTPT to maintain balanced carbon flow. Arabidopsis also up-regulates the expression of AtSUC1, and to lesser extent AtSUC2, as down-stream components facilitate sucrose transport in leaves that develop at low temperatures.

  14. A role for tungsten in the biology of Campylobacter jejuni: tungstate stimulates formate dehydrogenase activity and is transported via an ultra-high affinity ABC system distinct from the molybdate transporter.

    PubMed

    Smart, Jonathan P; Cliff, Matthew J; Kelly, David J

    2009-11-01

    The food-borne pathogen Campylobacter jejuni possesses no known tungstoenzymes, yet encodes two ABC transporters (Cj0300-0303 and Cj1538-1540) homologous to bacterial molybdate (ModABC) uptake systems and the tungstate transporter (TupABC) of Eubacterium acidaminophilum respectively. The actual substrates and physiological role of these transporters were investigated. Tryptophan fluorescence spectroscopy and isothermal titration calorimetry of the purified periplasmic binding proteins of each system revealed that while Cj0303 is unable to discriminate between molybdate and tungstate (K(D) values for both ligands of 4-8 nM), Cj1540 binds tungstate with a K(D) of 1.0 +/- 0.2 pM; 50 000-fold more tightly than molybdate. Induction-coupled plasma mass spectroscopy of single and double mutants showed that this large difference in affinity is reflected in a lower cellular tungsten content in a cj1540 (tupA) mutant compared with a cj0303c (modA) mutant. Surprisingly, formate dehydrogenase (FDH) activity was decreased approximately 50% in the tupA strain, and supplementation of the growth medium with tungstate significantly increased FDH activity in the wild type, while inhibiting known molybdoenzymes. Our data suggest that C. jejuni possesses a specific, ultra-high affinity tungstate transporter that supplies tungsten for incorporation into FDH. Furthermore, possession of two MoeA paralogues may explain the formation of both molybdopterin and tungstopterin in this bacterium.

  15. Sucrose Transporter ZmSut1 Expression and Localization Uncover New Insights into Sucrose Phloem Loading1[OPEN

    PubMed Central

    Baker, R. Frank; Leach, Kristen A.; Boyer, Nathanial R.; Skopelitis, Tara; Jackson, David; Braun, David M.

    2016-01-01

    Sucrose transporters (SUTs) translocate sucrose (Suc) across cellular membranes, and in eudicots, multiple SUTs are known to function in Suc phloem loading in leaves. In maize (Zea mays), the Sucrose Transporter1 (ZmSut1) gene has been implicated in Suc phloem loading based upon RNA expression in leaves, electrophysiological experiments, and phenotypic analysis of zmsut1 mutant plants. However, no previous studies have examined the cellular expression of ZmSut1 RNA or the subcellular localization of the ZmSUT1 protein to assess the gene’s hypothesized function in Suc phloem loading or to evaluate its potential roles, such as phloem unloading, in nonphotosynthetic tissues. To this end, we performed RNA in situ hybridization experiments, promoter-reporter gene analyses, and ZmSUT1 localization studies to elucidate the cellular expression pattern of the ZmSut1 transcript and protein. These data showed that ZmSut1 was expressed in multiple cell types throughout the plant and indicated that it functions in phloem companion cells to load Suc and also in other cell types to retrieve Suc from the apoplasm to prevent its accumulation and loss to the transpiration stream. Additionally, by comparing a phloem-mobile tracer with ZmSut1 expression, we determined that developing maize leaves dynamically switch from symplasmic to apoplasmic phloem unloading, reconciling previously conflicting reports, and suggest that ZmSut1 does not have an apparent function in either unloading process. A model for the dual roles for ZmSut1 function (phloem loading and apoplasmic recycling), Sut1 evolution, and its possible use to enhance Suc export from leaves in engineering C3 grasses for C4 photosynthesis is discussed. PMID:27621426

  16. Inverse pH Regulation of Plant and Fungal Sucrose Transporters: A Mechanism to Regulate Competition for Sucrose at the Host/Pathogen Interface?

    PubMed Central

    Wippel, Kathrin; Wittek, Anke; Hedrich, Rainer; Sauer, Norbert

    2010-01-01

    Background Plant sucrose transporter activities were shown to respond to changes in the extracellular pH and redox status, and oxidizing compounds like glutathione (GSSG) or H2O2 were reported to effect the subcellular targeting of these proteins. We hypothesized that changes in both parameters might be used to modulate the activities of competing sucrose transporters at a plant/pathogen interface. We, therefore, compared the effects of redox-active compounds and of extracellular pH on the sucrose transporters UmSRT1 and ZmSUT1 known to compete for extracellular sucrose in the Ustilago maydis (corn smut)/Zea mays (maize) pathosystem. Methodology/Principal Findings We present functional analyses of the U. maydis sucrose transporter UmSRT1 and of the plant sucrose transporters ZmSUT1 and StSUT1 in Saccharomyces cerevisiae or in Xenopus laevis oocytes in the presence of different extracellular pH-values and redox systems, and study the possible effects of these treatments on the subcellular targeting. We observed an inverse regulation of host and pathogen sucrose transporters by changes in the apoplastic pH. Under none of the conditions analyzed, we could confirm the reported effects of redox-active compounds. Conclusions/Significance Our data suggest that changes in the extracellular pH but not of the extracellular redox status might be used to oppositely adjust the transport activities of plant and fungal sucrose transporters at the host/pathogen interface. PMID:20865151

  17. Proton-associated sucrose transport of mammalian solute carrier family 45: an analysis in Saccharomyces cerevisiae.

    PubMed

    Bartölke, Rabea; Heinisch, Jürgen J; Wieczorek, Helmut; Vitavska, Olga

    2014-12-01

    The members of the solute carrier 45 (SLC45) family have been implicated in the regulation of glucose homoeostasis in the brain (SLC45A1), with skin and hair pigmentation (SLC45A2), and with prostate cancer and myelination (SLC45A3). However, apart from SLC45A1, a proton-associated glucose transporter, the function of these proteins is still largely unknown, although sequence similarities to plant sucrose transporters mark them as a putative sucrose transporter family. Heterologous expression of the three members SLC45A2, SLC45A3 and SLC45A4 in Saccharomyces cerevisiae confirmed that they are indeed sucrose transporters. [(14)C]Sucrose-uptake measurements revealed intermediate transport affinities with Km values of approximately 5 mM. Transport activities were best under slightly acidic conditions and were inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone, demonstrating an H(+)-coupled transport mechanism. Na(+), on the other hand, had no effect on sucrose transport. Competitive inhibition assays indicated a possible transport also of glucose and fructose. Real-time PCR of mouse tissues confirmed mRNA expression of SLC45A2 in eyes and skin and of SLC45A3 primarily in the prostate, but also in other tissues, whereas SLC45A4 showed a predominantly ubiquitous expression. Altogether the results provide new insights into the physiological significance of SLC45 family members and challenge existing concepts of mammalian sugar transport, as they (i) transport a disaccharide, and (ii) perform secondary active transport in a proton-dependent manner.

  18. Sucrose transport and phloem unloading in peach fruit: potential role of two transporters localized in different cell types.

    PubMed

    Zanon, Laura; Falchi, Rachele; Santi, Simonetta; Vizzotto, Giannina

    2015-06-01

    Several complex physiological processes, which include long-distance translocation in the phloem and unloading in sink tissues, govern the partitioning of sugars in economically important organs, such as peach fruit. In this study, we took advantage of a symplastic tracer, carboxyfluorescein (CF), providing evidence for an apoplastic sucrose transfer in the early (SI) and middle (SIII) phases of peach fruit development. Moreover, using a combination of in situ hybridization and laser microdissection-assisted expression analysis, three putative sucrose transporters encoding genes (PpSUT1, PpSUT2, PpSUT4) were transcriptionally analyzed to relate their expression with sucrose storage in this organ. Our study revealed that PpSUT2 and PpSUT4 are the genes predominantly expressed in fruit flesh, and the detailed analysis of their expression pattern in the different cell types enabled us to suggest a specialized role in sucrose distribution. Both PpSUTs transporters could be involved in the retrieval of sucrose lost from the symplastic continuum of the phloem and, when expressed in parenchyma cells, they could be active in the import of sucrose into sink tissues, via symport from the apoplast. An alternative hypothesis has been proposed and discussed for PpSUT4 because of its putative tonoplastic localization. Taken together, our results provide new insights into the molecular mechanisms underpinning sucrose unloading and accumulation in peach fruit.

  19. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9.

    PubMed

    Lin, I Winnie; Sosso, Davide; Chen, Li-Qing; Gase, Klaus; Kim, Sang-Gyu; Kessler, Danny; Klinkenberg, Peter M; Gorder, Molly K; Hou, Bi-Huei; Qu, Xiao-Qing; Carter, Clay J; Baldwin, Ian T; Frommer, Wolf B

    2014-04-24

    Angiosperms developed floral nectaries that reward pollinating insects. Although nectar function and composition have been characterized, the mechanism of nectar secretion has remained unclear. Here we identify SWEET9 as a nectary-specific sugar transporter in three eudicot species: Arabidopsis thaliana, Brassica rapa (extrastaminal nectaries) and Nicotiana attenuata (gynoecial nectaries). We show that SWEET9 is essential for nectar production and can function as an efflux transporter. We also show that sucrose phosphate synthase genes, encoding key enzymes for sucrose biosynthesis, are highly expressed in nectaries and that their expression is also essential for nectar secretion. Together these data are consistent with a model in which sucrose is synthesized in the nectary parenchyma and subsequently secreted into the extracellular space via SWEET9, where sucrose is hydrolysed by an apoplasmic invertase to produce a mixture of sucrose, glucose and fructose. The recruitment of SWEET9 for sucrose export may have been a key innovation, and could have coincided with the evolution of core eudicots and contributed to the evolution of nectar secretion to reward pollinators.

  20. The Raf-like Kinase ILK1 and the High Affinity K+ Transporter HAK5 Are Required for Innate Immunity and Abiotic Stress Response1[OPEN

    PubMed Central

    Brauer, Elizabeth K.; Ahsan, Nagib; Kato, Naohiro; Coluccio, Alison E.; Thelen, Jay J.

    2016-01-01

    Plant perception of pathogen-associated molecular patterns (PAMPs) and other environmental stresses trigger transient ion fluxes at the plasma membrane. Apart from the role of Ca2+ uptake in signaling, the regulation and significance of PAMP-induced ion fluxes in immunity remain unknown. We characterized the functions of INTEGRIN-LINKED KINASE1 (ILK1) that encodes a Raf-like MAP2K kinase with functions insufficiently understood in plants. Analysis of ILK1 mutants impaired in the expression or kinase activity revealed that ILK1 contributes to plant defense to bacterial pathogens, osmotic stress sensitivity, and cellular responses and total ion accumulation in the plant upon treatment with a bacterial-derived PAMP, flg22. The calmodulin-like protein CML9, a negative modulator of flg22-triggered immunity, interacted with, and suppressed ILK1 kinase activity. ILK1 interacted with and promoted the accumulation of HAK5, a putative (H+)/K+ symporter that mediates a high-affinity uptake during K+ deficiency. ILK1 or HAK5 expression was required for several flg22 responses including gene induction, growth arrest, and plasma membrane depolarization. Furthermore, flg22 treatment induced a rapid K+ efflux at both the plant and cellular levels in wild type, while mutants with impaired ILK1 or HAK5 expression exhibited a comparatively increased K+ loss. Taken together, our results position ILK1 as a link between plant defense pathways and K+ homeostasis. PMID:27208244

  1. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport.

    PubMed

    Chen, Li-Qing; Qu, Xiao-Qing; Hou, Bi-Huei; Sosso, Davide; Osorio, Sonia; Fernie, Alisdair R; Frommer, Wolf B

    2012-01-13

    Plants transport fixed carbon predominantly as sucrose, which is produced in mesophyll cells and imported into phloem cells for translocation throughout the plant. It is not known how sucrose migrates from sites of synthesis in the mesophyll to the phloem, or which cells mediate efflux into the apoplasm as a prerequisite for phloem loading by the SUT sucrose-H(+) (proton) cotransporters. Using optical sucrose sensors, we identified a subfamily of SWEET sucrose efflux transporters. AtSWEET11 and 12 localize to the plasma membrane of the phloem. Mutant plants carrying insertions in AtSWEET11 and 12 are defective in phloem loading, thus revealing a two-step mechanism of SWEET-mediated export from parenchyma cells feeding H(+)-coupled import into the sieve element-companion cell complex. We discuss how restriction of intercellular transport to the interface of adjacent phloem cells may be an effective mechanism to limit the availability of photosynthetic carbon in the leaf apoplasm in order to prevent pathogen infections.

  2. Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation

    USDA-ARS?s Scientific Manuscript database

    In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the Zn/Cd hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization ...

  3. In vivo transport of three radioactive [(18)F]-fluorinated deoxysucrose analogs by the maize sucrose transporter ZmSUT1.

    PubMed

    Tran, Thu M; Hampton, Carissa S; Brossard, Tom W; Harmata, Michael; Robertson, J David; Jurisson, Silvia S; Braun, David M

    2017-03-08

    Sucrose transporter (SUT) proteins translocate sucrose across cell membranes; however, mechanistic aspects of sucrose binding by SUTs are not well resolved. Specific hydroxyl groups in sucrose participate in hydrogen bonding with SUT proteins. We previously reported that substituting a radioactive fluorine-18 [(18)F] at the C-6' position within the fructosyl moiety of sucrose did not affect sucrose transport by the maize (Zea mays) ZmSUT1 protein. To determine how (18)F substitution of hydroxyl groups at two other positions within sucrose, the C-1' in the fructosyl moiety or the C-6 in the glucosyl moiety, impact sucrose transport, we synthesized 1'-[F(18)]fluoro-1'-deoxysucrose and 6-[F(18)]fluoro-6-deoxysucrose ([(18)F]FDS) analogs. Each [(18)F]FDS derivative was independently introduced into wild-type or sut1 mutant plants, which are defective in sucrose phloem loading. All three (1'-, 6'-, and 6-) [(18)F]FDS derivatives were efficiently and equally translocated, similarly to carbon-14 [(14)C]-labeled sucrose. Hence, individually replacing the hydroxyl groups at these positions within sucrose does not interfere with substrate recognition, binding, or membrane transport processes, and hydroxyl groups at these three positions are not essential for hydrogen bonding between sucrose and ZmSUT1. [(18)F]FDS imaging afforded several advantages compared to [(14)C]-sucrose detection. We calculated that 1'-[(18)F]FDS was transported at approximately a rate of 0.90 ± 0.15 m.h-1 in wild-type leaves, and at 0.68 ± 0.25 m.h-1 in sut1 mutant leaves. Collectively, our data indicated that [(18)F]FDS analogs are valuable tools to probe sucrose-SUT interactions and to monitor sucrose transport in plants.

  4. Sucrose transport and phloem unloading in stem of Vicia faba: possible involvement of a sucrose carrier and osmotic regulation

    SciTech Connect

    Aloni, B.; Wyse, R.E.; Griffith, S.

    1986-06-01

    After pulse labeling of a source leaf with /sup 14/CO/sub 2/, stem sections of Vicia faba plants were cut and the efflux characteristics of /sup 14/C-labeled sugars into various buffered solutions were determined. Radiolabeled sucrose was shown to remain localized in the phloem and adjacent phloem parenchyma tissues after a 2-hour chase. Therefore, sucrose leakage from stem segments prepared following a 75-minute chase period was assumed to be characteristic of phloem unloading. The efflux of /sup 14/C assimilates from the phloem was enhanced by 1 millimolar p-chloromercuribenzene sulfonic acid (PCMBS) and by 5 micromolar carbonyl cyanide m-chlorophenly hydrazone (CCCP). However, PCMBS inhibited and CCCP enhanced general leakage of nonradioactive sugars from the stem segments. Sucrose at concentrations of 50 millimolar in the free space increased efflux of (/sup 14/C)sucrose, presumably through an exchange mechanism. This exchange was inhibited by PCMBS and abolished by 0.2 molar mannitol. Increasing the osmotic concentration of the efflux medium with mannitol reduced (/sup 14/C)sucrose efflux. However, this inhibition seems not to be specific to sucrose unloading since leakage of total sugars, nonlabeled sucrose, glucose, and amino acids from the bulk of the tissue was reduced in a similar manner. The data suggest that phloem unloading in cut stem segments is consistent with passive efflux of sucrose from the phloem to the apoplast and that sucrose exchange via a membrane carrier may be involved.

  5. A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators.

    PubMed

    Zhou, Yuchan; Qu, Hongxia; Dibley, Katherine E; Offler, Christina E; Patrick, John W

    2007-02-01

    A suite of newly discovered sucrose transporter genes, PsSUF1, PsSUF4, PvSUT1 and PvSUF1, were isolated from the coats of developing pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) seeds. Sequence analysis indicated that deduced proteins encoded by PsSUF1, PvSUT1 and PvSUF1 clustered in a separate sub-group under sucrose transporter Clade I, whereas the deduced protein encoded by PsSUF4 clustered in Clade II. When expressed in yeast, these genes were shown to encode sucrose transporters with apparent Michaelis Menten constant (Km) values ranging from 8.9 to 99.8 mm. PvSUT1 exhibited functional characteristics of a sucrose/H+ symporter. In contrast, PsSUF1, PvSUF1 and PsSUF4 supported the pH- and energy independent transport of sucrose that was shown to be bi-directional. These transport properties, together with that of counter transport, indicated that PsSUF1, PvSUF1 and PsSUF4 function as carriers that support the facilitated diffusion of sucrose. Carrier function was unaffected by diethylpyrocarbonate and by maltose competition, suggesting that the sucrose binding sites of these transporters differed from those of known sucrose/H+ symporters. All sucrose transporters were expressed throughout the plant and, of greatest interest, were co-expressed in cells considered responsible for sucrose efflux from seed coats. The possible roles played by the novel facilitators in sucrose efflux from seed coats are discussed.

  6. SUT Sucrose and MST Monosaccharide Transporter Inventory of the Selaginella Genome

    PubMed Central

    Lalonde, Sylvie; Frommer, Wolf B.

    2012-01-01

    Most metazoa use hexose transporters to acquire hexoses from their diet and as a transport form for distributing carbon and energy within their bodies; insects use trehalose, and plants use sucrose as their major form for translocation. Plant genomes contain at least three families of mono- and disaccharide transporters: monosaccharide/polyol transporters that are evolutionary closely related to the yeast and human glucose transporters, sucrose transporters of the SUT family, which similar to the hexose transporters belong to the major facilitator superfamily, but share only minimal amino acid sequence homology with the hexose transporters, and the family of SWEET sugar transporters conserved between animals and plants. Recently, the genome sequence of the spikemoss Selaginella has been determined. In order to study the evolution of sugar transport in plants, we carefully annotated of the complement of sugar transporters in Selaginella. We review the current knowledge regarding sugar transport in spikemoss and provide phylogenetic analyses of the complement of MST and SUT homologs in Selaginella (and Physcomitrella). PMID:22645575

  7. SUT Sucrose and MST Monosaccharide Transporter Inventory of the Selaginella Genome.

    PubMed

    Lalonde, Sylvie; Frommer, Wolf B

    2012-01-01

    Most metazoa use hexose transporters to acquire hexoses from their diet and as a transport form for distributing carbon and energy within their bodies; insects use trehalose, and plants use sucrose as their major form for translocation. Plant genomes contain at least three families of mono- and disaccharide transporters: monosaccharide/polyol transporters that are evolutionary closely related to the yeast and human glucose transporters, sucrose transporters of the SUT family, which similar to the hexose transporters belong to the major facilitator superfamily, but share only minimal amino acid sequence homology with the hexose transporters, and the family of SWEET sugar transporters conserved between animals and plants. Recently, the genome sequence of the spikemoss Selaginella has been determined. In order to study the evolution of sugar transport in plants, we carefully annotated of the complement of sugar transporters in Selaginella. We review the current knowledge regarding sugar transport in spikemoss and provide phylogenetic analyses of the complement of MST and SUT homologs in Selaginella (and Physcomitrella).

  8. High-affinity nitrate/nitrite transporters NrtA and NrtB of Aspergillus nidulans exhibit high specificity and different inhibitor sensitivity

    PubMed Central

    Akhtar, Naureen; Karabika, Eugenia; Kinghorn, James R.; Glass, Anthony D.M.; Unkles, Shiela E.

    2015-01-01

    The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters. PMID:25855763

  9. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumour suppressor in colon that protects against colitis and colon cancer under low-fibre dietary conditions.

    PubMed

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2015-07-15

    Mammalian colon harbours trillions of bacteria under physiological conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. In the present study we show that Slc5a8 (solute carrier gene family 5a, member 8), a Na(+)-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T-cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naive T-cells into immunosuppressive forkhead box P3(+) (FoxP3(+)) Tregs (regulatory T-cells) and suppress conversion of naive T-cells into pro-inflammatory interferon (IFN)-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T-cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T-cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fibre intake but not when dietary fibre intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fibre and mucosal immune system via the bacterial metabolite butyrate and that this transporter is a conditional tumour suppressor in colon linked to dietary fibre content.

  10. Slc5a8, a Na+-coupled high-affinity transporter for short-chain fatty acids, is a conditional tumor suppressor in colon that protects against colitis and colon cancer under low-fiber dietary conditions

    PubMed Central

    Gurav, Ashish; Sivaprakasam, Sathish; Bhutia, Yangzom D.; Boettger, Thomas; Singh, Nagendra; Ganapathy, Vadivel

    2016-01-01

    Summary Mammalian colon harbors trillions of bacteria under physiologic conditions; this symbiosis is made possible because of a tolerized response from the mucosal immune system. The mechanisms underlying this tolerogenic phenomenon remain poorly understood. Here we show that Slc5a8, a Na+-coupled high-affinity transporter in colon for the bacterial fermentation product butyrate, plays a critical role in this process. Among various immune cells in colon, dendritic cells (DCs) are unique not only in their accessibility to luminal contents but also in their ability to induce tolerogenic phenotype in T cells. We found that DCs exposed to butyrate express the immunosuppressive enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and aldehyde dehydrogenase 1A2 (Aldh1A2), promote conversion of naïve T cells into immunosuppressive FoxP3+ Tregs, and suppress conversion of naïve T cells into pro-inflammatory IFN-γ-producing cells. Slc5a8-null DCs do not induce IDO1 and Aldh1A2 and do not generate Tregs or suppress IFN-γ-producing T cells in response to butyrate. We also provide in vivo evidence for an obligatory role for Slc5a8 in suppression of IFN-γ-producing T cells. Furthermore, Slc5a8 protects against colitis and colon cancer under conditions of low-fiber intake but not when dietary fiber intake is optimal. This agrees with the high-affinity nature of the transporter to mediate butyrate entry into cells. We conclude that Slc5a8 is an obligatory link between dietary fiber and mucosal immune system via the bacterial metabolite butyrate, and that this transporter is a conditional tumor suppressor in colon linked to dietary fiber content. PMID:25984582

  11. Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza.

    PubMed

    Boldt, Katja; Pörs, Yvonne; Haupt, Bastian; Bitterlich, Michael; Kühn, Christina; Grimm, Bernhard; Franken, Philipp

    2011-07-15

    Arbuscular mycorrhizal fungi enhance CO₂ assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO₂. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant. Copyright © 2011 Elsevier GmbH. All rights reserved.

  12. Activation of sucrose transport in defoliated Lolium perenne L.: an example of apoplastic phloem loading plasticity.

    PubMed

    Berthier, Alexandre; Desclos, Marie; Amiard, Véronique; Morvan-Bertrand, Annette; Demmig-Adams, Barbara; Adams, William W; Turgeon, Robert; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie

    2009-07-01

    The pathway of carbon phloem loading was examined in leaf tissues of the forage grass Lolium perenne. The effect of defoliation (leaf blade removal) on sucrose transport capacity was assessed in leaf sheaths as the major carbon source for regrowth. The pathway of carbon transport was assessed via a combination of electron microscopy, plasmolysis experiments and plasma membrane vesicles (PMVs) purified by aqueous two-phase partitioning from the microsomal fraction. Results support an apoplastic phloem loading mechanism. Imposition of an artificial proton-motive force to PMVs from leaf sheaths energized an active, transient and saturable uptake of sucrose (Suc). The affinity of Suc carriers for Suc was 580 microM in leaf sheaths of undefoliated plants. Defoliation induced a decrease of K(m) followed by an increase of V(max). A transporter was isolated from stubble (including leaf sheaths) cDNA libraries and functionally expressed in yeast. The level of L.perenne SUcrose Transporter 1 (LpSUT1) expression increased in leaf sheaths in response to defoliation. Taken together, the results indicate that Suc transport capacity increased in leaf sheaths of L. perenne in response to leaf blade removal. This increase might imply de novo synthesis of Suc transporters, including LpSUT1, and may represent one of the mechanisms contributing to rapid refoliation.

  13. Characterization of the high affinity Zn transporter from Noccaea caerulescens, NcZNT1, and dissection of its promoter for its role in Zn uptake and hyperaccumulation.

    PubMed

    Milner, Matthew J; Craft, Eric; Yamaji, Naoki; Koyama, Emi; Ma, Jian Feng; Kochian, Leon V

    2012-07-01

    • In this paper, we conducted a detailed analysis of the ZIP family transporter, NcZNT1, in the zinc (Zn)/cadmium (Cd) hyperaccumulating plant species, Noccaea caerulescens, formerly known as Thlaspi caerulescens. NcZNT1 was previously suggested to be the primary root Zn/Cd uptake transporter. Both a characterization of NcZNT1 transport function in planta and in heterologous systems, and an analysis of NcZNT1 gene expression and NcZNT1 protein localization were carried out. • We show that NcZNT1 is not only expressed in the root epidermis, but also is highly expressed in the root and shoot vasculature, suggesting a role in long-distance metal transport. Also, NcZNT1 was found to be a plasma membrane transporter that mediates Zn but not Cd, iron (Fe), manganese (Mn) or copper (Cu) uptake into plant cells. • Two novel regions of the NcZNT1 promoter were identified which may be involved in both the hyperexpression of NcZNT1 and its ability to be regulated by plant Zn status. • In conclusion, we demonstrate here that NcZNT1 plays a role in Zn and not Cd uptake from the soil, and based on its strong expression in the root and shoot vasculature, could be involved in long-distance transport of Zn from the root to the shoot via the xylem. No claim to original US government works. New Phytologist © 2012 New Phytologist Trust.

  14. Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

    PubMed Central

    Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

    2012-01-01

    Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

  15. Cloning of a high-affinity K+ transporter gene PutHKT2;1 from Puccinellia tenuiflora and its functional comparison with OsHKT2;1 from rice in yeast and Arabidopsis

    PubMed Central

    Ardie, Sintho Wahyuning; Xie, Lina; Takahashi, Ryuichi; Liu, Shenkui; Takano, Tetsuo

    2009-01-01

    A high-affinity K+ transporter PutHKT2;1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. Expression of PutHKT2;1 was induced by both 300 mM NaCl and K+-starvation stress in roots, but only slightly regulated by those stresses in shoots. PutHKT2;1 transcript levels in 300 mM NaCl were doubled by the depletion of potassium. Yeast transformed with PutHKT2;1, like those transformed with PhaHKT2;1 from salt-tolerant reed plants (Phragmites australis), (i) were able to take up K+ in low K+ concentration medium or in the presence of NaCl, and (ii) were permeable to Na+. This suggests that PutHKT2;1 has a high affinity K+-Na+ symport function in yeast. Arabidopsis over-expressing PutHKT2;1 showed increased sensitivities to Na+, K+, and Li+, while Arabidopsis over-expressing OsHKT2;1 from rice (Oryza sativa) showed increased sensitivity only to Na+. In contrast to OsHKT2;1, which functions in Na+-uptake at low external K+ concentrations, PutHKT2;1 functions in Na+-uptake at higher external K+ concentrations. These results show that the modes of action of PutHKT2;1 in transgenic yeast and Arabidopsis differ from the mode of action of the closely related OsHKT2;1 transporter. PMID:19528529

  16. Identification of Membrane Protein Associated with Sucrose Transport Into Cells of Developing Soybean Cotyledons 1

    PubMed Central

    Ripp, Kevin G.; Viitanen, Paul V.; Hitz, William D.; Franceschi, Vincent R.

    1988-01-01

    The photolyzable sucrose derivative 6′-deoxy-6′-(4-azido-2-hydroxy)-benzamidosucrose (6′-HABS), competitively inhibited the influx of [14C] sucrose into protoplasts from developing soybean (Glycine max L. Merr cv Wye) cotyledons. Photolysis of 125I-labeled 6′-HABS in the presence of 10 millimolar dithiothreitol and microsomal preparations from developing soybean cotyledons led to label incorporation into a moderately abundant membrane protein with an apparent molecular mass of about 62 kilodalton (kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 62 kD protein was partially protected from labeling by the inclusion of 100 millimolar sucrose in the photolysis medium and also by the inclusion of 10 millimolar phenyl α-d-thioglucopyranoside. Glucose, raffinose, or phenyl α-d-3-deoxy-3-fluoroglucopyranoside did not afford even partial protection from labeling. When the photolyzable moiety of 6′-HABS was attached to 6-deoxy-6-aminoglucose and 125I labeled, the resulting photoprobe did not label the 62 kD protein above background. The labeled protein at 62 kD is therefore apparently a specific, sucrose binding protein. Sucrose influx into cotlyedons of less than 25 milligrams fresh weight (approximately 10 days after flowering) occurred by passive processes, but metabolically dependent uptake became dominant over the next 5 to 7 days of development. Both the Coomassie staining protein at 62 kD and label incorporation at that position in analysis of membrane proteins appeared concomitant with the onset of active sucrose influx. Polyclonal antibodies to the purified 62 kD protein bound specifically to a protein in the plasmalemma of thin sections prepared from cotyledons and density stained with colloidal gold-protein A. The results suggest that the 62 kD membrane protein is associated with sucrose transport and may be the plasmalemma sucrose transporter. Images Fig. 2 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Fig. 11 Fig. 12 Fig. 13 Fig. 14

  17. Partitioning and transport of the translocates mannitol and sucrose in the light and dark in celery (Apium Graveolens L. )

    SciTech Connect

    Davis, J.M.; Loescher, W.H.

    1987-04-01

    Sucrose and mannitol are major photosynthetic products and translocates in celery. Assimilate partitioning and transport were studied by pulse-labeling leaves with /sup 14/CO/sub 2/ followed by different length chases in ambient air. After a 2 h chase in the light there was more /sup 14/C in sucrose than mannitol in source leaves and their petioles. In contrast after a 2 h dark chase leaves contained more /sup 14/C in mannitol than sucrose but petioles had more /sup 14/C in sucrose than mannitol. After a 15 h chase (6 h light; 9 h dark) labeled sucrose was higher in source petiole vascular bundles than in adjacent parenchyma tissue but label in glucose and fructose was higher in the parenchyma tissue. After the 15 h chase most of the /sup 14/C remaining in developing sink leaves and their petioles was in mannitol. Although in the light mannitol:sucrose ratios are the same in leaf and petiole tissues, in the dark sucrose is initially the major translocate with mannitol becoming more important as leaf sucrose pools are depleted. When synthesized, sucrose is rapidly transported and then metabolized to hexose sugars whereas mannitol is used both for transport and storage.

  18. Characterisation of [¹¹C]PR04.MZ in Papio anubis baboon: a selective high-affinity radioligand for quantitative imaging of the dopamine transporter.

    PubMed

    Riss, Patrick J; Hooker, Jacob M; Shea, Colleen; Xu, Youwen; Carter, Pauline; Warner, Donald; Ferrari, Valentina; Kim, Sung-Won; Aigbirhio, Franklin I; Fowler, Joanna S; Roesch, Frank

    2012-01-01

    N-(4-fluorobut-2-yn-1-yl)-2β-carbomethoxy-3β-(4'-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [(11)C]PR04.MZ ([(11)C]-1) has been developed using GMP compliant equipment. An adult female Papio anubis baboon was studied using a test-retest protocol with [(11)C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (f(P)), plasma input functions and metabolic degradation of the radiotracer [(11)C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (V(T)) and non-displaceable binding potentials (BP(ND)) for various brain regions and the blood were obtained from kinetic modelling. [(11)C]-1 shows promising results as a selective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Characterisation of [11C]PR04.MZ in Papio anubis baboon: A selective high-affinity radioligand for quantitative imaging of the dopamine transporter

    SciTech Connect

    Riss P. J.; Fowler J.; Riss, P.J.; Hooker, J.M.; Shea, C.; Xu, Y.; Carter, P.; Warner, D.; Ferrari V.; Kim, S.W.; Aigbirhio, F.I.; Fowler, J.S.; Roesch, F.

    2011-10-25

    N-(4-fluorobut-2-yn-1-yl)-2{beta}-carbomethoxy-3{beta}-(4{prime}-tolyl)nortropane (PR04.MZ, 1) is a PET radioligand for the non-invasive exploration of the function of the cerebral dopamine transporter (DAT). A reliable automated process for routine production of the carbon-11 labelled analogue [{sup 11}C]PR04.MZ ([{sup 11}C]-1) has been developed using GMP compliant equipment. An adult female Papioanubis baboon was studied using a test-retest protocol with [{sup 11}C]-1 in order to assess test-retest reliability, metabolism and CNS distribution profile of the tracer in non-human primates. Blood sampling was performed throughout the studies for determination of the free fraction in plasma (fP), plasma input functions and metabolic degradation of the radiotracer [{sup 11}C]-1. Time-activity curves were derived for the putamen, the caudate nucleus, the ventral striatum, the midbrain and the cerebellum. Distribution volumes (VT) and non-displaceable binding potentials (BPND) for various brain regions and the blood were obtained from kinetic modelling. [{sup 11}C]-1 shows promising results as aselective marker of the presynaptic dopamine transporter. With the reliable visualisation of the extra-striatal dopaminergic neurons and no indication on labelled metabolites, the tracer provides excellent potential for translation into man.

  20. Sucrose Transporter AtSUC9 Mediated by a Low Sucrose Level is Involved in Arabidopsis Abiotic Stress Resistance by Regulating Sucrose Distribution and ABA Accumulation.

    PubMed

    Jia, Wanqiu; Zhang, Lijun; Wu, Di; Liu, Shan; Gong, Xue; Cui, Zhenhai; Cui, Na; Cao, Huiying; Rao, Longbing; Wang, Che

    2015-08-01

    Sucrose (Suc) transporters (SUCs or SUTs) are important regulators in plant growth and stress tolerance. However, the mechanism of SUCs in plant abiotic stress resistance remains to be dietermined. Here, we found that AtSUC9 expression was induced by abiotic stress, including salt, osmotic and cold stress conditions. Disruption of AtSUC9 led to sensitive responses to abiotic stress during seed germination and seedling growth. Further analyses indicated that the sensitivity phenotype of Atsuc9 mutants resulted from higher Suc content in shoots and lower Suc content in roots, as compared with that in wild-type (WT) plants. In addition, we found that the expression of AtSUC9 is induced in particular by low levels of exogenous and endogenous Suc, and deletion of AtSUC9 affected the expression of the low Suc level-responsive genes. AtSUC9 also showed an obvious response to treatments with low concentrations of exogenous Suc during seed germination, seedling growth and Suc distribution, and Atsuc9 mutants hardly grew in abiotic stress treatments without exogenous Suc. Moreover, our results illustrated not only that deletion of AtSUC9 blocks abiotic stress-inducible ABA accumulation but also that Atsuc9 mutants had a lower content of endogenous ABA in stress conditions than in normal conditions. Deletion of AtSUC9 also inhibited the expression of many ABA-inducible genes (SnRk2.2/3/6, ABF2/3/4, ABI1/3/4, RD29A, KIN1 and KIN2). These results indicate that AtSUC9 is induced in particular by low Suc levels then mediates the balance of Suc distribution and promotes ABA accumulation to enhance Arabidopsis abiotic stress resistance. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Overexpression of a Grapevine Sucrose Transporter (VvSUC27) in Tobacco Improves Plant Growth Rate in the Presence of Sucrose In vitro.

    PubMed

    Cai, Yumeng; Tu, Wenrui; Zu, Yunyun; Jing, Yan; Xu, Zimo; Lu, Jiang; Zhang, Yali

    2017-01-01

    The import of sugar from source leaves and it further accumulation in grape berries are considerably high during ripening, and this process is mediated via sucrose transporters. In this study, a grape sucrose transporter (SUT) gene, VvSUC27, located at the plasma membrane, was transferred to tobacco (Nicotiana tabacum). The transformants were more sensitive to sucrose and showed more rapid development, especially roots, when cultured on MS agar medium containing sucrose, considering that the shoot/root dry weight ratio was only half that of the control. Moreover, all transformed plants exhibited light-colored leaves throughout their development, which indicated chlorosis and an associated reduction in photosynthesis. The total sugar content in the roots and stems of transformants was higher than that in control plants. No significant difference was observed in the leaves between the transformants and control plants. The levels of growth-promoting hormones were increased, and those of stress-mediating hormones were reduced in transgenic tobacco plants. The qRT-PCR analysis revealed that the expression of VvSUC27 was 1,000 times higher than that of the autologous tobacco sucrose transporter, which suggested that the markedly increased growth rate of transformants was because of the heterogeneously expressed gene. The transgenic tobacco plants showed resistance to abiotic stresses. Strikingly, the overexpression of VvSUC27 leaded to the up regulation of most reactive oxygen species scavengers and abscisic acid-related genes that might enable transgenic plants to overcome abiotic stress. Taken together, these results revealed an important role of VvSUC27 in plant growth and response to abiotic stresses, especially in the presence of sucrose in vitro.

  2. Molecular cloning and expression analysis of a gene for a sucrose transporter in maize (Zea mays L.).

    PubMed

    Aoki, N; Hirose, T; Takahashi, S; Ono, K; Ishimaru, K; Ohsugi, R

    1999-10-01

    Here we report the cloning of a sucrose transporter cDNA from maize (Zea mays L.) and an analysis of the expression of the gene. A cDNA clone (ZmSUT1) was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. Based on the results of genomic Southern hybridization, ZmSUT1 appears to be a single copy gene. A Northern blot analysis of seedlings and leaf blades suggests that the sucrose transporter is involved in the export of accumulated carbohydrates from source leaf blades. From the measurements of transcript levels and carbohydrate contents in mature leaf blades, we propose that the expression of the gene for the maize sucrose transporter is positively regulated by the amounts of soluble sugars such as sucrose and glucose in source leaves of maize. In addition, based on the tissue specificity of the expression of the gene in maize plants at the reproductive stage, it is possible that the sucrose transporter acts in sink tissues such as pedicles as well as in source tissues such as leaf blades.

  3. MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2013-11-01

    The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC.

  4. Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain.

    PubMed Central

    Moores, J C; Magazin, M; Ditta, G S; Leong, J

    1984-01-01

    A gene bank of DNA from plant growth-promoting Pseudomonas sp. strain B10 was constructed using the broad host-range conjugative cosmid pLAFR1. The recombinant cosmids contained insert DNA averaging 21.5 kilobase pairs in length. Nonfluorescent mutants of Pseudomonas sp. strain B10 were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, or UV light and were defective in the biosynthesis of its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin. No yellow-green, fluorescent mutants defective in the production of pseudobactin were identified. Nonfluorescent mutants were individually complemented by mating the gene bank en masse and identifying fluorescent transconjugants. Eight recombinant cosmids were sufficient to complement 154 nonfluorescent mutants. The pattern of complementation suggests that a minimum of 12 genes arranged in four gene clusters is required for the biosynthesis of pseudobactin. This minimum number of genes seems reasonable considering the structural complexity of pseudobactin. Images PMID:6690426

  5. Transcriptional coordination and abscisic acid mediated regulation of sucrose transport and sucrose-to-starch metabolism related genes during grain filling in wheat (Triticum aestivum L.).

    PubMed

    Mukherjee, Shalini; Liu, Aihua; Deol, Kirandeep K; Kulichikhin, Konstanin; Stasolla, Claudio; Brûlé-Babel, Anita; Ayele, Belay T

    2015-11-01

    Combining physiological, molecular and biochemical approaches, this study investigated the transcriptional coordination and abscisic acid (ABA) mediated regulation of genes involved in sucrose import and its conversion to starch during grain filling in wheat. Sucrose import appears to be mediated by seed localized TaSUT1, mainly TaSUT1D, while sucrose cleavage by TaSuSy2. Temporal overlapping of the transcriptional activation of AGPL1 and AGPS1a that encode AGPase with that of the above genes suggests their significance in the synthesis of ADP-glucose; TaAGPL1A and TaAGPL1D contributing the majority of AGPL1 transcripts. ABA induced repressions of TaSUT1, TaSuSy2, TaAGPL1 and TaAGPS1a imply that ABA negatively regulates sucrose import into the endosperm and its subsequent metabolism to ADP-glucose, the substrate for starch synthesis. The formations of amyloses and amylopectin from ADP-glucose appear to be mediated by specific members of GBSS, and SS, SBE and DBE gene families, and the ABA-induced transcriptional change in most of these genes implies that ABA regulates amylose and amylopectin synthesis. The findings provide insights into the molecular mechanisms underlying the coordination and ABA mediated regulation of sucrose transport into the developing endosperm and its subsequent metabolism to starch during grain filling in wheat.

  6. Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco.

    PubMed

    Lemoine, R; Bürkle, L; Barker, L; Sakr, S; Kühn, C; Regnacq, M; Gaillard, C; Delrot, S; Frommer, W B

    1999-07-09

    Pollen cells are symplasmically isolated during maturation and germination. Pollen therefore needs to take up nutrients via membrane carriers. Physiological measurements on pollen indicate sucrose transport in the pollen tube. A cDNA encoding a pollen-specific sucrose transporter-like protein NtSUT3 was isolated from a tobacco pollen cDNA library. NtSUT3 expression is detected only in pollen and is restricted to late pollen development, pollen germination and pollen tube growth. Altogether these data indicate that pollen is supplied not only with glucose, but also with sucrose through a specific sucrose transporter. The respective contribution of each transport pathway may change during pollen tube growth.

  7. Endolysosomal transport of newly-synthesized cathepsin D in a sucrose model of lysosomal storage.

    PubMed

    Hamer, Isabelle; Jadot, Michel

    2005-10-01

    Newly-synthesized soluble lysosomal enzymes are transported from the trans-Golgi network to lysosomes by a mannose 6-phosphate receptor-mediated pathway. Lysosomal storage of indigestible material has been reported to perturb the biosynthesis and the fate of lysosomal hydrolases. In this study, we have focused our attention on the last steps in the transport of newly-synthesized cathepsin D to lysosomes in sucrose-treated WI-38 fibroblasts. Pulse-chase experiments indicate that, in sucrose-treated cells, cathepsin D maturation is delayed by 2 to 4 h. By subcellular fractionation, we show that newly-synthesized cathepsin D precursors transit through organelles endowed with a high sedimentation coefficient. These organelles are recovered in the dense region of a self-forming Percoll density gradient while the bulk of hydrolytic activities is redistributed to the low density region. Only later, are the precursors delivered to organelles containing the bulk of active hydrolases. There, procathepsin D is proteolytically processed into its 31 kDa-mature form. Our results suggest that when sucrose is present, the delayed maturation of procathepsin D is related to the delivery of the polypeptides into an organelle behaving in centrifugation like lysosomes but which is poorly efficient in proteolytic processing of procathepsin D. This low proteolytic activity of this organelle could be due to its poor ability to interact with hydrolase-containing structures.

  8. Transport and Sorting of the Solanum tuberosum Sucrose Transporter SUT1 Is Affected by Posttranslational Modification[W

    PubMed Central

    Krügel, Undine; Veenhoff, Liesbeth M.; Langbein, Jennifer; Wiederhold, Elena; Liesche, Johannes; Friedrich, Thomas; Grimm, Bernhard; Martinoia, Enrico; Poolman, Bert; Kühn, Christina

    2008-01-01

    The plant sucrose transporter SUT1 from Solanum tuberosum revealed a dramatic redox-dependent increase in sucrose transport activity when heterologously expressed in Saccharomyces cerevisiae. Plant plasma membrane vesicles do not show any change in proton flux across the plasma membrane in the presence of redox reagents, indicating a SUT1-specific effect of redox reagents. Redox-dependent sucrose transport activity was confirmed electrophysiologically in Xenopus laevis oocytes with SUT1 from maize (Zea mays). Localization studies of green fluorescent protein fusion constructs showed that an oxidative environment increased the targeting of SUT1 to the plasma membrane where the protein concentrates in 200- to 300-nm raft-like microdomains. Using plant plasma membranes, St SUT1 can be detected in the detergent-resistant membrane fraction. Importantly, in yeast and in plants, oxidative reagents induced a shift in the monomer to dimer equilibrium of the St SUT1 protein and increased the fraction of dimer. Biochemical methods confirmed the capacity of SUT1 to form a dimer in plants and yeast cells in a redox-dependent manner. Blue native PAGE, chemical cross-linking, and immunoprecipitation, as well as the analysis of transgenic plants with reduced expression of St SUT1, confirmed the dimerization of St SUT1 and Sl SUT1 (from Solanum lycopersicum) in planta. The ability to form homodimers in plant cells was analyzed by the split yellow fluorescent protein technique in transiently transformed tobacco (Nicotiana tabacum) leaves and protoplasts. Oligomerization seems to be cell type specific since under native-like conditions, a phloem-specific reduction of the dimeric form of the St SUT1 protein was detectable in SUT1 antisense plants, whereas constitutively inhibited antisense plants showed reduction only of the monomeric form. The role of redox control of sucrose transport in plants is discussed. PMID:18790827

  9. Investigating polymorphisms in membrane-associated transporter protein SLC45A2, using sucrose transporters as a model.

    PubMed

    Reinders, Anke; Ward, John M

    2015-07-01

    Solute carrier family 45 member 2 encodes the melanosomal membrane protein, membrane-associated transporter protein (MATP), of unknown function, that is required for normal melanin synthesis. The present study analyzed the effects of two human MATP mutations, D93N, which causes oculocutaneous albinism 4 (OCA4), and L374F, which is correlated with light pigmentation in European populations. Corresponding mutations were produced in the related and well-characterized sucrose transporter from rice, OsSUT1, and transport activity was measured by heterologous expression in Xenopus laevis oocytes, in addition to 14C-sucrose uptake in yeast. The mutation corresponding to D93N resulted in a complete loss of transport activity. The mutation corresponding to L374F resulted in a 90% decrease in transport activity, although the substrate affinity was unaffected. The results indicated that the D93N mutation causes OCA4 as a result of loss of MATP transport activity, and that the F374 allele confers significantly lower transport activity than L374.

  10. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  11. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior.

    PubMed

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-08-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker's yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  12. Characterization, localization, and seasonal changes of the sucrose transporter FeSUT1 in the phloem of Fraxinus excelsior

    PubMed Central

    Öner-Sieben, Soner; Rappl, Christine; Sauer, Norbert; Stadler, Ruth; Lohaus, Gertrud

    2015-01-01

    Trees are generally assumed to be symplastic phloem loaders. A typical feature for most wooden species is an open minor vein structure with symplastic connections between mesophyll cells and phloem cells, which allow sucrose to move cell-to-cell through the plasmodesmata into the phloem. Fraxinus excelsior (Oleaceae) also translocates raffinose family oligosaccharides in addition to sucrose. Sucrose concentration was recently shown to be higher in the phloem sap than in the mesophyll cells. This suggests the involvement of apoplastic steps and the activity of sucrose transporters in addition to symplastic phloem-loading processes. In this study, the sucrose transporter FeSUT1 from F. excelsior was analysed. Heterologous expression in baker’s yeast showed that FeSUT1 mediates the uptake of sucrose. Immunohistochemical analyses revealed that FeSUT1 was exclusively located in phloem cells of minor veins and in the transport phloem of F. excelsior. Further characterization identified these cells as sieve elements and possibly ordinary companion cells but not as intermediary cells. The localization and expression pattern point towards functions of FeSUT1 in phloem loading of sucrose as well as in sucrose retrieval. FeSUT1 is most likely responsible for the observed sucrose gradient between mesophyll and phloem. The elevated expression level of FeSUT1 indicated an increased apoplastic carbon export activity from the leaves during spring and late autumn. It is hypothesized that the importance of apoplastic loading is high under low-sucrose conditions and that the availability of two different phloem-loading mechanisms confers advantages for temperate woody species like F. excelsior. PMID:26022258

  13. Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth.

    PubMed

    Eom, Joon-Seob; Cho, Jung-Il; Reinders, Anke; Lee, Sang-Won; Yoo, Youngchul; Tuan, Pham Quoc; Choi, Sang-Bong; Bang, Geul; Park, Youn-Il; Cho, Man-Ho; Bhoo, Seong Hee; An, Gynheung; Hahn, Tae-Ryong; Ward, John M; Jeon, Jong-Seong

    2011-09-01

    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.

  14. SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation.

    PubMed

    Pommerrenig, Benjamin; Popko, Jennifer; Heilmann, Mareike; Schulmeister, Sylwia; Dietel, Katharina; Schmitt, Bianca; Stadler, Ruth; Feussner, Ivo; Sauer, Norbert

    2013-02-01

    The Arabidopsis SUC5 protein represents a classical sucrose/H(+) symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. © 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.

  15. SUCROSE TRANSPORTER 5 supplies Arabidopsis embryos with biotin and affects triacylglycerol accumulation

    PubMed Central

    Pommerrenig, Benjamin; Popko, Jennifer; Heilmann, Mareike; Schulmeister, Sylwia; Dietel, Katharina; Schmitt, Bianca; Stadler, Ruth; Feussner, Ivo; Sauer, Norbert

    2013-01-01

    The Arabidopsis SUC5 protein represents a classical sucrose/H+ symporter. Functional analyses previously revealed that SUC5 also transports biotin, an essential co-factor for fatty acid synthesis. However, evidence for a dual role in transport of the structurally unrelated compounds sucrose and biotin in plants was lacking. Here we show that SUC5 localizes to the plasma membrane, and that the SUC5 gene is expressed in developing embryos, confirming the role of the SUC5 protein as substrate carrier across apoplastic barriers in seeds. We show that transport of biotin but not of sucrose across these barriers is impaired in suc5 mutant embryos. In addition, we show that SUC5 is essential for the delivery of biotin into the embryo of biotin biosynthesis-defective mutants (bio1 and bio2). We compared embryo and seedling development as well as triacylglycerol accumulation and fatty acid composition in seeds of single mutants (suc5, bio1 or bio2), double mutants (suc5 bio1 and suc5 bio2) and wild-type plants. Although suc5 mutants were like the wild-type, bio1 and bio2 mutants showed developmental defects and reduced triacylglycerol contents. In suc5 bio1 and suc5 bio2 double mutants, developmental defects were severely increased and the triacylglycerol content was reduced to a greater extent in comparison to the single mutants. Supplementation with externally applied biotin helped to reduce symptoms in both single and double mutants, but the efficacy of supplementation was significantly lower in double than in single mutants, showing that transport of biotin into the embryo is lower in the absence of SUC5. PMID:23031218

  16. Transport and metabolism of a sucrose analog (1'-fluorosucrose) into Zea mays L. Endosperm without invertase hydrolysis

    SciTech Connect

    Schmalstig, J.G.; Hitz, W.D.

    1987-12-01

    1'-fluorosucrose (FS), a sucrose analog resistant to hydrolysis by invertase, was transported from husk leaves into maize (Zea mays L.) kernels with the same magnitude and kinetics as sucrose. /sup 14/C-Label from (/sup 14/C)FS and (/sup 14/C)sucrose in separate experiments was distributed similarly between the pedicel, endosperm, and embryo with time. FS passed through maternal tissue and was adsorbed intact into the endosperm where it was metabolized and used in synthesis of sucrose and methanol-chloroform-water insolubles. Accumulation of (/sup 14/C)sucrose from supplied (/sup 14/C)glucosyl-FS indicated that the glucose moiety from the breakdown of sucrose (here FS), which normally occurs in the process of starch synthesis in maize endosperm, was available to the pool of substrates for resynthesis of sucrose. Uptake of FS into maize endosperm without hydrolysis suggest that despite the presence of invertase in maternal tissues and the hydrolysis of a large percentage of sucrose unloaded form the phloem, hexoses are not specifically needed for uptake into maize endosperm.

  17. Structure, phylogeny, allelic haplotypes and expression of sucrose transporter gene families in Saccharum.

    PubMed

    Zhang, Qing; Hu, Weichang; Zhu, Fan; Wang, Liming; Yu, Qingyi; Ming, Ray; Zhang, Jisen

    2016-02-01

    Sugarcane is an economically important crop contributing to about 80% of the world sugar production. Increasing efforts in molecular biological studies have been performed for improving the sugar yield and other relevant important agronomic traits. However, due to sugarcane's complicated genomes, it is still challenging to study the genetic basis of traits, such as sucrose accumulation. Sucrose transporters (SUTs) are critical for both phloem loading in source tissue and sucrose uptaking in sink tissue, and are considered to be the control points for regulating sucrose storage. However, no genomic study for sugarcane sucrose transporter (SsSUT) families has been reported up to date. By using comparative genomics and bacterial artificial chromosomes (BACs), six SUT genes were identified and characterized in S. spontaenum. Phylogenetic analyses revealed that the two pairs SsSUTs (SsSUT1/SsSUT3 and SsSUT5/SsSUT6) could be clustered together into two separate monocot specific SUT groups, while SsSUT2 and SsSUT4 were separated into the other two groups, with members from both dicot and monocot species. Gene structure comparison demonstrated that the number and position of exons/introns in SUTs were highly conserved among the close orthologs; in contrast, there were variations among the paralogous SUTs in Sacchuarm. Though with the high polyploidy level, gene allelic haplotype comparative analysis showed that the examined four SsSUT members exhibited conservations of gene structures and amino acid sequences among the allelic haplotypes accompanied by variations of intron sizes. Gene expression analyses were performed for tissues from seedlings under drought stress and mature plants of three Saccharum species (S.officinarnum, S.spotaneum and S.robustum). Both SUT1 and SUT4 expressed abundantly at different conditions. SUT2 had similar expression level in all of the examined tissues, but SUT3 was undetectable. Both of SUT5 and SUT6 had lower expression level than other

  18. Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.

    PubMed

    Liu, Xiaoqin; Huang, Daimin; Tao, Jinyuan; Miller, Anthony J; Fan, Xiaorong; Xu, Guohua

    2014-10-01

    A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.

  19. Proton-driven sucrose symport and antiport are provided by the vacuolar transporters SUC4 and TMT1/2.

    PubMed

    Schulz, Alexander; Beyhl, Diana; Marten, Irene; Wormit, Alexandra; Neuhaus, Ekkehard; Poschet, Gernot; Büttner, Michael; Schneider, Sabine; Sauer, Norbert; Hedrich, Rainer

    2011-10-01

    The vacuolar membrane is involved in solute uptake into and release from the vacuole, which is the largest plant organelle. In addition to inorganic ions and metabolites, large quantities of protons and sugars are shuttled across this membrane. Current models suggest that the proton gradient across the membrane drives the accumulation and/or release of sugars. Recent studies have associated AtSUC4 with the vacuolar membrane. Some members of the SUC family are plasma membrane proton/sucrose symporters. In addition, the sugar transporters TMT1 and TMT2, which are localized to the vacuolar membrane, have been suggested to function in proton-driven glucose antiport. Here we used the patch-clamp technique to monitor carrier-mediated sucrose transport by AtSUC4 and AtTMTs in intact Arabidopsis thaliana mesophyll vacuoles. In the whole-vacuole configuration with wild-type material, cytosolic sucrose-induced proton currents were associated with a proton/sucrose antiport mechanism. To identify the related transporter on one hand, and to enable the recording of symporter-mediated currents on the other hand, we electrophysiologically characterized vacuolar proteins recognized by Arabidopsis mutants of partially impaired sugar compartmentation. To our surprise, the intrinsic sucrose/proton antiporter activity was greatly reduced when vacuoles were isolated from plants lacking the monosaccharide transporter AtTMT1/TMT2. Transient expression of AtSUC4 in this mutant background resulted in proton/sucrose symport activity. From these studies, we conclude that, in the natural environment within the Arabidopsis cell, AtSUC4 most likely catalyses proton-coupled sucrose export from the vacuole. However, TMT1/2 probably represents a proton-coupled antiporter capable of high-capacity loading of glucose and sucrose into the vacuole. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  20. Essential role of K(+) uptake permease (Kup) for resistance to sucrose-induced stress in Gluconacetobacter diazotrophicus PAl 5.

    PubMed

    de Oliveira, Marcos V V; Intorne, Aline C; Vespoli, Luciano de S; Andrade, Leandro F; Pereira, Leandro de M; Rangel, Patrícia L; de Souza Filho, Gonçalo A

    2017-04-01

    Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K(+) uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K(+) transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  1. Aspen SUCROSE TRANSPORTER3 Allocates Carbon into Wood Fibers1[C][W

    PubMed Central

    Mahboubi, Amir; Ratke, Christine; Gorzsás, András; Kumar, Manoj; Mellerowicz, Ewa J.; Niittylä, Totte

    2013-01-01

    Wood formation in trees requires carbon import from the photosynthetic tissues. In several tree species, including Populus species, the majority of this carbon is derived from sucrose (Suc) transported in the phloem. The mechanism of radial Suc transport from phloem to developing wood is not well understood. We investigated the role of active Suc transport during secondary cell wall formation in hybrid aspen (Populus tremula × Populus tremuloides). We show that RNA interference-mediated reduction of PttSUT3 (for Suc/H+ symporter) during secondary cell wall formation in developing wood caused thinner wood fiber walls accompanied by a reduction in cellulose and an increase in lignin. Suc content in the phloem and developing wood was not significantly changed. However, after 13CO2 assimilation, the SUT3RNAi lines contained more 13C than the wild type in the Suc-containing extract of developing wood. Hence, Suc was transported into developing wood, but the Suc-derived carbon was not efficiently incorporated to wood fiber walls. A yellow fluorescent protein:PttSUT3 fusion localized to plasma membrane, suggesting that reduced Suc import into developing wood fibers was the cause of the observed cell wall phenotype. The results show the importance of active Suc transport for wood formation in a symplasmically phloem-loading tree species and identify PttSUT3 as a principal transporter for carbon delivery into secondary cell wall-forming wood fibers. PMID:24170204

  2. Synthesis and molecular modeling of new 1-aryl-3-[4-arylpiperazin-1-yl]-1-propane derivatives with high affinity at the serotonin transporter and at 5-HT(1A) receptors.

    PubMed

    Orús, Lara; Pérez-Silanes, Silvia; Oficialdegui, Ana-M; Martínez-Esparza, Javier; Del Castillo, Juan-C; Mourelle, Marisa; Langer, Thierry; Guccione, Salvatore; Donzella, Giuseppina; Krovat, Eva M; Poptodorov, Konstantin; Lasheras, Berta; Ballaz, Santiago; Hervías, Isabel; Tordera, Rosa; Del Río, Joaquín; Monge, Antonio

    2002-09-12

    It has been proposed that 5-HT(1A) receptor antagonists augment the antidepressant efficacy of selective serotonin (5-HT) reuptake inhibitors. In a search toward new and efficient antidepressants, 1-(aryl)-3-[4-arylpiperazin-1-yl]-1-propane molecular hybrids were designed, synthesized, and evaluated for 5-HT reuptake inhibition and 5-HT(1A) receptor affinity. The design was based in coupling structural moieties related to inhibition of serotonin reuptake, such as benzo[b]thiophene derivatives to arylpiperazines, typical 5-HT(1A) receptor ligands. In binding studies, several compounds showed affinity at the 5-HT transporter and at 5-HT(1A) receptors. Molecular modeling studies predicted the pharmacophore elements required for high affinity binding and the features that enable to discriminate between agonist, partial agonist, or antagonist action at 5-HT(1A) receptors and 5-HT transporter inhibition. Solvent interactions in desolvation prior to the binding step along with enthalpy and enthropy compensations might be responsible to explain agonist, partial agonist, and antagonist character. Hydrogen-bonding capability seems to be important to break hydrogen interhelical hydrogen bonds or alternatively to form other bonds upon ligand binding. Partial agonists and antagonists are unable to do this as the full agonist, which interacts closely by long-range forces or directly. The compounds showing the higher affinity at both the 5-HT transporter (K(i) < 50 nM) and the 5-HT(1A) receptors (K(i) < 20 nM) were further explored for their ability to stimulate [(35)S]GTPgammaS binding or to antagonize 8-hydroxy-2-di-n-propylamino-tetralin (8-OH-DPAT)-stimulated [(35)]GTPgammaS binding to rat hippocampal membranes, an index of agonist/antagonist action at 5-HT(1A) receptors, respectively. Compound 8g exhibited agonist activity (EC(50) = 30 nM) in this assay, whereas compounds 7g and 8h,i behaved as weak partial agonists and 7h-j and 8j,l antagonized the R(+)-8-OH

  3. Molecular cloning and expression analysis of a gene for sucrose transporter from pear (Pyrus bretschneideri Rehd.) fruit.

    PubMed

    Zhang, Huping; Zhang, Shujun; Qin, Gaihua; Wang, Lifen; Wu, Tao; Qi, Kaijie; Zhang, Shaoling

    2013-12-01

    Here we report the cloning of a sucrose transporter cDNA from pear (Pyrus bretschneideri Rehd. cv 'Yali') fruit and an analysis of the expression of the gene. A cDNA clone, designated PbSUT1 was identified as a sucrose transporter cDNA from its sequence homology at the amino acid level to sucrose transporters that have been cloned from other higher plant species. PbSUT1 potentially encoded a protein of 499 amino acid residues with a predicted molecular mass of 53.4 kDa and an isoelectric point (pI) of 9.21. Phylogenetic analysis revealed that the PbSUT1 belonged to type III SUTs and was more closely related to the MdSUT1 from apple fruit. Some major facilitator superfamily (MFS)-specific sequence motifs were found in the predicted PbSUT1 peptides, and an MFS_1 domain was located at the amino acid positions of 29-447 of the sequence. A study of gene expression along fruit development showed that PbSUT1 transcripts are present at all stages but significantly increase before fruit enlargement and during the ripening process with increasing sucrose levels. In contrast, the expression levels don't change much during the period of rapid fruit growth. This work shows that sucrose transporter may play a role in the accumulation of sugars during maturation and in maintaining the internal cellular distribution.

  4. Molecular cloning and expression analysis of the sucrose transporter gene family from Theobroma cacao L.

    PubMed

    Li, Fupeng; Wu, Baoduo; Qin, Xiaowei; Yan, Lin; Hao, Chaoyun; Tan, Lehe; Lai, Jianxiong

    2014-08-10

    In this study, we performed cloning and expression analysis of six putative sucrose transporter genes, designated TcSUT1, TcSUT2, TcSUT3, TcSUT4, TcSUT5 and TcSUT6, from the cacao genotype 'TAS-R8'. The combination of cDNA and genomic DNA sequences revealed that the cacao SUT genes contained exon numbers ranging from 1 to 14. The average molecular mass of all six deduced proteins was approximately 56 kDa (range 52 to 66 kDa). All six proteins were predicted to exhibit typical features of sucrose transporters with 12 trans-membrane spanning domains. Phylogenetic analysis revealed that TcSUT2 and TcSUT4 belonged to Group 2 SUT and Group 4 SUT, respectively, and the other TcSUT proteins were belonging to Group 1 SUT. Real-time PCR was conducted to investigate the expression pattern of each member of the SUT family in cacao. Our experiment showed that TcSUT1 was expressed dominantly in pods and that, TcSUT3 and TcSUT4 were highly expressed in both pods and in bark with phloem. Within pods, TcSUT1 and TcSUT4 were expressed more in the seed coat and seed from the pod enlargement stage to the ripening stage. TcSUT5 expression sharply increased to its highest expression level in the seed coat during the ripening stage. Expression pattern analysis indicated that TcSUT genes may be associated with photoassimilate transport into developing seeds and may, therefore, have an impact on seed production. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

    PubMed Central

    Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; Harding, Scott A.

    2016-01-01

    Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism in root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability. PMID:27641356

  6. Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression.

    PubMed

    Xue, Liang-Jiao; Frost, Christopher J; Tsai, Chung-Jui; Harding, Scott A

    2016-09-19

    Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism in root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.

  7. Molecular regulation of sucrose catabolism and sugar transport for development, defence and phloem function.

    PubMed

    Li, Jun; Wu, Limin; Foster, Ryan; Ruan, Yong-Ling

    2017-05-01

    Sucrose (Suc) is the major end product of photosynthesis in mesophyll cells of most vascular plants. It is loaded into phloem of mature leaves for long-distance translocation to non-photosynthetic organs where it is unloaded for diverse uses. Clearly, Suc transport and metabolism is central to plant growth and development and the functionality of the entire vascular system. Despite vast information in the literature about the physiological roles of individual sugar metabolic enzymes and transporters, there is a lack of systematic evaluation about their molecular regulation from transcriptional to post-translational levels. Knowledge on this topic is essential for understanding and improving plant development, optimizing resource distribution and increasing crop productivity. We therefore focused our analyses on molecular control of key players in Suc metabolism and transport, including: (i) the identification of promoter elements responsive to sugars and hormones or targeted by transcription factors and microRNAs degrading transcripts of target genes; and (ii) modulation of enzyme and transporter activities through protein-protein interactions and other post-translational modifications. We have highlighted major remaining questions and discussed opportunities to exploit current understanding to gain new insights into molecular control of carbon partitioning for improving plant performance. © 2017 Institute of Botany, Chinese Academy of Sciences.

  8. Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12.

    PubMed Central

    Gehring, K; Hofnung, M; Nikaido, H

    1990-01-01

    Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells. Glutamine transport via a low-affinity system was not affected. Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids. Maltose transport was strongly inhibited by osmotic stress. PMID:2198275

  9. The Medicago truncatula sucrose transporter family: characterization and implication of key members in carbon partitioning towards arbuscular mycorrhizal fungi.

    PubMed

    Doidy, Joan; van Tuinen, Diederik; Lamotte, Olivier; Corneillat, Marion; Alcaraz, Gérard; Wipf, Daniel

    2012-11-01

    We identified de novo sucrose transporter (SUT) genes involved in long-distance transport of sucrose from photosynthetic source leaves towards sink organs in the model leguminous species Medicago truncatula. The identification and functional analysis of sugar transporters provide key information on mechanisms that underlie carbon partitioning in plant-microorganism interactions. In that way, full-length sequences of the M. truncatula SUT (MtSUT) family were retrieved and biochemical characterization of MtSUT members was performed by heterologous expression in yeast. The MtSUT family now comprises six genes which distribute among Dicotyledonous clades. MtSUT1-1 and MtSUT4-1 are key members in regard to their expression profiles in source leaves and sink roots and were characterized as functional H(+)/sucrose transporters. Physiological and molecular responses to phosphorus supply and inoculation by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was studied by gene expression and sugar quantification analyses. Sucrose represents the main sugar transport form in M. truncatula and the expression profiles of MtSUT1-1, MtSUT2, and MtSUT4-1 highlight a fine-tuning regulation for beneficial sugar fluxes towards the fungal symbiont. Taken together, these results suggest distinct functions for proteins from the SUT1, SUT2, and SUT4 clades in plant and in biotrophic interactions.

  10. Sucrose Transporter Localization and Function in Phloem Unloading in Developing Stems.

    PubMed

    Milne, Ricky J; Perroux, Jai M; Rae, Anne L; Reinders, Anke; Ward, John M; Offler, Christina E; Patrick, John W; Grof, Christopher P L

    2017-02-01

    How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5-SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Sucrose Transporter Localization and Function in Phloem Unloading in Developing Stems1[OPEN

    PubMed Central

    Rae, Anne L.; Reinders, Anke

    2017-01-01

    How sucrose transporters (SUTs) regulate phloem unloading in monocot stems is poorly understood and particularly so for species storing high Suc concentrations. To this end, Sorghum bicolor SUTs SbSUT1 and SbSUT5 were characterized by determining their transport properties heterologously expressed in yeast or Xenopus laevis oocytes, and their in planta cellular and subcellular localization. The plasma membrane-localized SbSUT1 and SbSUT5 exhibited a strong selectivity for Suc and high Suc affinities in X. laevis oocytes at pH 5—SbSUT1, 6.3 ± 0.7 mm, and SbSUT5, 2.4 ± 0.5 mm Suc. The Suc affinity of SbSUT1 was dependent on membrane potential and pH. In contrast, SbSUT5 Suc affinity was independent of membrane potential and pH but supported high transport rates at neutral pH. Suc transport by the tonoplast localized SbSUT4 could not be detected using yeast or X. laevis oocytes. Across internode development, SUTs, other than SbSUT4, were immunolocalized to sieve elements, while for elongating and recently elongated internodes, SUTs also were detected in storage parenchyma cells. We conclude that apoplasmic Suc unloading from de-energized protophloem sieve elements in meristematic zones may be mediated by reversal of SbSUT1 and/or by uniporting SWEETs. Storage parenchyma localized SbSUT1 and SbSUT5 may accumulate Suc from the stem apoplasms of elongating and recently elongated internodes, whereas SbSUT4 may function to release Suc from vacuoles. Transiting from an apoplasmic to symplasmic unloading pathway as the stem matures, SbSUT1 and SbSUT5 increasingly function in Suc retrieval into metaphloem sieve elements to maintain a high turgor to drive symplasmic unloading by bulk flow. PMID:27986867

  12. Sucrose Concentration Gradients along the Post-Phloem Transport Pathway in the Maternal Tissues of Developing Wheat Grains.

    PubMed Central

    Fisher, D. B.; Wang, N.

    1995-01-01

    Sucrose concentrations were measured in serial frozen sections of the post-phloem transport pathway in developing wheat (Triticum aestivum L.) grains. In normally importing grains, there was an approximately linear concentration gradient along the pathway, with a difference between the ends of the pathway of about 180 mM. This indicates an unusually low resistance for cell-to-cell transport, due perhaps to the large size-exclusion limit for the pathway. However, the existence of concentration gradients raises presently unresolvable questions about the relative contributions of diffusion versus bulk flow to transport within the symplast. The concentration gradient disappeared when sucrose movement ceased (i.e. in excised grains or when endosperm cavities of attached grains were perfused with p-chloromercuribenzene sulfonate [PCMBS] or with 1660 mOsm sorbitol). PCMBS appeared to block solute release into the endosperm cavity, whereas the sorbitol treatment, previously shown to cause localized plasmolysis in the chalaza, appeared to block movement across the chalaza. Sieve element/companion cell unloading appears to be an important control point for assimilate import. The sucrose concentration gradient and, probably, turgor and osmotic gradients are extremely steep there. PCMBS blocked import without affecting the sucrose concentration in the vascular parenchyma around the phloem. Thus, blockage of unloading was more complex than a simple "backing up" of solutes in the vascular parenchyma. PMID:12228615

  13. Sucrose transport into plasma membrane vesicles from tobacco leaves by H+ symport or counter exchange does not display a linear component.

    PubMed

    Borstlap, A C; Schuurmans, J A M J

    2004-03-01

    Uptake of [14C]sucrose by plasma membrane vesicles from leaves of tobacco (Nicotiana tabacum L.) was measured after the imposition of an inwardly directed proton gradient (delta pH = 2) and an electrical gradient (delta psi = -68 mV, inside negative) across the vesicle membrane. The vesicles were isolated from a microsomal fraction by two-phase partitioning using media that contained 330 mM of either sorbitol or sucrose. Sucrose transport into vesicles isolated using the sorbitol-containing media showed the hallmarks of electrogenic H+-symport, as it was highly dependent on delta pH, could be increased three- to four-fold by delta psi, and was abolished by carbonylcyanide m-chlorophenylhydrazone (CCCP). Transport of [14C]sucrose into vesicles that were isolated using the sucrose-containing media apparently occurred by counter exchange. Its initial influx also depended on a low external pH, but it was insensitive to CCCP and hardly stimulated by delta psi. Both symport and counter exchange obeyed simple Michaelis-Menten kinetics. Transport that depends linearly on the external sucrose concentration could not be detected, indicating that the 'linear' component that has been observed in sucrose uptake by leaf tissues does not represent a transport route that is provided by the sucrose symporter. The potential role of H+/sucrose-symporters in phloem unloading is briefly discussed.

  14. Functional characterization of a plant-like sucrose transporter from the beneficial fungus Trichoderma virens. Regulation of the symbiotic association with plants by sucrose metabolism inside the fungal cells.

    PubMed

    Vargas, Walter A; Crutcher, Frankie K; Kenerley, Charles M

    2011-02-01

    Sucrose exuded by plants into the rhizosphere is a crucial component for the symbiotic association between the beneficial fungus Trichoderma and plant roots. In this article we sought to identify and characterize the molecular basis of sucrose uptake into the fungal cells. • Several bioinformatics tools enabled us to identify a plant-like sucrose transporter in the genome of Trichoderma virens Gv29-8 (TvSut). Gene expression profiles in the fungal cells were analyzed by Northern blotting and quantitative real-time PCR (qRT-PCR). Biochemical and physiological studies were conducted on Gv29-8 and fungal strains impaired in the expression of TvSut. • TvSut exhibits biochemical properties similar to those described for sucrose symporters from plants. The null expression of tvsut caused a detrimental effect on fungal growth when sucrose was the sole source of carbon in the medium, and also affected the expression of genes involved in the symbiotic association. • Similar to plants, T. virens contains a highly specific sucrose/H(+) symporter that is induced in the early stages of root colonization. Our results suggest an active sucrose transference from the plant to the fungal cells during the beneficial associations. In addition, our expression experiments suggest the existence of a sucrose-dependent network in the fungal cells that regulates the symbiotic association. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

  15. AmSUT1, a sucrose transporter in collection and transport phloem of the putative symplastic phloem loader Alonsoa meridionalis.

    PubMed

    Knop, Christian; Stadler, Ruth; Sauer, Norbert; Lohaus, Gertrud

    2004-01-01

    A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80-91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading.

  16. Sucrose transporter NtSUT4 from tobacco BY-2 involved in plant cell shape during miniprotoplast culture.

    PubMed

    Okubo-Kurihara, Emiko; Higaki, Takumi; Kurihara, Yukio; Kutsuna, Natsumaro; Yamaguchi, Junji; Hasezawa, Seiichiro

    2011-05-01

    Sucrose plays an important role in several cellular processes since it is a general source of metabolic energy, serves as a precursor for starch and cellulose synthesis, and is a metabolic starting point for carboxylate- and amino acid synthesis. While plant vacuole is the main cellular storage pool, where sucrose accumulates to high concentrations, only a small number of vacuolar sugar transporters have been identified and characterized to date. We initially identified a vacuolar sucrose transporter (NtSUT4) from tobacco BY-2 cells and established transgenic tobacco BY-2 cell lines that overexpress NtSUT4-GFP (BY-SUTG cells). Using a model system for synchronous cell elongation in miniprotoplasts (evacuolated cells) prepared from tobacco BY-2 cells, we found that NtSUT4-GFP overexpression inhibited cell growth towards the cell major axis. Moreover, under the same conditions, we found that the cell walls were well stained by calcofluor in BY-SUTG cells than in wild type BY-2 cells. These results suggest that NtSUT4 is involved in cell shape via sucrose homeostasis in plant cells.

  17. Identification of a new sucrose transporter in rye-grass (LpSUT2): effect of defoliation and putative fructose sensing.

    PubMed

    Berthier, Alexandre; Meuriot, Frédéric; Dédaldéchamp, Fabienne; Lemoine, Rémi; Prud'homme, Marie-Pascale; Noiraud-Romy, Nathalie

    2014-11-01

    Rye-grass fast regrowth after defoliation results from an efficient mobilization of C reserves which are transported as sucrose towards regrowing leaves, and which can be supported by one or several sucrose transporters (SUTs) like LpSUT1. Therefore, our objectives were to isolate, identify, characterize and immunolocalize such sucrose transporters. A protein (LpSUT2) showing a twelve spanning trans-membrane domain, extended N terminal and internal cytoplasmic loop, and kinetic properties consistent with well-known sucrose transporters, was isolated and successfully characterized. Along with LpSUT1, it was mainly localized in mesophyll cells of leaf sheaths and elongating leaf bases. These transporters were also found in parenchyma bundle sheath (PBS) cells but they were not detected in the sieve element/companion cell complex of the phloem. Unlike LpSUT1 transcript levels which increased as a response to defoliation in source and sink tissues, LpSUT2 transcript levels were unaffected by defoliation and weakly expressed. Interestingly, sucrose transport by LpSUT2 was inhibited by fructose. LpSUT1 and LpSUT2 appeared to have different functions. LpSUT1 is proposed to play a key role in C storage and mobilization by allowing sucrose transport between PBS and mesophyll cells, depending on the plant C status. LpSUT2 could be involved in sucrose/fructose sensing at sub-cellular level. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  18. Organ and Tissue-specific Sucrose Transporters. Important Hubs in Gene and Metabolite Networks Regulating Carbon Use in Wood-forming Tissues of Populus

    SciTech Connect

    Harding, Scott A.; Tsai, Chung-Jui

    2016-01-04

    The overall project objective was to probe the relationship between sucrose transporters and plant productivity in the biomass for biofuels woody perennial, Populus. At the time the proposal was written, sucrose transporters had already been investigated in many plant model systems, primarily with respect to the export of photosynthate sucrose from source leaves, and the uptake of sucrose in storage organs and seeds. Preliminary findings by the PI found that in Populus, sucrose transporter genes (SUTs) were well expressed in wood-forming tissues that comprise the feedstock for biofuels production. Because sucrose comprises by far the predominant form in which photosynthate is delivered from source organs to sink organs like roots and wood-forming tissues, SUTs control a gate that nominally at least could impact the allocation or partitioning of sucrose for potentially competing end uses like growth (stem biomass) and storage. In addition, water use might be conditioned by the way in which sucrose is distributed throughout the plant, and/or by the way in which sucrose is partitioned intracellularly. Several dozen transgenic lines were produced in year 1 of the project to perturb the expression ratio of multiple plasma membrane (PM) SUTs (intercellular trafficking), versus the single tonoplast membrane (TM) sucrose transporter that effectively regulates intracellular trafficking of sucrose. It was possible to obtain transgenic lines with dual SUT gene knockdown using the 35S promoter, but not the wood-specific TUA1 promoter. By the end of project year 2, a decision was made to work with the 35S plants while archiving the TUA1 plants. The PhD candidate charged with producing the transgenic lines abandoned the project during its second year, substantially contributing to the decision to operate with just the 35S lines. That student’s interests ranged more toward evolutionary topics, and a report on SUT gene evolution was published (Peng et al 2014).

  19. Putative role of the H(+)/sucrose symporter SLC45A3 as an osmolyte transporter in the kidney.

    PubMed

    Vitavska, Olga; Edemir, Bayram; Wieczorek, Helmut

    2016-08-01

    The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown. Here, we report that SLC45A3 occurs in the kidney and is highly expressed in the medullary collecting duct (IMCD), a part of the kidney responsible for final urine concentration and faced to hyperosmotic environment. Moreover, messenger RNA (mRNA) expression of endogenous SLC45A3 in rat IMCD cells as well as in NRK52E cells increased up to four-fold under hyperosmotic conditions at 600 mOsmol/kg. Using NRK52E cells as an experimental model, we investigated the proton-coupled sugar transport and found that the uptake of sucrose or glucose was enhanced by hyperosmolarity. Down-regulation of expression by small interfering RNA (siRNA) decreased the osmotically inducible part of sucrose uptake and confirmed the involvement of SLC45A3 in this process. Furthermore, we observed an up to four-fold elevation of sucrose uptake triggered by hyperosmolarity across the apical membrane of NRK52E cells, while uptake across the basolateral membrane was not affected. Due to this finding, we conclude that SLC45A3 may occur at the luminal side of kidney epithelial cells and thus may take up solutes from the tubular fluid. Altogether, we show that SLC45A3 is a novel sugar transporter in kidney and hypothesise that the disaccharide sucrose, and probably the monosaccharides glucose and fructose, may serve as compatible osmolytes in urine.

  20. A novel sucrose/H+ symport system and an intracellular sucrase in Leishmania donovani.

    PubMed

    Singh, Arpita; Mandal, Debjani

    2011-07-01

    The flagellated form of pathogenic parasitic protozoa Leishmania, resides in the alimentary tract of its sandfly vector, where sucrose serves as a major nutrient source. In this study we report the presence of a sucrose transport system in Leishmania donovani promastigotes. The kinetics of sucrose uptake in promastigotes are biphasic in nature with both high affinity K(m) (K(m) of ∼ 75 μM) and low affinity K(m) (K(m)∼ 1.38 mM) components. By contrast the virulent amastigotes take up sucrose via a low affinity process with a K(m) of 2.5mM. The transport of sucrose into promastigotes leads to rapid intracellular acidification, as indicated by changes in the fluorescence of the pH indicator 2',7'-bis-(2-carboxyethyl)-5-(6) Carboxyfluorescein (BCECF). In experiments with right side-out plasma membrane vesicles derived from L. donovani promastigotes, an artificial pH gradient was able to drive the active accumulation of sucrose. These data are consistent with the operation of a H(+)-sucrose symporter. The symporter was shown to be independent of Na(+) and to be insensitive to cytochalasin B, to the flavonoid phloretin and to the Na(+)/K(+) ATPase inhibitor ouabain. However, the protonophore carbonylcyanide P- (trifluromethoxy) phenylhydrazone (FCCP) and a number of thiol reagents caused significant inhibition of sucrose uptake. Evidence was also obtained for the presence of a stable intracellular pool of the sucrose splitting enzyme, sucrase, in promastigote stage parasites. The results are consistent with the hypothesis that L. donovani promastigotes take up sucrose via a novel H(+)-sucrose symport system and that, on entering the cell, the sucrose is hydrolysed to its component monosaccharides by an intracellular sucrase, thereby providing an energy source for the parasites. Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  1. Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza.

    PubMed

    Bitterlich, Michael; Krügel, Undine; Boldt-Burisch, Katja; Franken, Philipp; Kühn, Christina

    2014-01-01

    Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d(x) defective in BR synthesis (1) showed significantly reduced mycorrhization parameters. (2) The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.

  2. Functional characterisation and cell specificity of BvSUT1, the transporter that loads sucrose into the phloem of sugar beet (Beta vulgaris L.) source leaves.

    PubMed

    Nieberl, P; Ehrl, C; Pommerrenig, B; Graus, D; Marten, I; Jung, B; Ludewig, F; Koch, W; Harms, K; Flügge, U-I; Neuhaus, H E; Hedrich, R; Sauer, N

    2017-05-01

    Sugar beet (Beta vulgaris L.) is one of the most important sugar-producing plants worldwide and provides about one third of the sugar consumed by humans. Here we report on molecular characterisation of the BvSUT1 gene and on the functional characterisation of the encoded transporter. In contrast to the recently identified tonoplast-localised sucrose transporter BvTST2.1 from sugar beet taproots, which evolved within the monosaccharide transporter (MST) superfamily, BvSUT1 represents a classical sucrose transporter and is a typical member of the disaccharide transporter (DST) superfamily. Transgenic Arabidopsis plants expressing the β-GLUCURONIDASE (GUS) reporter gene under control of the BvSUT1-promoter showed GUS histochemical staining of their phloem; an anti-BvSUT1-antiserum identified the BvSUT1 transporter specifically in phloem companion cells. After expression of BvSUT1 cDNA in bakers' yeasts (Saccharomyces cerevisiae) uptake characteristics of the BvSUT1 protein were studied. Moreover, the sugar beet transporter was characterised as a proton-coupled sucrose symporter in Xenopus laevis oocytes. Our findings indicate that BvSUT1 is the sucrose transporter that is responsible for loading of sucrose into the phloem of sugar beet source leaves delivering sucrose to the storage tissue in sugar beet taproot sinks.

  3. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    PubMed

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  4. Switching the mode of sucrose utilization by Saccharomyces cerevisiae

    PubMed Central

    Badotti, Fernanda; Dário, Marcelo G; Alves, Sergio L; Cordioli, Maria Luiza A; Miletti, Luiz C; de Araujo, Pedro S; Stambuk, Boris U

    2008-01-01

    Background Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H+ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures. Results We have deleted from the genome of a S. cerevisiae strain lacking invertase the high-affinity sucrose-H+ symporter encoded by the AGT1 gene. This strain could still grow efficiently on sucrose due to a low-affinity and low-capacity sucrose-H+ symport activity mediated by the MALx1 maltose permeases, and its further intracellular hydrolysis by cytoplasmic maltases. Although sucrose consumption by this engineered yeast strain was slower than with the parental yeast strain, the cells grew efficiently on sucrose due to an increased respiration of the carbon source. Consequently, this engineered yeast strain produced less ethanol and 1.5 to 2 times more biomass when cultivated in simple batch mode using 20 g/L sucrose as the carbon source. Conclusion Higher cell densities during batch cultures on 20 g/L sucrose were achieved by using a S. cerevisiae strain engineered in the sucrose uptake system. Such result was accomplished by effectively reducing sucrose

  5. High-affinity neuropeptide Y receptor antagonists.

    PubMed Central

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J

    1995-01-01

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats. PMID:7568074

  6. High-affinity neuropeptide Y receptor antagonists.

    PubMed

    Daniels, A J; Matthews, J E; Slepetis, R J; Jansen, M; Viveros, O H; Tadepalli, A; Harrington, W; Heyer, D; Landavazo, A; Leban, J J; Spaltenstein, A

    1995-09-26

    Neuropeptide Y (NPY) is one of the most abundant peptide transmitters in the mammalian brain. In the periphery it is costored and coreleased with norepinephrine from sympathetic nerve terminals. However, the physiological functions of this peptide remain unclear because of the absence of specific high-affinity receptor antagonists. Three potent NPY receptor antagonists were synthesized and tested for their biological activity in in vitro, ex vivo, and in vivo functional assays. We describe here the effects of these antagonists inhibiting specific radiolabeled NPY binding at Y1 and Y2 receptors and antagonizing the effects of NPY in human erythroleukemia cell intracellular calcium mobilization perfusion pressure in the isolated rat kidney, and mean arterial blood pressure in anesthetized rats.

  7. A cascade of sequentially expressed sucrose transporters in the seed coat and endosperm provides nutrition for the Arabidopsis embryo.

    PubMed

    Chen, Li-Qing; Lin, I Winnie; Qu, Xiao-Qing; Sosso, Davide; McFarlane, Heather E; Londoño, Alejandra; Samuels, A Lacey; Frommer, Wolf B

    2015-03-01

    Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a "wrinkled" seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential. © 2015 American Society of Plant Biologists. All rights reserved.

  8. Sulla carnosa modulates root invertase activity in response to the inhibition of long-distance sucrose transport under magnesium deficiency.

    PubMed

    Farhat, N; Smaoui, A; Maurousset, L; Porcheron, B; Lemoine, R; Abdelly, C; Rabhi, M

    2016-11-01

    Being the principal product of photosynthesis, sucrose is involved in many metabolic processes in plants. As magnesium (Mg) is phloem mobile, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed. Mg deficiency effects on carbohydrate contents and invertase activities were determined in Sulla carnosa Desf. Plants were grown hydroponically at different Mg concentrations (0.00, 0.01, 0.05 and 1.50 mM Mg) for one month. Mineral analysis showed that Mg contents were drastically diminished in shoots and roots mainly at 0.01 and 0.00 mM Mg. This decline was adversely associated with a significant increase of sucrose, fructose and mainly glucose in shoots of plants exposed to severe deficiency. By contrast, sugar contents were severely reduced in roots of these plants indicating an alteration of carbohydrate partitioning between shoots and roots of Mg-deficient plants. Cell wall invertase activity was highly enhanced in roots of Mg-deficient plants, while the vacuolar invertase activity was reduced at 0.00 mM Mg. This decrease of vacuolar invertase activity may indicate the sensibility of roots to Mg starvation resulting from sucrose transport inhibition. (14) CO2 labeling experiments were in accordance with these findings showing an inhibition of sucrose transport from source leaves to sink tissues (roots) under Mg depletion. The obtained results confirm previous findings about Mg involvement in photosynthate loading into phloem and add new insights into mechanisms evolved by S. carnosa to cope with Mg shortage in particular the increase of the activity of cell wall invertase.

  9. Direct measurement of equilibrium constants for high-affinity hemoglobins.

    PubMed

    Kundu, Suman; Premer, Scott A; Hoy, Julie A; Trent, James T; Hargrove, Mark S

    2003-06-01

    The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

  10. Identification and characterization of the three homeologues of a new sucrose transporter in hexaploid wheat (Triticum aestivum L.)

    PubMed Central

    2013-01-01

    Background Sucrose transporters (SUTs) play important roles in regulating the translocation of assimilates from source to sink tissues. Identification and characterization of new SUTs in economically important crops such as wheat provide insights into their role in determining seed yield. To date, however, only one SUT of wheat has been reported and functionally characterized. The present study reports the isolation and characterization of a new SUT, designated as TaSUT2, and its homeologues (TaSUT2A, TaSUT2B and TaSUT2D) in hexaploid wheat (Triticum aestivum L.). Results TaSUT2A and TaSUT2B genes each encode a protein with 506 amino acids, whereas TaSUT2D encodes a protein of 508 amino acids. The molecular mass of these proteins is predicted to be ~ 54 kDA. Topological analysis of the amino acid sequences of the three homeologues revealed that they contain 12 transmembrane spanning helices, which are described as distinct characteristic features of glycoside-pentoside-hexuronide cation symporter family that includes all known plant SUTs, and a histidine residue that appears to be localized at and associated conformationally with the sucrose binding site. Yeast SUSY7/ura3 strain cells transformed with TaSUT2A, TaSUT2B and TaSUT2D were able to uptake sucrose and grow on a medium containing sucrose as a sole source of carbon; however, our subcellular localization study with plant cells revealed that TaSUT2 is localized to the tonoplast. The expression of TaSUT2 was detected in the source, including flag leaf blade, flag leaf sheath, peduncle, glumes, palea and lemma, and sink (seed) tissues. The relative contributions of the three genomes of wheat to the total expression of TaSUT2 appear to differ with tissues and developmental stages. At the cellular level, TaSUT2 is expressed mainly in the vein of developing seeds and subepidermal mesophyll cells of the leaf blade. Conclusion This study demonstrated that TaSUT2 is a new wheat SUT protein. Given that TaSUT2 is

  11. The sucrose transporter SlSUT2 from tomato interacts with brassinosteroid functioning and affects arbuscular mycorrhiza formation.

    PubMed

    Bitterlich, Michael; Krügel, Undine; Boldt-Burisch, Katja; Franken, Philipp; Kühn, Christina

    2014-06-01

    Mycorrhizal plants benefit from the fungal partners by getting better access to soil nutrients. In exchange, the plant supplies carbohydrates to the fungus. The additional carbohydrate demand in mycorrhizal plants was shown to be balanced partially by higher CO2 assimilation and increased C metabolism in shoots and roots. In order to test the role of sucrose transport for fungal development in arbuscular mycorrhizal (AM) tomato, transgenic plants with down-regulated expression of three sucrose transporter genes were analysed. Plants that carried an antisense construct of SlSUT2 (SlSUT2as) repeatedly exhibited increased mycorrhizal colonization and the positive effect of plants to mycorrhiza was abolished. Grafting experiments between transgenic and wild-type rootstocks and scions indicated that mainly the root-specific function of SlSUT2 has an impact on colonization of tomato roots with the AM fungus. Localization of SISUT2 to the periarbuscular membrane indicates a role in back transport of sucrose from the periarbuscular matrix into the plant cell thereby affecting hyphal development. Screening of an expression library for SlSUT2-interacting proteins revealed interactions with candidates involved in brassinosteroid (BR) signaling or biosynthesis. Interaction of these candidates with SlSUT2 was confirmed by bimolecular fluorescence complementation. Tomato mutants defective in BR biosynthesis were analysed with respect to mycorrhizal symbiosis and showed indeed decreased mycorrhization. This finding suggests that BRs affect mycorrhizal infection and colonization. If the inhibitory effect of SlSUT2 on mycorrhizal growth involves components of BR synthesis and of the BR signaling pathway is discussed. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  12. A New Subfamily of Sucrose Transporters, SUT4, with Low Affinity/High Capacity Localized in Enucleate Sieve Elements of Plants

    PubMed Central

    Weise, Andreas; Barker, Laurence; Kühn, Christina; Lalonde, Sylvie; Buschmann, Henrik; Frommer, Wolf B.; Ward, John M.

    2000-01-01

    A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The Km for sucrose uptake by AtSUT4 of 11.6 ± 0.6 mM was ∼10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements. PMID:10948254

  13. A new subfamily of sucrose transporters, SUT4, with low affinity/high capacity localized in enucleate sieve elements of plants.

    PubMed

    Weise, A; Barker, L; Kühn, C; Lalonde, S; Buschmann, H; Frommer, W B; Ward, J M

    2000-08-01

    A new subfamily of sucrose transporters from Arabidopsis (AtSUT4), tomato (LeSUT4), and potato (StSUT4) was isolated, demonstrating only 47% similarity to the previously characterized SUT1. SUT4 from two plant species conferred sucrose uptake activity when expressed in yeast. The K(m) for sucrose uptake by AtSUT4 of 11.6 +/- 0.6 mM was approximately 10-fold greater than for all other plant sucrose transporters characterized to date. An ortholog from potato had similar kinetic properties. Thus, SUT4 corresponds to the low-affinity/high-capacity saturable component of sucrose uptake found in leaves. In contrast to SUT1, SUT4 is expressed predominantly in minor veins in source leaves, where high-capacity sucrose transport is needed for phloem loading. In potato and tomato, SUT4 was immunolocalized specifically to enucleate sieve elements, indicating that like SUT1, macromolecular trafficking is required to transport the mRNA or the protein from companion cells through plasmodesmata into the sieve elements.

  14. The potato sucrose transporter StSUT1 interacts with a DRM-associated protein disulfide isomerase.

    PubMed

    Krügel, Undine; He, Hong-Xia; Gier, Konstanze; Reins, Jana; Chincinska, Izabela; Grimm, Bernhard; Schulze, Waltraud X; Kühn, Christina

    2012-01-01

    Organization of proteins into complexes is crucial for many cellular functions. Recently, the SUT1 protein was shown to form homodimeric complexes, to be associated with lipid raft-like microdomains in yeast as well as in plants and to undergo endocytosis in response to brefeldin A. We therefore aimed to identify SUT1-interacting proteins that might be involved in dimerization, endocytosis, or targeting of SUT1 to raft-like microdomains. Therefore, we identified potato membrane proteins, which are associated with the detergent-resistant membrane (DRM) fraction. Among the proteins identified, we clearly confirmed StSUT1 as part of DRM in potato source leaves. We used the yeast two-hybrid split ubiquitin system (SUS) to systematically screen for interaction between the sucrose transporter StSUT1 and other membrane-associated or soluble proteins in vivo. The SUS screen was followed by immunoprecipitation using affinity-purified StSUT1-specific peptide antibodies and mass spectrometric analysis of co-precipitated proteins. A large overlap was observed between the StSUT1-interacting proteins identified in the co-immunoprecipitation and the detergent-resistant membrane fraction. One of the SUT1-interacting proteins, a protein disulfide isomerase (PDI), interacts also with other sucrose transporter proteins. A potential role of the PDI as escort protein is discussed.

  15. The F130S point mutation in the Arabidopsis high-affinity K+ transporter AtHAK5 increases K+ over Na+ and Cs+ selectivity and confers Na+ and Cs+ tolerance to yeast under heterologous expression

    PubMed Central

    Alemán, Fernando; Caballero, Fernando; Ródenas, Reyes; Rivero, Rosa M.; Martínez, Vicente; Rubio, Francisco

    2014-01-01

    Potassium (K+) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K+ acquisition by plant roots at low external K+ concentrations. Certain abiotic stress conditions such as salinity or Cs+-polluted soils may jeopardize plant K+ nutrition because HAK5-mediated K+ transport is inhibited by Na+ and Cs+. Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na+ tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs+ tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K+ and reduces the Ki values for Na+ and Cs+, suggesting that the F130 residue may contribute to the structure of the pore region involved in K+ binding. In addition, this mutation increases the Vmax for K+. All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. PMID:25228905

  16. Expression of Sucrose Transporter cDNAs Specifically in Companion Cells Enhances Phloem Loading and Long-Distance Transport of Sucrose but Leads to an Inhibition of Growth and the Perception of a Phosphate Limitation1[W][OPEN

    PubMed Central

    Dasgupta, Kasturi; Khadilkar, Aswad S.; Sulpice, Ronan; Pant, Bikram; Scheible, Wolf-Rüdiger; Fisahn, Joachim; Stitt, Mark; Ayre, Brian G.

    2014-01-01

    Sucrose (Suc) is the predominant form of carbon transported through the phloem from source to sink organs and is also a prominent sugar for short-distance transport. In all streptophytes analyzed, Suc transporter genes (SUTs or SUCs) form small families, with different subgroups evolving distinct functions. To gain insight into their capacity for moving Suc in planta, representative members of each clade were first expressed specifically in companion cells of Arabidopsis (Arabidopsis thaliana) and tested for their ability to rescue the phloem-loading defect caused by the Suc transporter mutation, Atsuc2-4. Sequence similarity was a poor indicator of ability: Several genes with high homology to AtSUC2, some of which have phloem-loading functions in other eudicot species, did not rescue the Atsuc2-4 mutation, whereas a more distantly related gene, ZmSUT1 from the monocot Zea mays, did restore phloem loading. Transporter complementary DNAs were also expressed in the companion cells of wild-type Arabidopsis, with the aim of increasing productivity by enhancing Suc transport to growing sink organs and reducing Suc-mediated feedback inhibition on photosynthesis. Although enhanced Suc loading and long-distance transport was achieved, growth was diminished. This growth inhibition was accompanied by increased expression of phosphate (P) starvation-induced genes and was reversed by providing a higher supply of external P. These experiments suggest that efforts to increase productivity by enhancing sugar transport may disrupt the carbon-to-P homeostasis. A model for how the plant perceives and responds to changes in the carbon-to-P balance is presented. PMID:24777345

  17. High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

    PubMed Central

    Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

    2001-01-01

    In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

  18. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    USDA-ARS?s Scientific Manuscript database

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  19. Apple Sucrose Transporter SUT1 and Sorbitol Transporter SOT6 Interact with Cytochrome b5 to Regulate Their Affinity for Substrate Sugars1[W][OA

    PubMed Central

    Fan, Ren-Chun; Peng, Chang-Cao; Xu, Yan-Hong; Wang, Xiao-Fang; Li, Yan; Shang, Yi; Du, Shu-Yuan; Zhao, Rui; Zhang, Xiao-Yan; Zhang, Ling-Yun; Zhang, Da-Peng

    2009-01-01

    Sugar transporters are central machineries to mediate cross-membrane transport of sugars into the cells, and sugar availability may serve as a signal to regulate the sugar transporters. However, the mechanisms of sugar transport regulation by signal sugar availability remain unclear in plant and animal cells. Here, we report that a sucrose transporter, MdSUT1, and a sorbitol transporter, MdSOT6, both localized to plasma membrane, were identified from apple (Malus domestica) fruit. Using a combination of the split-ubiquitin yeast two-hybrid, immunocoprecipitation, and bimolecular fluorescence complementation assays, the two distinct sugar transporters were shown to interact physically with an apple endoplasmic reticulum-anchored cytochrome b5 MdCYB5 in vitro and in vivo. In the yeast systems, the two different interaction complexes function to up-regulate the affinity of the sugar transporters, allowing cells to adapt to sugar starvation. An Arabidopsis (Arabidopsis thaliana) homolog of MdCYB5, AtCYB5-A, also interacts with the two sugar transporters and functions similarly. The point mutations leucine-73 → proline in MdSUT1 and leucine-117 → proline in MdSOT6, disrupting the bimolecular interactions but without significantly affecting the transporter activities, abolish the stimulating effects of the sugar transporter-cytochrome b5 complex on the affinity of the sugar transporters. However, the yeast (Saccharomyces cerevisiae) cytochrome b5 ScCYB5, an additional interacting partner of the two plant sugar transporters, has no function in the regulation of the sugar transporters, indicating that the observed biological functions in the yeast systems are specific to plant cytochrome b5s. These findings suggest a novel mechanism by which the plant cells tailor sugar uptake to the surrounding sugar availability. PMID:19502355

  20. Sugar Uptake and Transport in Rice Embryo. Expression of Companion Cell-Specific Sucrose Transporter (OsSUT1) Induced by Sugar and Light1

    PubMed Central

    Matsukura, Chi-aki; Saitoh, Toshikazu; Hirose, Toshiro; Ohsugi, Ryu; Perata, Pierdomenico; Yamaguchi, Junji

    2000-01-01

    We investigated sugar uptake and transport in rice (Oryza sativa) embryo during grain germination. Endogenous sugar levels, accumulation of starch granules, and gene expression of a rice sucrose transporter (OsSUT1) were examined using rice embryos germinated with or without exogenous sugar supply. Starch granules remarkably accumulated in the cells around vascular bundles as a consequence of the sugar taken up by the embryos, indicating that the taken-up sugars are transiently converted into starch. In situ detection for OsSUT1 mRNA indicated its localization in the phloem companion cells. Furthermore, northern-blot and in situ hybridization analyses showed that OsSUT1 expression is not detectable in embryos subjected to sugar starvation conditions, whereas its expression is enhanced by an increased endogenous sugar level. Overall results indicate that the expression of companion cell-specific sucrose transporter, OsSUT1 is regulated by the endogenous sugar status as well as light exposure. PMID:10982424

  1. Linking Expression of Fructan Active Enzymes, Cell Wall Invertases and Sucrose Transporters with Fructan Profiles in Growing Taproot of Chicory (Cichorium intybus): Impact of Hormonal and Environmental Cues

    PubMed Central

    Wei, Hongbin; Bausewein, Anja; Steininger, Heike; Su, Tao; Zhao, Hongbo; Harms, Karsten; Greiner, Steffen; Rausch, Thomas

    2016-01-01

    In chicory taproot, the inulin-type fructans serve as carbohydrate reserve. Inulin metabolism is mediated by fructan active enzymes (FAZYs): sucrose:sucrose 1-fructosyltransferase (1-SST; fructan synthesis), fructan:fructan-1-fructosyltransferase (1-FFT; fructan synthesis and degradation), and fructan 1-exohydrolases (1-FEH1/2a/2b; fructan degradation). In developing taproot, fructan synthesis is affected by source-to-sink sucrose transport and sink unloading. In the present study, expression of FAZYs, sucrose transporter and CWI isoforms, vacuolar invertase and sucrose synthase was determined in leaf blade, petiole and taproot of young chicory plants (taproot diameter: 2 cm) and compared with taproot fructan profiles for the following scenarios: (i) N-starvation, (ii) abscisic acid (ABA) treatment, (iii) ethylene treatment (via 1-aminoyclopropane-1-carboxylic acid [ACC]), and (iv) cold treatment. Both N-starvation and ABA treatment induced an increase in taproot oligofructans. However, while under N-starvation this increase reflected de novo synthesis, under ABA treatment gene expression profiles indicated a role for both de novo synthesis and degradation of long-chain fructans. Conversely, under ACC and cold treatment oligofructans slightly decreased, correlating with reduced expression of 1-SST and 1-FFT and increased expression of FEHs and VI. Distinct SUT and CWI expression profiles were observed, indicating a functional alignment of SUT and CWI expression with taproot fructan metabolism under different source-sink scenarios. PMID:27994611

  2. His-65 in the proton–sucrose symporter is an essential amino acid whose modification with site-directed mutagenesis increases transport activity

    PubMed Central

    Lu, Jade M.-Y.; Bush, Daniel R.

    1998-01-01

    The proton–sucrose symporter that mediates phloem loading is a key component of assimilate partitioning in many higher plants. Previous biochemical investigations showed that a diethyl pyrocarbonate-sensitive histidine residue is at or near the substrate-binding site of the symporter. Among the proton–sucrose symporters cloned to date, only the histidine residue at position 65 of AtSUC1 from Arabidopsis thaliana is conserved across species. To test whether His-65 is involved in the transport reaction, we have used site-directed mutagenesis and functional expression in yeast to determine the significance of this residue in the reaction mechanism. Symporters with mutations at His-65 exhibited a range of activities; for example, the H65C mutant resulted in the complete loss of transport capacity, whereas H65Q was almost as active as wild type. Surprisingly, the H65K and H65R symporters transport sucrose at significantly higher rates (increased Vmax) than the wild-type symporter, suggesting His-65 may be associated with a rate-limiting step in the transport reaction. RNA gel blot and protein blot analyses showed that, with the exception of H65C, the variation in transport activity was not because of alterations in steady-state levels of mRNA or symporter protein. Significantly, those symporters with substitutions of His-65 that remained transport competent were no longer sensitive to inactivation by diethyl pyrocarbonate, demonstrating that this is the inhibitor-sensitive histidine residue. Taken together with our previous results, these data show that His-65 is involved in sucrose binding, and increased rates of transport implicate this region of the protein in the transport reaction. PMID:9671798

  3. A mathematical model of phloem sucrose transport as a new tool for designing rice panicle structure for high grain yield.

    PubMed

    Seki, Motohide; Feugier, François Gabriel; Song, Xian-Jun; Ashikari, Motoyuki; Nakamura, Haruka; Ishiyama, Keiki; Yamaya, Tomoyuki; Inari-Ikeda, Mayuko; Kitano, Hidemi; Satake, Akiko

    2015-04-01

    Rice (Oryza sativa) is one of the most important food crops in the world. Numerous quantitative trait loci or genes controlling panicle architecture have been identified to increase grain yield. Yet grain yield, defined as the product of the number of well-ripened grains and their weight, is a complex trait that is determined by multiple factors such as source, sink and translocation capacity. Mechanistic modelling capturing capacities of source, sink and transport will help in the theoretical design of crop ideotypes that guarantee high grain yield. Here we present a mathematical model simulating sucrose transport and grain growth within a complex phloem network. The model predicts that the optimal panicle structure for high yield shows a simple grain arrangement with few higher order branches. In addition, numerical analyses revealed that inefficient delivery of carbon to panicles with higher order branches prevails regardless of source capacity, indicating the importance of designing grain arrangement and phloem structure. Our model highlights the previously unexplored effect of grain arrangement on the yield, and provides numerical solutions for optimal panicle structure under various source and sink capacities. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. A transgenic study on affecting potato tuber yield by expressing the rice sucrose transporter genes OsSUT5Z and OsSUT2M.

    PubMed

    Sun, Aijun; Dai, Yan; Zhang, Xinsheng; Li, Chunmin; Meng, Kun; Xu, Honglin; Wei, Xiaoli; Xiao, Guifang; Ouwerkerk, Pieter B F; Wang, Mei; Zhu, Zhen

    2011-07-01

    In many plants, sucrose transporters are essential for both sucrose exports from sources and imports into sinks, indicating a function in assimilate partitioning. To investigate whether sucrose transporters can improve the yield of starch plant, potato plants (Solanum tuberosum L. cv. Désirée) were transformed with cDNAs of the rice sucrose transporter genes OsSUT5Z and OsSUT2M under the control of a tuber-specific, class-I patatin promoter. Compared to the controls, the average fructose content of OsSUT5Z transgenic tubers significantly increased. However, the content of the sugars and starch in the OsSUT2M transgenic potato tubers showed no obvious difference. Correspondingly, the average tuber yield, average number of tubers per plant and average weight of single tuber showed no significant difference in OsSUT2M transgenic tubers with controls. In the OsSUT5Z transgenic lines, the average tuber yield per plant was 1.9-fold higher than the controls, and the average number of tubers per plant increased by more than 10 tubers on average, whereas the average weight of a single tuber did not increase significantly. These results suggested that the average number of tubers per plant showed more contribution than the average weight of a single tuber to the tuber yield per plant.

  5. Targeting the AtCWIN1 Gene to Explore the Role of Invertases in Sucrose Transport in Roots and during Botrytis cinerea Infection

    PubMed Central

    Veillet, Florian; Gaillard, Cécile; Coutos-Thévenot, Pierre; La Camera, Sylvain

    2016-01-01

    Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink, or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H+ symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e., STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and AtVIN genes

  6. Targeting the AtCWIN1 Gene to Explore the Role of Invertases in Sucrose Transport in Roots and during Botrytis cinerea Infection.

    PubMed

    Veillet, Florian; Gaillard, Cécile; Coutos-Thévenot, Pierre; La Camera, Sylvain

    2016-01-01

    Cell wall invertases (CWIN) cleave sucrose into glucose and fructose in the apoplast. CWINs are key regulators of carbon partitioning and source/sink relationships during growth, development and under biotic stresses. In this report, we monitored the expression/activity of Arabidopsis cell wall invertases in organs behaving as source, sink, or subjected to a source/sink transition after infection with the necrotrophic fungus Botrytis cinerea. We showed that organs with different source/sink status displayed differential CWIN activities, depending on carbohydrate needs or availabilities in the surrounding environment, through a transcriptional and posttranslational regulation. Loss-of-function mutation of the Arabidopsis cell wall invertase 1 gene, AtCWIN1, showed that the corresponding protein was the main contributor to the apoplastic sucrose cleaving activity in both leaves and roots. The CWIN-deficient mutant cwin1-1 exhibited a reduced capacity to actively take up external sucrose in roots, indicating that this process is mainly dependent on the sucrolytic activity of AtCWIN1. Using T-DNA and CRISPR/Cas9 mutants impaired in hexose transport, we demonstrated that external sucrose is actively absorbed in the form of hexoses by a sugar/H(+) symport system involving the coordinated activity of AtCWIN1 with several Sugar Transporter Proteins (STP) of the plasma membrane, i.e., STP1 and STP13. Part of external sucrose was imported without apoplastic cleavage into cwin1-1 seedling roots, highlighting an alternative AtCWIN1-independent pathway for the assimilation of external sucrose. Accordingly, we showed that several genes encoding sucrose transporters of the plasma membrane were expressed. We also detected transcript accumulation of vacuolar invertase (VIN)-encoding genes and high VIN activities. Upon infection, AtCWIN1 was responsible for all the Botrytis-induced apoplastic invertase activity. We detected a transcriptional activation of several AtSUC and At

  7. Photoperiodic regulation of the sucrose transporter StSUT4 affects the expression of circadian-regulated genes and ethylene production

    PubMed Central

    Chincinska, Izabela; Gier, Konstanze; Krügel, Undine; Liesche, Johannes; He, Hongxia; Grimm, Bernhard; Harren, Frans J. M.; Cristescu, Simona M.; Kühn, Christina

    2012-01-01

    Several recent publications reported different subcellular localization of the sucrose transporters belonging to the SUT4 subfamily. The physiological function of the SUT4 sucrose transporters requires clarification, because down-regulation of the members of the SUT4 clade had different effects in rice, poplar, and potato. Here, we provide new data for the localization and function of the Solanaceous StSUT4 protein, further elucidating involvement in the onset of flowering, tuberization and in the shade avoidance syndrome of potato plants. Induction of an early flowering and a tuberization in the SUT4-inhibited potato plants correlates with increased sucrose export from leaves and increased sucrose and starch accumulation in terminal sink organs, such as developing tubers. SUT4 affects expression of the enzymes involved in gibberellin and ethylene biosynthesis, as well as the rate of ethylene biosynthesis in potato. In the SUT4-inhibited plants, the ethylene production no longer follows a diurnal rhythm. Thus it was concluded that StSUT4 controls circadian gene expression, potentially by regulating sucrose export from leaves. Furthermore, SUT4 expression affects clock-regulated genes such as StFT, StSOC1, and StCO, which might be also involved in a photoperiod-dependent tuberization. A model is proposed in which StSUT4 controls a phloem-mobile signaling molecule generated in leaves, which together with enhanced sucrose export affects developmental switches in apical meristems. SUT4 seems to link photoreceptor-perceived information about the light quality and day length with phytohormone biosynthesis and the expression of circadian-regulated genes. PMID:23429841

  8. Genome-Wide Analysis and Expression Profiling of the SUC and SWEET Gene Families of Sucrose Transporters in Oilseed Rape (Brassica napus L.).

    PubMed

    Jian, Hongju; Lu, Kun; Yang, Bo; Wang, Tengyue; Zhang, Li; Zhang, Aoxiang; Wang, Jia; Liu, Liezhao; Qu, Cunmin; Li, Jiana

    2016-01-01

    Sucrose is the principal transported product of photosynthesis from source leaves to sink organs. SUTs/SUCs (sucrose transporters or sucrose carriers) and SWEETs (Sugars Will Eventually be Exported Transporters) play significant central roles in phloem loading and unloading. SUTs/SUCs and SWEETs are key players in sucrose translocation and are associated with crop yields. The SUT/SUC and SWEET genes have been characterized in several plant species, but a comprehensive analysis of these two gene families in oilseed rape has not yet been reported. In our study, 22 and 68 members of the SUT/SUCs and SWEET gene families, respectively, were identified in the oilseed rape (Brassica napus) genome through homology searches. An analysis of the chromosomal distribution, phylogenetic relationships, gene structures, motifs and the cis-acting regulatory elements in the promoters of BnSUC and BnSWEET genes were analyzed. Furthermore, we examined the expression of the 18 BnSUC and 16 BnSWEET genes in different tissues of "ZS11" and the expression of 9 BnSUC and 7 BnSWEET genes in "ZS11" under various conditions, including biotic stress (Sclerotinia sclerotiorum), abiotic stresses (drought, salt and heat), and hormone treatments (abscisic acid, auxin, cytokinin, brassinolide, gibberellin, and salicylic acid). In conclusion, our study provides the first comprehensive analysis of the oilseed rape SUC and SWEET gene families. Information regarding the phylogenetic relationships, gene structure and expression profiles of the SUC and SWEET genes in the different tissues of oilseed rape helps to identify candidates with potential roles in specific developmental processes. Our study advances our understanding of the important roles of sucrose transport in oilseed rape.

  9. Genome-Wide Analysis and Expression Profiling of the SUC and SWEET Gene Families of Sucrose Transporters in Oilseed Rape (Brassica napus L.)

    PubMed Central

    Jian, Hongju; Lu, Kun; Yang, Bo; Wang, Tengyue; Zhang, Li; Zhang, Aoxiang; Wang, Jia; Liu, Liezhao; Qu, Cunmin; Li, Jiana

    2016-01-01

    Sucrose is the principal transported product of photosynthesis from source leaves to sink organs. SUTs/SUCs (sucrose transporters or sucrose carriers) and SWEETs (Sugars Will Eventually be Exported Transporters) play significant central roles in phloem loading and unloading. SUTs/SUCs and SWEETs are key players in sucrose translocation and are associated with crop yields. The SUT/SUC and SWEET genes have been characterized in several plant species, but a comprehensive analysis of these two gene families in oilseed rape has not yet been reported. In our study, 22 and 68 members of the SUT/SUCs and SWEET gene families, respectively, were identified in the oilseed rape (Brassica napus) genome through homology searches. An analysis of the chromosomal distribution, phylogenetic relationships, gene structures, motifs and the cis-acting regulatory elements in the promoters of BnSUC and BnSWEET genes were analyzed. Furthermore, we examined the expression of the 18 BnSUC and 16 BnSWEET genes in different tissues of “ZS11” and the expression of 9 BnSUC and 7 BnSWEET genes in “ZS11” under various conditions, including biotic stress (Sclerotinia sclerotiorum), abiotic stresses (drought, salt and heat), and hormone treatments (abscisic acid, auxin, cytokinin, brassinolide, gibberellin, and salicylic acid). In conclusion, our study provides the first comprehensive analysis of the oilseed rape SUC and SWEET gene families. Information regarding the phylogenetic relationships, gene structure and expression profiles of the SUC and SWEET genes in the different tissues of oilseed rape helps to identify candidates with potential roles in specific developmental processes. Our study advances our understanding of the important roles of sucrose transport in oilseed rape. PMID:27733861

  10. Conformational Changes Represent the Rate-Limiting Step in the Transport Cycle of Maize SUCROSE TRANSPORTER1[C][W

    PubMed Central

    Derrer, Carmen; Wittek, Anke; Bamberg, Ernst; Carpaneto, Armando; Dreyer, Ingo; Geiger, Dietmar

    2013-01-01

    Proton-driven Suc transporters allow phloem cells of higher plants to accumulate Suc to more than 1 M, which is up to ∼1000-fold higher than in the surrounding extracellular space. The carrier protein can accomplish this task only because proton and Suc transport are tightly coupled. This study provides insights into this coupling by resolving the first step in the transport cycle of the Suc transporter SUT1 from maize (Zea mays). Voltage clamp fluorometry measurements combining electrophysiological techniques with fluorescence-based methods enable the visualization of conformational changes of SUT1 expressed in Xenopus laevis oocytes. Using the Suc derivate sucralose, binding of which hinders conformational changes of SUT1, the association of protons to the carrier could be dissected from transport-associated movements of the protein. These combined approaches enabled us to resolve the binding of protons to the carrier and its interrelationship with the alternating movement of the protein. The data indicate that the rate-limiting step of the reaction cycle is determined by the accessibility of the proton binding site. This, in turn, is determined by the conformational change of the SUT1 protein, alternately exposing the binding pockets to the inward and to the outward face of the membrane. PMID:23964025

  11. Conformational changes represent the rate-limiting step in the transport cycle of maize sucrose transporter1.

    PubMed

    Derrer, Carmen; Wittek, Anke; Bamberg, Ernst; Carpaneto, Armando; Dreyer, Ingo; Geiger, Dietmar

    2013-08-01

    Proton-driven Suc transporters allow phloem cells of higher plants to accumulate Suc to more than 1 M, which is up to ~1000-fold higher than in the surrounding extracellular space. The carrier protein can accomplish this task only because proton and Suc transport are tightly coupled. This study provides insights into this coupling by resolving the first step in the transport cycle of the Suc transporter SUT1 from maize (Zea mays). Voltage clamp fluorometry measurements combining electrophysiological techniques with fluorescence-based methods enable the visualization of conformational changes of SUT1 expressed in Xenopus laevis oocytes. Using the Suc derivate sucralose, binding of which hinders conformational changes of SUT1, the association of protons to the carrier could be dissected from transport-associated movements of the protein. These combined approaches enabled us to resolve the binding of protons to the carrier and its interrelationship with the alternating movement of the protein. The data indicate that the rate-limiting step of the reaction cycle is determined by the accessibility of the proton binding site. This, in turn, is determined by the conformational change of the SUT1 protein, alternately exposing the binding pockets to the inward and to the outward face of the membrane.

  12. AmSUT1, a Sucrose Transporter in Collection and Transport Phloem of the Putative Symplastic Phloem Loader Alonsoa meridionalis1

    PubMed Central

    Knop, Christian; Stadler, Ruth; Sauer, Norbert; Lohaus, Gertrud

    2004-01-01

    A sucrose (Suc) transporter cDNA has been cloned from Alonsoa meridionalis, a member of the Scrophulariaceae. This plant species has an open minor vein configuration and translocates mainly raffinose and stachyose in addition to Suc in the phloem (C. Knop, O. Voitsekhovskaja, G. Lohaus [2001] Planta 213: 80–91). These are typical properties of symplastic phloem loaders. For functional characterization, AmSUT1 cDNA was expressed in bakers' yeast (Saccharomyces cerevisiae). Substrate and inhibitor specificities, energy dependence, and Km value of the protein agree well with the properties measured for other Suc transporters of apoplastic phloem loaders. A polyclonal antiserum against the 17 N-terminal amino acids of the A. meridionalis Suc transporter AmSUT1 was used to determine the cellular localization of the AmSUT1 protein. Using fluorescence labeling on sections from A. meridionalis leaves and stems, AmSUT1 was localized exclusively in phloem cells. Further histological characterization identified these cells as companion cells and sieve elements. p-Chloromercuribenzenesulfonic acid affected the sugar exudation of cut leaves in such a way that the exudation rates of Suc and hexoses decreased, whereas those of raffinose and stachyose increased. The data presented indicate that phloem loading of Suc and retrieval of Suc in A. meridionalis are at least partly mediated by the activity of AmSUT1 in addition to symplastic phloem loading. PMID:14730068

  13. Neurotransmitter/sodium symporter orthologue LeuT has a single high-affinity substrate site.

    PubMed

    Piscitelli, Chayne L; Krishnamurthy, Harini; Gouaux, Eric

    2010-12-23

    Neurotransmitter/sodium symporters (NSSs) couple the uptake of neurotransmitter with one or more sodium ions, removing neurotransmitter from the synaptic cleft. NSSs are essential to the function of chemical synapses, are associated with multiple neurological diseases and disorders, and are the targets of therapeutic and illicit drugs. LeuT, a prokaryotic orthologue of the NSS family, is a model transporter for understanding the relationships between molecular mechanism and atomic structure in a broad range of sodium-dependent and sodium-independent secondary transporters. At present there is a controversy over whether there are one or two high-affinity substrate binding sites in LeuT. The first-reported crystal structure of LeuT, together with subsequent functional and structural studies, provided direct evidence for a single, high-affinity, centrally located substrate-binding site, defined as the S1 site. Recent binding, flux and molecular simulation studies, however, have been interpreted in terms of a model where there are two high-affinity binding sites: the central, S1, site and a second, the S2 site, located within the extracellular vestibule. Furthermore, it was proposed that the S1 and S2 sites are allosterically coupled such that occupancy of the S2 site is required for the cytoplasmic release of substrate from the S1 site. Here we address this controversy by performing direct measurement of substrate binding to wild-type LeuT and to S2 site mutants using isothermal titration calorimetry, equilibrium dialysis and scintillation proximity assays. In addition, we perform uptake experiments to determine whether the proposed allosteric coupling between the putative S2 site and the S1 site manifests itself in the kinetics of substrate flux. We conclude that LeuT harbours a single, centrally located, high-affinity substrate-binding site and that transport is well described by a simple, single-substrate kinetic mechanism.

  14. Immunological Evidence for the Existence of a Carrier Protein for Sucrose Transport in Tonoplast Vesicles from Red Beet (Beta vulgaris L.) Root Storage Tissue.

    PubMed Central

    Getz, H. P.; Grosclaude, J.; Kurkdjian, A.; Lelievre, F.; Maretzki, A.; Guern, J.

    1993-01-01

    Monoclonal antibodies were raised in mice against a highly purified tonoplast fraction from isolated red beet (Beta vulgaris L. ssp. conditiva) root vacuoles. Positive hybridoma clones and sub-clones were identified by prescreening using an enzyme-linked immunosorbent assay (ELISA) and by postscreening using a functional assay. This functional assay consisted of testing the impact of hybridoma supernatants and antibody-containing ascites fluids on basal and ATP-stimulated sugar uptake in vacuoles, isolated from protoplasts, as well as in tonoplast vesicles, prepared from tissue homogenates of red beet roots. Antibodies from four clones were particularly positive in ELISAs and they inhibited sucrose uptake significantly. These antibodies were specific inhibitors of sucrose transport, but they exhibited relatively low membrane and species specificity since uptake into red beet root protoplasts and sugarcane tonoplast vesicles was inhibited as well. Fast protein liquid chromatography assisted size exclusion chromatography on Superose 6 columns yielded two major peaks in the 55 to 65-kD regions and in the 110- to 130-kD regions of solubilized proteins from red beet root tonoplasts, which reacted positively in immunoglobulin-M(IgM)-specific ELISAs with anti-sugarcane tonoplast monoclonal IgM antibodies. Only reconstituted proteoliposomes containing polypeptides from the 55- to 65-kD band took up [14C]-sucrose with linear rates for 2 min, suggesting that this fraction contains the tonoplast sucrose carrier. PMID:12231863

  15. Characterization of Sucrose transporter alleles and their association with seed yield-related traits in Brassica napus L

    PubMed Central

    2011-01-01

    Background Sucrose is the primary photosynthesis product and the principal translocating form within higher plants. Sucrose transporters (SUC/SUT) play a critical role in phloem loading and unloading. Photoassimilate transport is a major limiting factor for seed yield. Our previous research demonstrated that SUT co-localizes with yield-related quantitative trait loci. This paper reports the isolation of BnA7.SUT1 alleles and their promoters and their association with yield-related traits. Results Two novel BnA7.SUT1 genes were isolated from B. napus lines 'Eagle' and 'S-1300' and designated as BnA7.SUT1.a and BnA7.SUT1.b, respectively. The BnA7.SUT1 protein exhibited typical SUT features and showed high amino acid homology with related species. Promoters of BnA7.SUT1.a and BnA7.SUT1.b were also isolated and classified as pBnA7.SUT1.a and pBnA7.SUT1.b, respectively. Four dominant sequence-characterized amplified region markers were developed to distinguish BnA7.SUT1.a and BnA7.SUT1.b. The two genes were estimated as alleles with two segregating populations (F2 and BC1) obtained by crossing '3715'×'3769'. BnA7.SUT1 was mapped to the A7 linkage group of the TN doubled haploid population. In silico analysis of 55 segmental BnA7.SUT1 alleles resulted three BnA7.SUT1 clusters: pBnA7.SUT1.a- BnA7.SUT1.a (type I), pBnA7.SUT1.b- BnA7.SUT1.a (type II), and pBnA7.SUT1.b- BnA7.SUT1.b (type III). Association analysis with a diverse panel of 55 rapeseed lines identified single nucleotide polymorphisms (SNPs) in promoter and coding domain sequences of BnA7.SUT1 that were significantly associated with one of three yield-related traits: number of effective first branches (EFB), siliques per plant (SP), and seed weight (n = 1000) (TSW) across all four environments examined. SNPs at other BnA7.SUT1 sites were also significantly associated with at least one of six yield-related traits: EFB, SP, number of seeds per silique, seed yield per plant, block yield, and TSW. Expression levels

  16. Suppressing a Putative Sterol Carrier Gene Reduces Plasmodesmal Permeability and Activates Sucrose Transporter Genes during Cotton Fiber Elongation.

    PubMed

    Zhang, Zhiyuan; Ruan, Yong-Ling; Zhou, Na; Wang, Fang; Guan, Xueying; Fang, Lei; Shang, Xiaoguang; Guo, Wangzhen; Zhu, Shuijin; Zhang, Tianzhen

    2017-08-01

    Plasmodesmata (PDs) play vital roles in cell-to-cell communication and plant development. Emerging evidence suggests that sterols are involved in PD activity during cytokinesis. However, whether sterols contribute to PD gating between established cells remains unknown. Here, we isolated GhSCP2D, a putative sterol carrier protein gene from elongating cotton (Gossypium hirsutum) fibers. In contrast to wild-type fiber PDs, which opened at 5 to 10 d postanthesis (DPA) and closed only at 15 to 25 DPA, plants with suppressed GhSCP2D expression had reduced sterol contents and closed PDs at 5 through 25 DPA The GhSCP2D-suppressed fibers exhibited callose deposition at the PDs, likely due to reduced expression of GhPdBG3-2A/D, which encodes a PD-targeting β-1,3-glucanase. Both GhPdBG3-2A/D expression and callose deposition were sensitive to a sterol biosynthesis inhibitor. Moreover, suppressing GhSCP2D upregulated a cohort of SUT and SWEET sucrose transporter genes in fiber cells. Collectively, our results indicate that (1) GhSCP2D is required for GhPdBG3-2A/D expression to degrade callose at the PD, thereby contributing to the establishment of the symplasmic pathway; and (2) blocking the symplasmic pathway by downregulating GhSCP2D activates or increases the expression of SUTs and SWEETs, leading to the switch from symplasmic to apoplasmic pathways. © 2017 American Society of Plant Biologists. All rights reserved.

  17. New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L. (Pomel)

    PubMed Central

    Péron, Thomas; Candat, Adrien; Montiel, Grégory; Veronesi, Christophe; Macherel, David; Delavault, Philippe; Simier, Philippe

    2017-01-01

    The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in

  18. New Insights into Phloem Unloading and Expression of Sucrose Transporters in Vegetative Sinks of the Parasitic Plant Phelipanche ramosa L. (Pomel).

    PubMed

    Péron, Thomas; Candat, Adrien; Montiel, Grégory; Veronesi, Christophe; Macherel, David; Delavault, Philippe; Simier, Philippe

    2016-01-01

    The plant-parasitic plant interaction is a interesting model to study sink-source relationship and phloem unloading. The parasitic plants, such as the achlorophyllous plant Phelipanche ramosa, connect to the host phloem through the haustorium and act as supernumerary sinks for the host-derived photoassimilates, primarily sucrose. The application of the fluorescent symplastic tracer, carboxyfluorescein (CF) derived from carboxyfluorescein diacetate (CFDA), to the leaves of the host plant (Brassica napus) showed direct phloem connections at the host-parasite interface. These experiments also evidenced the dominant apoplastic pathway for phloem unloading in major vegetative sinks of the parasite, including tubercles and shoots, except the adventitious root apices. The CF experiments showed also the symplastic isolation of the phloem tissues from the sink tissues in tubercle and shoot of the parasite, then suggesting the pivotal role of sucrose transporters in sucrose unloading in P. ramosa sinks. Three cDNAs encoding sucrose transporters (PrSUT) were isolated from the parasitic plant. PrSUT1 transcripts accumulated at the same level in the tubercle throughout the parasite growth while a significant increase in transcript accumulation occurred after emergence in the flowering shoot, notably in the growing apical part. The in situ hybridization experiments revealed the PrSUT1 transcript accumulation in the mature phloem cells of both subterranean and flowering shoots, as well as in shoot terminal sinks corresponding to apical meristem, scale leaf primordia and immature vasculature. The transient expression experiments in Arabidopsis protoplasts showed that PrSUT1 was localized at the plasma membrane, suggesting its role in phloem functioning and sucrose uptake by the sink cells in P. ramosa. Conversely, the PrSUT2 transcript accumulation was constantly low in tubercles and shoots but PrSUT3 transcripts accumulated markedly in the subterranean and flowering shoots, in

  19. Tissue-specific transcriptome analysis within the maturing sugarcane stalk reveals spatial regulation in the expression of cellulose synthase and sucrose transporter gene families.

    PubMed

    Casu, Rosanne E; Rae, Anne L; Nielsen, Janine M; Perroux, Jai M; Bonnett, Graham D; Manners, John M

    2015-12-01

    Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.

  20. Transport of amino acids (L-valine, L-lysine, L-glutamic acid) and sucrose into plasma membrane vesicles isolated from cotyledons of developing pea seeds.

    PubMed

    de Jong, A; Borstlap, A C

    2000-10-01

    Transport of the amino acids L-valine, L-lysine, and L-glutamic acid and of sucrose was studied in plasma membrane vesicles isolated from developing cotyledons of pea (Pisum sativum L. cv. Marzia). The vesicles were obtained by aqueous polymer two-phase partitioning of a microsomal fraction and the uptake was determined after the imposition of a H(+)-gradient (DeltapH, inside alkaline) and/or an electrical gradient (Deltapsi, inside negative) across the vesicle membrane. In the absence of gradients, a distinct, time-dependent uptake of L-valine was measured, which could be enhanced about 2-fold by the imposition of DeltapH. The imposition of Deltapsi stimulated the influx of valine by 20%, both in the absence and in the presence of DeltapH. Uptake of L-lysine was more strongly stimulated by Deltapsi than by DeltapH, and its DeltapH-dependent uptake was enhanced about 6-fold by the simultaneous imposition of Deltapsi. In the absence of gradients the uptake of L-glutamic acid was about 2-fold higher than that of L-valine, but it was not detectably affected by DeltapH or Deltapsi. Although the transport of sucrose was very low, a stimulating effect of DeltapH could be clearly demonstrated. The results lend further support to the contention that during seed development cotyledonary cells employ H(+)-symporters for the active uptake of sucrose and amino acids.

  1. A Cascade of Sequentially Expressed Sucrose Transporters in the Seed Coat and Endosperm Provides Nutrition for the Arabidopsis Embryo[OPEN

    PubMed Central

    Chen, Li-Qing; Lin, I Winnie; Qu, Xiao-Qing; Sosso, Davide; McFarlane, Heather E.; Londoño, Alejandra; Samuels, A. Lacey; Frommer, Wolf B.

    2015-01-01

    Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a “wrinkled” seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential. PMID:25794936

  2. Radiosynthesis of 6’-Deoxy-6’[18F]Fluorosucrose via Automated Synthesis and Its Utility to Study In Vivo Sucrose Transport in Maize (Zea mays) Leaves

    PubMed Central

    Ying, Weijiang; Gaddam, Vikram; Harmata, Michael; Robertson, J. David; Swyers, Michael; Jurisson, Silvia S.

    2015-01-01

    Sugars produced from photosynthesis in leaves are transported through the phloem tissues within veins and delivered to non-photosynthetic organs, such as roots, stems, flowers, and seeds, to support their growth and/or storage of carbohydrates. However, because the phloem is located internally within the veins, it is difficult to access and to study the dynamics of sugar transport. Radioactive tracers have been extensively used to study vascular transport in plants and have provided great insights into transport dynamics. To better study sucrose partitioning in vivo, a novel radioactive analog of sucrose was synthesized through a completely chemical synthesis route by substituting fluorine-18 (half-life 110 min) at the 6’ position to generate 6’-deoxy-6’[18F]fluorosucrose (18FS). This radiotracer was then used to compare sucrose transport between wild-type maize plants and mutant plants lacking the Sucrose transporter1 (Sut1) gene, which has been shown to function in sucrose phloem loading. Our results demonstrate that 18FS is transported in vivo, with the wild-type plants showing a greater rate of transport down the leaf blade than the sut1 mutant plants. A similar transport pattern was also observed for universally labeled [U-14C]sucrose ([U-14C]suc). Our findings support the proposed sucrose phloem loading function of the Sut1 gene in maize, and additionally demonstrate that the 18FS analog is a valuable, new tool that offers imaging advantages over [U-14C]suc for studying phloem transport in plants. PMID:26024520

  3. Structural origins of high-affinity biotin binding to streptavidin.

    PubMed

    Weber, P C; Ohlendorf, D H; Wendoloski, J J; Salemme, F R

    1989-01-06

    The high affinity of the noncovalent interaction between biotin and streptavidin forms the basis for many diagnostic assays that require the formation of an irreversible and specific linkage between biological macromolecules. Comparison of the refined crystal structures of apo and a streptavidin:biotin complex shows that the high affinity results from several factors. These factors include the formation of multiple hydrogen bonds and van der Waals interactions between biotin and the protein, together with the ordering of surface polypeptide loops that bury the biotin in the protein interior. Structural alterations at the biotin binding site produce quaternary changes in the streptavidin tetramer. These changes apparently propagate through cooperative deformations in the twisted beta sheets that link tetramer subunits.

  4. High affinity choline uptake: an early index of cholinergic innervation in rat brain.

    PubMed

    Sorimachi, M; Kataoka, K

    1975-08-29

    The uptake of [3H]choline was investigated in nuclei-free homogenates or crude synaptosomal fractions (P2) from rat brain under various stages of development. A comparable sensitivity of uptake to treatment by hyposmotic shock suggested the involvement of synaptosomal populations in choline uptake in immature as well as in adult brains. However, significant changes in the "apparent" Km for the high affinity transport system and quantitative differences in the Na ion requirement for maximal uptake at 0.43 muM choline concentration were found during development; facts which suggested a greater contribution of the low affinity system in the more immature brains. Assuming that the uptake with high and low sensitivity to Na+ reduction reflected that via the high and low affinity system reslectively, we have attempted to obtain real Km values for the high affinity system. These Km values changed less than those measured directly, suggesting that the affinity constant for the high affinity system does not change during development. On these assumptions, the developmental changes of cholinergic synaptogenesis were examined in 5 distinct regions of the brain. It was found that the synaptogenesis begins several days earlier than the increase of choline acetyltransferase (ChAc) level in the frontal cortex, the hippocampus, the superior colliculus and the cerebellum. These regions may be included among the terminal-rich regions according to available evidence related to cholinergic systems. On the other hand, synaptogenesis accompanied the concomitant ChAc increase in the striatum, where the cholinergic interneurons are present. It is concluded that the increase of ChAc in the terminal-rich regions is delayed by the axoplasmic flow; therefore, the earlier index of cholinergic synaptogenesis in these regions is the high affinity uptake activity rather than the enzyme activity.

  5. Selective high affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2010-02-16

    This invention provides novel polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each bind different region son the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  6. The Control of Single-Celled Cotton Fiber Elongation by Developmentally Reversible Gating of Plasmodesmata and Coordinated Expression of Sucrose and K+ Transporters and Expansin

    PubMed Central

    Ruan, Yong-Ling; Llewellyn, Danny J.; Furbank, Robert T.

    2001-01-01

    Each cotton fiber is a single cell that elongates to 2.5 to 3.0 cm from the seed coat epidermis within ∼16 days after anthesis (DAA). To elucidate the mechanisms controlling this rapid elongation, we studied the gating of fiber plasmodesmata and the expression of the cell wall–loosening gene expansin and plasma membrane transporters for sucrose and K+, the major osmotic solutes imported into fibers. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed that the fiber plasmodesmata were initially permeable to CF (0 to 9 DAA), but closed at ∼10 DAA and re-opened at 16 DAA. A developmental switch from simple to branched plasmodesmata was also observed in fibers at 10 DAA. Coincident with the transient closure of the plasmodesmata, the sucrose and K+ transporter genes were expressed maximally in fibers at 10 DAA with sucrose transporter proteins predominately localized at the fiber base. Consequently, fiber osmotic and turgor potentials were elevated, driving the rapid phase of elongation. The level of expansin mRNA, however, was high at the early phase of elongation (6 to 8 DAA) and decreased rapidly afterwards. The fiber turgor was similar to the underlying seed coat cells at 6 to 10 DAA and after 16 DAA. These results suggest that fiber elongation is initially achieved largely by cell wall loosening and finally terminated by increased wall rigidity and loss of higher turgor. To our knowledge, this study provides an unprecedented demonstration that the gating of plasmodesmata in a given cell is developmentally reversible and is coordinated with the expression of solute transporters and the cell wall–loosening gene. This integration of plasmodesmatal gating and gene expression appears to control fiber cell elongation. PMID:11158528

  7. The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis.

    PubMed

    Frost, Christopher J; Nyamdari, Batbayar; Tsai, Chung-Jui; Harding, Scott A

    2012-01-01

    The Populus sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of Populus, SUT4 is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in Populus. Here, we describe the effects of PtaSUT4-RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in Populus.

  8. Ethylene stimulation of latex yield depends on the expression of a sucrose transporter (HbSUT1B) in rubber tree (Hevea brasiliensis).

    PubMed

    Dusotoit-Coucaud, Anaïs; Kongsawadworakul, Panida; Maurousset, Laurence; Viboonjun, Unshira; Brunel, Nicole; Pujade-Renaud, Valérie; Chrestin, Hervé; Sakr, Soulaïman

    2010-12-01

    Hevea brasiliensis is an important industrial crop for natural rubber production. Latex biosynthesis occurs in the cytoplasm of highly specialized latex cells and requires sucrose as the unique precursor. Ethylene stimulation of latex production results in high sugar flow from the surrounding cells of inner bark towards the latex cells. The aim of this work was to understand the role of seven sucrose transporters (HbSUTs) and one hexose transporter (HbHXT1) in this process. Two Hevea clones were used: PB217 and PB260, respectively described as high and low yielding clones. The expression pattern of these sugar transporters (HbSUTs and HbHXT1) was monitored under different physiological conditions and found to be maximal in latex cells. HbSUT1, one of the most abundant isoforms, displayed the greatest response to ethylene treatment. In clone PB217, ethylene treatment led to a higher accumulation of HbSUT1B in latex cells than in the inner bark tissues. Conversely, stronger expression of HbSUT1B was observed in inner bark tissues than in latex cells of PB260. A positive correlation with HbSUT1B transcript accumulation and increased latex production was further supported by its lower expression in latex cells of the virgin clone PB217.

  9. Low and high affinity receptors mediate cellular uptake of heparanase

    PubMed Central

    Ben-Zaken, Olga; Shafat, Itay; Gingis-Velitski, Svetlana; Bangio, Haim; Kelson, Idil Kasuto; Alergand, Tal; Amor, Yehudit; Maya, Ruth Ben-Yakar; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated. PMID:17981072

  10. 01-ERD-111 - The Development of Synthetic High Affinity Ligands

    SciTech Connect

    Perkins, J; Balhorn, R; Cosman, M; Lightstone, F; Zeller, L

    2004-02-05

    The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus or botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.

  11. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    SciTech Connect

    Matt Vaughn Greg Harrington Daniel R Bush

    2002-08-06

    This project was based on our discovery that sucrose acts as a signaling molecule that regulates the activity of a proton-sucrose symporter in sugar beet leaf tissue. A major objective here was determining how sucrose transporter activity is being regulated. When sucrose accumulates in the phloem sucrose transport activity drops dramatically. Western blots of plasma membrane proteins isolated from sucrose treated leaves showed that the loss of sucrose transport activity was proportional to a decline in symporter abundance, demonstrating that sucrose transport is regulated by changes in the amount of BvSUT1 protein. BvSUT1 transcript levels decreased in parallel with the loss of sucrose transport activity. Nuclear run-on experiments demonstrated that BvSUT1 gene transcription was repressed significantly in nuclei from leaves fed 100 mM exogenous sucrose, showing that sucrose-dependent modulation of BvSUT1 mRNA levels is mediated by changes in transcription. To identify which secondary messenger systems might be involved in regulating symporter activity, we used a variety of pharmacological agents to probe for a role of calcium or protein phosphorylation in sucrose signaling. In a detailed analysis, only okadaic acid altered sucrose transport activity. These results suggest a protein phosphatase is involved. We hypothesized that protein kinase inhibitors would have a neutral affect or increase symporter transcription. Transpirational feeding of the protein kinase inhibitor staurosporine had no impact on sucrose transport while calphostin C, an inhibitor of protein kinase C, caused a 60% increase. These data provided good evidence that protein phosphorylation plays a central role in regulating sucrose symporter expression and sucrose transport activity. To determine whether protein phosphorylation is involved in sucrose regulation of proton-sucrose symporter activity, we pre-fed leaves with staurosporine for 4 h and then fed the treated leaves water or 100 mM sucrose

  12. The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum

    PubMed Central

    Li, Yan; Wang, Yannan; Zhang, Huan; Zhang, Qian; Zhai, Hong; Liu, Qingchang; He, Shaozhen

    2017-01-01

    SWEET (Sugars Will Eventually be Exported Transporter) proteins, a novel family of sugar transporters, mediate the diffusion of sugars across cell membranes and acts as key players in sucrose phloem loading. Manipulation of SWEET genes in plants leads to various effects on resistance to biotic and abiotic stresses due to disruption of sugar efflux and changes in sugar distribution. In this study, a member of the SWEET gene family, IbSWEET10, was cloned from the sweet potato line ND98. mRNA expression analysis in sweet potato and promoter β-Glucuronidase analysis in Arabidopsis showed that IbSWEET10 is highly expressed in leaves, especially in vascular tissue. Transient expression in tobacco epidermal cells revealed plasma membrane localization of IbSWEET10, and heterologous expression assays in yeast indicated that IbSWEET10 encodes a sucrose transporter. The expression level of IbSWEET10 was significantly up-regulated in sweet potato infected with Fusarium oxysporum Schlecht. f. sp. batatas. Further characterization revealed IbSWEET10-overexpressing sweet potato lines to be more resistant to F. oxysporum, exhibiting better growth after infection compared with the control; conversely, RNA interference (RNAi) lines showed the opposite results. Additionally, the sugar content of IbSWEET10-overexpression sweet potato was significantly reduced, whereas that in RNAi plants was significantly increased compared with the control. Therefore, we suggest that the reduction in sugar content caused by IbSWEET10 overexpression is the major reason for the enhanced F. oxysporum resistance of the transgenic plants. This is the first report that the IbSWEET10 transporter contributes to the resistance of sweet potato to F. oxysporum. The IbSWEET10 gene has the great potential to be used for improving the resistance to F. oxysporum in sweet potato and other plants. PMID:28261250

  13. The Plasma Membrane-Localized Sucrose Transporter IbSWEET10 Contributes to the Resistance of Sweet Potato to Fusarium oxysporum.

    PubMed

    Li, Yan; Wang, Yannan; Zhang, Huan; Zhang, Qian; Zhai, Hong; Liu, Qingchang; He, Shaozhen

    2017-01-01

    SWEET (Sugars Will Eventually be Exported Transporter) proteins, a novel family of sugar transporters, mediate the diffusion of sugars across cell membranes and acts as key players in sucrose phloem loading. Manipulation of SWEET genes in plants leads to various effects on resistance to biotic and abiotic stresses due to disruption of sugar efflux and changes in sugar distribution. In this study, a member of the SWEET gene family, IbSWEET10, was cloned from the sweet potato line ND98. mRNA expression analysis in sweet potato and promoter β-Glucuronidase analysis in Arabidopsis showed that IbSWEET10 is highly expressed in leaves, especially in vascular tissue. Transient expression in tobacco epidermal cells revealed plasma membrane localization of IbSWEET10, and heterologous expression assays in yeast indicated that IbSWEET10 encodes a sucrose transporter. The expression level of IbSWEET10 was significantly up-regulated in sweet potato infected with Fusarium oxysporum Schlecht. f. sp. batatas. Further characterization revealed IbSWEET10-overexpressing sweet potato lines to be more resistant to F. oxysporum, exhibiting better growth after infection compared with the control; conversely, RNA interference (RNAi) lines showed the opposite results. Additionally, the sugar content of IbSWEET10-overexpression sweet potato was significantly reduced, whereas that in RNAi plants was significantly increased compared with the control. Therefore, we suggest that the reduction in sugar content caused by IbSWEET10 overexpression is the major reason for the enhanced F. oxysporum resistance of the transgenic plants. This is the first report that the IbSWEET10 transporter contributes to the resistance of sweet potato to F. oxysporum. The IbSWEET10 gene has the great potential to be used for improving the resistance to F. oxysporum in sweet potato and other plants.

  14. Rice sucrose transporter1 (OsSUT1) up-regulation in xylem parenchyma is caused by aphid feeding on rice leaf blade vascular bundles.

    PubMed

    Ibraheem, O; Botha, C E J; Bradley, G; Dealtry, G; Roux, S

    2014-07-01

    The role of the sucrose transporter OsSUT1 in assimilate retrieval via the xylem, as a result of damage to and leakage from punctured phloem was examined after rusty plum aphid (Hysteroneura setariae, Thomas) infestation on leaves from 3-week-old rice (Oryza sativa L. cv Nipponbare) plants. Leaves were examined over a 1- to 10-day infestation time course, using a combination of gene expression and β-glucuronidase (GUS) reporter gene analyses. qPCR and Western blot analyses revealed differential expression of OsSUT1 during aphid infestation. Wide-field fluorescence microscopy was used to confirm the expression of OsSUT1-promoter::GUS reporter gene in vascular parenchyma associated with xylem elements, as well as in companion cells associated with phloem sieve tubes of large, intermediate and small vascular bundles within the leaf blade, in regions where the aphids had settled and were feeding. Of great interest was up-regulation of OsSUT1 expression associated with the xylem parenchyma cells, abutting the metaxylem vessels, which confirmed that OsSUT1 was not only involved in loading of sugars into the phloem under normal physiological conditions, but was apparently involved in the retrieval of sucrose leaked into the xylem conduits, which occurred as a direct result of aphid feeding, probing and puncturing of vascular bundles. The up-regulation of OsSUT1 in xylem vascular parenchyma thus provides evidence in support of the location within the xylem parenchyma cells of an efficient mechanism to ensure sucrose recovery after loss to the apoplast (xylem) after aphid-related feeding damage and its transfer back to the symplast (phloem) in O. sativa leaves.

  15. HIGH AFFINITY, DSRNA BINDING BY DISCONNECTED INTERACTING PROTEIN 1†

    PubMed Central

    Catanese, Daniel J.; Matthews, Kathleen S.

    2010-01-01

    Disconnected Interacting Protein 1 (DIP1) appears from sequence analysis and preliminary binding studies to be a member of the dsRNA-binding protein family. Of interest, DIP1 was shown previously to interact with and influence multiple proteins involved in transcription regulation in Drosophila melanogaster. We show here that the longest isoform of this protein, DIP1-c, exhibits a 500-fold preference for dsRNA over dsDNA of similar nucleotide sequence. Further, DIP1-c demonstrated very high affinity for a subset of dsRNA ligands, with binding in the picomolar range for VA1 RNA and miR-iab-4 precursor stem-loop, a potential physiological RNA target involved in regulating expression of its protein partner, Ultrabithorax. PMID:20643095

  16. Complex high affinity interactions occur between MHCI and superantigens

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Herpich, A. R.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Staphylococcal enterotoxins A and C1 (SEA or SEC1) bound to major histocompatibility-I (MHCI) molecules with high affinity (binding constants ranging from 1.1 microM to 79 nM). SEA and SEC1 directly bound MHCI molecules that had been captured by monoclonal antibodies specific for H-2Kk, H-2Dk, or both. In addition, MHCI-specific antibodies inhibited the binding of SEC1 to LM929 cells and SEA competitively inhibited SEC1 binding; indicating that the superantigens bound to MHCI on the cell surface. The affinity and number of superantigen binding sites differed depending on whether MHCI was expressed in the membrane of LM929 cells or whether it was captured. These data support the hypothesis that MHCI molecules can serve as superantigen receptors.

  17. Molecular Control of Sucrose Utilization in Escherichia coli W, an Efficient Sucrose-Utilizing Strain

    PubMed Central

    Sabri, Suriana; Nielsen, Lars K.

    2013-01-01

    Sucrose is an industrially important carbon source for microbial fermentation. Sucrose utilization in Escherichia coli, however, is poorly understood, and most industrial strains cannot utilize sucrose. The roles of the chromosomally encoded sucrose catabolism (csc) genes in E. coli W were examined by knockout and overexpression experiments. At low sucrose concentrations, the csc genes are repressed and cells cannot grow. Removal of either the repressor protein (cscR) or the fructokinase (cscK) gene facilitated derepression. Furthermore, combinatorial knockout of cscR and cscK conferred an improved growth rate on low sucrose. The invertase (cscA) and sucrose transporter (cscB) genes are essential for sucrose catabolism in E. coli W, demonstrating that no other genes can provide sucrose transport or inversion activities. However, cscK is not essential for sucrose utilization. Fructose is excreted into the medium by the cscK-knockout strain in the presence of high sucrose, whereas at low sucrose (when carbon availability is limiting), fructose is utilized by the cell. Overexpression of cscA, cscAK, or cscAB could complement the WΔcscRKAB knockout mutant or confer growth on a K-12 strain which could not naturally utilize sucrose. However, phenotypic stability and relatively good growth rates were observed in the K-12 strain only when overexpressing cscAB, and full growth rate complementation in WΔcscRKAB also required cscAB. Our understanding of sucrose utilization can be used to improve E. coli W and engineer sucrose utilization in strains which do not naturally utilize sucrose, allowing substitution of sucrose for other, less desirable carbon sources in industrial fermentations. PMID:23124236

  18. Transport of sucrose-modified nanoscale zero-valent iron in saturated porous media: role of media size, injection rate and input concentration.

    PubMed

    Li, Hui; Zhao, Yong-sheng; Han, Zhan-tao; Hong, Mei

    2015-01-01

    The growing use of nanoscale zero-valent iron (NZVI) in the remediation of contaminated groundwater raises concerns regarding its transport in aquifers. Laboratory-scale sand-packed column experiments were conducted with bare and sucrose-modified NZVI (SM-NZVI) to improve our understanding of the transport of the nanoparticles in saturated porous media, as well as the role of media size, suspension injection rate and concentration on the nanoparticle behavior. As the main indicative parameters, the normalized effluent concentration was measured and the deposition rate coefficient (k) was calculated for different simulated conditions. Overall, compared to the high retention of bare NZVI in the saturated silica column, SM-NZVI suspension could travel through the coarse sand column easily. However, the transport of SM-NZVI particles was not very satisfactory in a smaller size granular matrix especially in fine silica sand. Furthermore, the value of k regularly decreased with the increasing injection rate of suspension but increased with suspension concentration, which could reflect the role of these factors in the SM-NZVI travel process. The calculation of k-value at the tests condition adequately described the experimental results from the point of deposition dynamics, which meant the assumption of first-order deposition kinetics for the transport of NZVI particles was reasonable and feasible.

  19. SNF3 as High Affinity Glucose Sensor and Its Function in Supporting the Viability of Candida glabrata under Glucose-Limited Environment

    PubMed Central

    Ng, Tzu Shan; Chew, Shu Yih; Rangasamy, Premmala; Mohd Desa, Mohd N.; Sandai, Doblin; Chong, Pei Pei; Than, Leslie Thian Lung

    2015-01-01

    Candida glabrata is an emerging human fungal pathogen that has efficacious nutrient sensing and responsiveness ability. It can be seen through its ability to thrive in diverse range of nutrient limited-human anatomical sites. Therefore, nutrient sensing particularly glucose sensing is thought to be crucial in contributing to the development and fitness of the pathogen. This study aimed to elucidate the role of SNF3 (Sucrose Non Fermenting 3) as a glucose sensor and its possible role in contributing to the fitness and survivability of C. glabrata in glucose-limited environment. The SNF3 knockout strain was constructed and subjected to different glucose concentrations to evaluate its growth, biofilm formation, amphotericin B susceptibility, ex vivo survivability and effects on the transcriptional profiling of the sugar receptor repressor (SRR) pathway-related genes. The CgSNF3Δ strain showed a retarded growth in low glucose environments (0.01 and 0.1%) in both fermentation and respiration-preferred conditions but grew well in high glucose concentration environments (1 and 2%). It was also found to be more susceptible to amphotericin B in low glucose environment (0.1%) and macrophage engulfment but showed no difference in the biofilm formation capability. The deletion of SNF3 also resulted in the down-regulation of about half of hexose transporters genes (four out of nine). Overall, the deletion of SNF3 causes significant reduction in the ability of C. glabrata to sense limited surrounding glucose and consequently disrupts its competency to transport and perform the uptake of this critical nutrient. This study highlighted the role of SNF3 as a high affinity glucose sensor and its role in aiding the survivability of C. glabrata particularly in glucose limited environment. PMID:26648919

  20. Phosphatidylserine Reversibly Binds Cu2+ with Extremely High Affinity

    PubMed Central

    Monson, Christopher F.; Cong, Xiao; Robison, Aaron; Pace, Hudson P.; Liu, Chunming; Poyton, Matthew F.; Cremer, Paul S.

    2012-01-01

    Phosphatidylserine (PS) embedded within supported lipid bilayers (SLBs) was found to bind Cu2+ from solution with extraordinarily high affinity. In fact, the equilibrium dissociation constant was in the femtomolar range. The resulting complex formed in a 1:2 Cu2+ to PS ratio and quenches a broad spectrum of lipid-bound fluorophores in a reversible and pH-dependent fashion. At acidic pH values, the fluorophores were almost completely unquenched, while at basic pH values significant quenching (85–90%) was observed. The pH at which the transition occurred was dependent on the PS concentration and ranged from approximately pH 5 to 8. The quenching kinetics was slow at low Cu2+ concentrations and basic values pH (up to several hours), while the unquenching reaction was orders of magnitude more rapid upon lowering the pH. This was consistent with diffusion limited complex formation at basic pH, but rapid dissociation under acidic conditions. The tight binding of Cu2+ to PS may have physiological consequences under certain circumstances. PMID:22548290

  1. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium

    PubMed Central

    1994-01-01

    Interactions between the plasma membrane and underlying actin-based cortex have been implicated in membrane organization and stability, the control of cell shape, and various motile processes. To ascertain the function of high affinity actin-membrane associations, we have disrupted by homologous recombination the gene encoding ponticulin, the major high affinity actin-membrane link in Dictyostelium discoideum amoebae. Cells lacking detectable amounts of ponticulin message and protein also are deficient in high affinity actin-membrane binding by several criteria. First, only 10-13% as much endogenous actin cosediments through sucrose and crude plasma membranes from ponticulin- minus cells, as compared with membranes from the parental strain. Second, purified plasma membranes exhibit little or no binding or nucleation of exogenous actin in vitro. Finally, only 10-30% as much endogenous actin partitions with plasma membranes from ponticulin-minus cells after these cells are mechanically unroofed with polylysine- coated coverslips. The loss of the cell's major actin-binding membrane protein appears to be surprisingly benign under laboratory conditions. Ponticulin-minus cells grow normally in axenic culture and pinocytose FITC-dextran at the same rate as do parental cells. The rate of phagocytosis of particles by ponticulin-minus cells in growth media also is unaffected. By contrast, after initiation of development, cells lacking ponticulin aggregate faster than the parental cells. Subsequent morphogenesis proceeds asynchronously, but viable spores can form. These results indicate that ponticulin is not required for cellular translocation, but apparently plays a role in cell patterning during development. PMID:8089176

  2. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  3. Water Deficit Enhances C Export to the Roots in Arabidopsis thaliana Plants with Contribution of Sucrose Transporters in Both Shoot and Roots1[OPEN

    PubMed Central

    Durand, Mickaël; Porcheron, Benoît; Maurousset, Laurence; Lemoine, Rémi; Pourtau, Nathalie

    2016-01-01

    Root high plasticity is an adaptation to its changing environment. Water deficit impairs growth, leading to sugar accumulation in leaves, part of which could be available to roots via sucrose (Suc) phloem transport. Phloem loading is widely described in Arabidopsis (Arabidopsis thaliana), while unloading in roots is less understood. To gain information on leaf-to-root transport, a soil-based culture system was developed to monitor root system architecture in two dimensions. Under water deficit (50% of soil water-holding capacity), total root length was strongly reduced but the depth of root foraging and the shape of the root system were less affected, likely to improve water uptake. 14CO2 pulse-chase experiments confirmed that water deficit enhanced carbon (C) export to the roots, as suggested by the increased root-to-shoot ratio. The transcript levels of AtSWEET11 (for sugar will eventually be exported transporter), AtSWEET12, and AtSUC2 (for Suc carrier) genes, all three involved in Suc phloem loading, were significantly up-regulated in leaves of water deficit plants, in accordance with the increase in C export from the leaves to the roots. Interestingly, the transcript levels of AtSUC2 and AtSWEET11 to AtSWEET15 were also significantly higher in stressed roots, underlying the importance of Suc apoplastic unloading in Arabidopsis roots and a putative role for these Suc transporters in Suc unloading. These data demonstrate that, during water deficit, plants respond to growth limitation by allocating relatively more C to the roots to maintain an efficient root system and that a subset of Suc transporters is potentially involved in the flux of C to and in the roots. PMID:26802041

  4. Water Deficit Enhances C Export to the Roots in Arabidopsis thaliana Plants with Contribution of Sucrose Transporters in Both Shoot and Roots.

    PubMed

    Durand, Mickaël; Porcheron, Benoît; Hennion, Nils; Maurousset, Laurence; Lemoine, Rémi; Pourtau, Nathalie

    2016-03-01

    Root high plasticity is an adaptation to its changing environment. Water deficit impairs growth, leading to sugar accumulation in leaves, part of which could be available to roots via sucrose (Suc) phloem transport. Phloem loading is widely described in Arabidopsis (Arabidopsis thaliana), while unloading in roots is less understood. To gain information on leaf-to-root transport, a soil-based culture system was developed to monitor root system architecture in two dimensions. Under water deficit (50% of soil water-holding capacity), total root length was strongly reduced but the depth of root foraging and the shape of the root system were less affected, likely to improve water uptake. (14)CO2 pulse-chase experiments confirmed that water deficit enhanced carbon (C) export to the roots, as suggested by the increased root-to-shoot ratio. The transcript levels of AtSWEET11 (for sugar will eventually be exported transporter), AtSWEET12, and AtSUC2 (for Suc carrier) genes, all three involved in Suc phloem loading, were significantly up-regulated in leaves of water deficit plants, in accordance with the increase in C export from the leaves to the roots. Interestingly, the transcript levels of AtSUC2 and AtSWEET11 to AtSWEET15 were also significantly higher in stressed roots, underlying the importance of Suc apoplastic unloading in Arabidopsis roots and a putative role for these Suc transporters in Suc unloading. These data demonstrate that, during water deficit, plants respond to growth limitation by allocating relatively more C to the roots to maintain an efficient root system and that a subset of Suc transporters is potentially involved in the flux of C to and in the roots. © 2016 American Society of Plant Biologists. All Rights Reserved.

  5. Reducing sucrose loss in sugarbeet storage

    USDA-ARS?s Scientific Manuscript database

    Sucrose loss in sugarbeet storage is a considerable problem which is negatively influenced by environmental conditions, stress on roots from disease problems in the field, rough handling during harvest and transport, and microbial growth. To reduce sucrose loss in sugarbeet storage, studies were in...

  6. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  7. Sucrose preload reduces snacking after mild mental stress in healthy participants as a function of 5-hydroxytryptamine transporter gene promoter polymorphism.

    PubMed

    Markus, C Rob; Jonkman, Lisa M; Capello, Aimee; Leinders, Sacha; Hüsch, Fabian

    2015-01-01

    Brain serotonin (5-hydroxytryptamine, 5-HT) dysfunction is considered to promote food intake and eating-related disturbances, especially under stress or negative mood. Vulnerability for 5-HT disturbances is considered to be genetically determined, including a short (S) allele polymorphism in the serotonin transporter gene (5-HTTLPR) that is associated with lower serotonin function. Since 5-HT function may be slightly increased by carbohydrate consumption, S-allele 5-HTTLPR carriers in particular may benefit from a sugar-preload due to their enhanced 5-HT vulnerability. The aim of the current study was to investigate whether a sugar-containing preload may reduce appetite and energy intake after exposure to stress to induce negative mood, depending on genetic 5-HT vulnerability. From a population of 771 healthy young male and female genotyped college students 31 S/S carriers (8 males, 23 females) and 26 long allele (L/L) carriers (9 males, 17 females) (mean ± S.D. 22 ± 1.6 years; body mass index, BMI, 18-33 kg/m(2)) were monitored for changes in appetite and snacking behavior after stress exposure. Results revealed an increased energy intake after mild mental stress (negative mood) mainly for high-fat sweet foods, which was significantly greater in S/S carriers, and only in these genotypes this intake was significantly reduced by a sucrose-containing preload. Although alternative explanations are possible, it is suggested that S/S participants may have enhanced brain (hypothalamic) 5-HT responsiveness to food that makes them more susceptible to the beneficial satiation effects of a sucrose-preload as well as to the negative effects of mild mental stress on weight gain.

  8. Inhibition of high-affinity gamma-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C.

    PubMed Central

    Gomeza, J; Casado, M; Gimenez, C; Aragon, C

    1991-01-01

    The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), on high-affinity Na(+)-dependent gamma-aminobutyric acid (GABA) uptake were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with PMA led to concentration- and time-dependent decreases in the GABA transport in glial cells. This effect could be completely suppressed by addition of the PKC inhibitor H7. The PMA effects could be mimicked by oleoylacetylglycerol, the diacylglycerol kinase inhibitor R59022 and exogenous phospholipase C. Treatment with PMA did not affect GABA transport in neuronal cells. PMID:1902665

  9. A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.

    PubMed Central

    Weber, H; Borisjuk, L; Heim, U; Sauer, N; Wobus, U

    1997-01-01

    To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability. PMID:9212465

  10. A role for sugar transporters during seed development: molecular characterization of a hexose and a sucrose carrier in fava bean seeds.

    PubMed

    Weber, H; Borisjuk, L; Heim, U; Sauer, N; Wobus, U

    1997-06-01

    To analyze sugar transport processes during seed development of fava bean, we cloned cDNAs encoding one sucrose and one hexose transporter, designated VfSUT1 and VfSTP1, respectively. sugar uptake activity was confirmed after heterologous expression in yeast. Gene expression was studied in relation to seed development. Transcripts were detected in both vegetative and seed tissues. In the embryo, VfSUT1 and VfSTP1 mRNAs were detected only in epidermal cells, but in a different temporal and spatial pattern. VfSTP1 mRNA accumulates during the midcotyledon stage in epidermal cells covering the mitotically active parenchyma, whereas the VfSUT1 transcript was specific to outer epidermal cells showing transfer cell morphology and covering the storage parenchyma. Transfer cells developed at the contact area of the cotyledonary epidermis and the seed coat, starting first at the early cotyledon stage and subsequently spreading to the abaxial region at the late cotyledon stage. Feeding high concentrations of sugars suppressed both VfSUT1 expression and transfer cell differentiation in vitro, suggesting a control by carbohydrate availability.

  11. High Affinity Binding of Indium and Ruthenium Ions by Gastrins

    PubMed Central

    Baldwin, Graham S.; George, Graham N.; Pushie, M. Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10−7 and 1.1 x 10−6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10−15 and 1.7 x 10−7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10−13 and 1.2 x 10−5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy. PMID:26457677

  12. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  13. Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.).

    PubMed

    Gu, Riliang; Duan, Fengying; An, Xia; Zhang, Fusuo; von Wirén, Nicolaus; Yuan, Lixing

    2013-09-01

    High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 μM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

  14. Sucrose carrier RcSCR1 is involved in sucrose retrieval, but not in sucrose unloading in growing hypocotyls of Ricinus communis L.

    PubMed

    Eisenbarth, D A; Weig, A R

    2005-01-01

    The transport of assimilates from source to sink tissues is mediated by the phloem. Along the vascular system the phloem changes its physiological function from loading phloem to transport and unloading phloem. Sucrose carrier proteins have been identified in the transport phloem, but it is unclear whether the physiological role of these transporters is phloem unloading of sucrose or retrieval of apoplasmic sucrose back into the sieve element/companion cell complex. Here, we describe the dynamic expression of the Ricinus communis sucrose carrier RcSCR1 in the hypocotyl at different sink strengths. Our results indicate that phloem unloading in castor bean is not catalysed by the phloem loader RcSCR1. However, this sucrose carrier represents the molecular basis of the sucrose retrieval mechanism along the transport phloem, which is dynamically adjusted to the sink strength. As a consequence, we assume that other release carrier(s) exist in sink tissues, such as the hypocotyl, in R. communis.

  15. Evidence that synaptosomal high-affinity carriers for amino acid neurotransmitters are glycosylated

    SciTech Connect

    Zaleska, M.M.; Erecinska, M.

    1987-05-01

    The effect of removal of surface sialic acid from synaptosomes on the high-affinity, Na/sup +/-dependent uptake systems for amino acid neurotransmitters was evaluated. Synaptosomes from rat forebrain were preincubated with neuraminidase from Vibrio cholerae for 20 min at 34/sup 0/. After washing and resuspension, their ability to transport /sup 14/C-GABA and the acidic amino acid, /sup 3/H-aspartate was studied. Pretreatment with neuraminidase resulted in a concentration-dependent inhibition of the uptake of both amino acids while the influx of /sup 3/H-L-leucine was unaffected. Inhibition was a function of the amount of sialic acid released from membranes. The analysis of the kinetic parameters of amino acid uptake revealed that inhibition resulted from a decrease of Vmax without any change in the Km. Neither the synaptosomal energy levels nor the internal concentration of potassium ions was affected by the pretreatment with neuraminidase. The maximum accumulation ratios for both amino acids remained largely unaltered. It is concluded that the GABA and acidic amino acid transporters are glycosylated and that sialic acid is involved in the operation of carrier proteins directly and not through modification of the driving forces responsible for amino acid transport.

  16. Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.

    PubMed

    Gabriel-Neumann, Elke; Neumann, Günter; Leggewie, Georg; George, Eckhard

    2011-06-15

    The sucrose transporter SUT1 functions in phloem loading of photoassimilates in solanaceous plant species. In the present study, wildtype and transgenic potato plants with either constitutive overexpression or antisense inhibition of SUT1 were grown under high or low phosphorus (P) fertilization levels in the presence or absence of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. At a low soil P fertilization level, the extent of AM fungal root colonization was not different among the genotypes. In all plants, the AM symbiosis contributed significantly to P uptake under these conditions. In response to a high soil P fertilization level, all genotypes showed a decrease in AM fungal root colonization, indicating that the expression level of SUT1 does not constitute a major mechanism of control over AM development in response to the soil P availability. However, plants with overexpression of SUT1 showed a higher extent of AM fungal root colonization compared with the other genotypes when the soil P availability was high. Whether an increased symbiotic C supply, alterations in the phytohormonal balance, or a decreased synthesis of antimicrobial compounds was the major cause for this effect requires further investigation. In plants with impaired phloem loading, a low C status of plant sink tissues did apparently not negatively affect plant C supply to the AM symbiosis. It is possible that, at least during vegetative and early generative growth, source rather than sink tissues exert control over amounts of C supplied to AM fungi. Copyright © 2011 Elsevier GmbH. All rights reserved.

  17. Uptake and metabolism of sucrose by Streptococcus lactis.

    PubMed

    Thompson, J; Chassy, B M

    1981-08-01

    Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS.

  18. Uptake and metabolism of sucrose by Streptococcus lactis.

    PubMed Central

    Thompson, J; Chassy, B M

    1981-01-01

    Transport and metabolism of sucrose in Streptococcus lactis K1 have been examined. Starved cells of S. lactis K1 grown previously on sucrose accumulated [14C]sucrose by a phosphoenolpyruvate-dependent phosphotransferase system (PTS) (sucrose-PTS; Km, 22 microM; Vmax, 191 mumol transported min-1 g of dry weight of cells-1). The product of group translocation was sucrose 6-phosphate (6-O-phosphoryl-D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside). A specific sucrose 6-phosphate hydrolase was identified which cleaved the disaccharide phosphate (Km, 0.10 mM) to glucose 6-phosphate and fructose. The enzyme did not cleave sucrose 6'-phosphate(D-glucopyranosyl-1-alpha-beta-2-D-fructofuranoside-6'-phosphate). Extracts prepared from sucrose-grown cells also contained an ATP-dependent mannofructokinase which catalyzed the conversion of fructose to fructose 6-phosphate (Km, 0.33 mM). The sucrose-PTS and sucrose 6-phosphate hydrolase activities were coordinately induced during growth on sucrose. Mannofructokinase appeared to be regulated independently of the sucrose-PTS and sucrose 6-phosphate hydrolase, since expression also occurred when S. lactis K1 was grown on non-PTS sugars. Expression of the mannofructokinase may be negatively regulated by a component (or a derivative) of the PTS. PMID:6267012

  19. Stable U(IV) complexes form at high-affinity mineral surface sites.

    PubMed

    Latta, Drew E; Mishra, Bhoopesh; Cook, Russell E; Kemner, Kenneth M; Boyanov, Maxim I

    2014-01-01

    Uranium (U) poses a significant contamination hazard to soils, sediments, and groundwater due to its extensive use for energy production. Despite advances in modeling the risks of this toxic and radioactive element, lack of information about the mechanisms controlling U transport hinders further improvements, particularly in reducing environments where U(IV) predominates. Here we establish that mineral surfaces can stabilize the majority of U as adsorbed U(IV) species following reduction of U(VI). Using X-ray absorption spectroscopy and electron imaging analysis, we find that at low surface loading, U(IV) forms inner-sphere complexes with two metal oxides, TiO2 (rutile) and Fe3O4 (magnetite) (at <1.3 U nm(-2) and <0.037 U nm(-2), respectively). The uraninite (UO2) form of U(IV) predominates only at higher surface loading. U(IV)-TiO2 complexes remain stable for at least 12 months, and U(IV)-Fe3O4 complexes remain stable for at least 4 months, under anoxic conditions. Adsorbed U(IV) results from U(VI) reduction by Fe(II) or by the reduced electron shuttle AH2QDS, suggesting that both abiotic and biotic reduction pathways can produce stable U(IV)-mineral complexes in the subsurface. The observed control of high-affinity mineral surface sites on U(IV) speciation helps explain the presence of nonuraninite U(IV) in sediments and has important implications for U transport modeling.

  20. The Sucrose Transporter StSUT1 Localizes to Sieve Elements in Potato Tuber Phloem and Influences Tuber Physiology and Development1[w

    PubMed Central

    Kühn, Christina; Hajirezaei, Mohammad-Reza; Fernie, Alisdair R.; Roessner-Tunali, Ute; Czechowski, Tomasz; Hirner, Brigitte; Frommer, Wolf B.

    2003-01-01

    The sucrose (Suc) H+-cotransporter StSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. Reduced SUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc- and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm. PMID:12529519

  1. Sucrose and Related Oligosaccharides

    NASA Astrophysics Data System (ADS)

    Eggleston, Gillian

    Sucrose (α-D-glucopyranosyl-(1↔2)-β-D-fructofuranoside) is the most common low-molecular-weight sugar found in the plant kingdom. It is ubiquitously known as common table sugar and primarily produced industrially from sugarcane (Saccharum officinarum) and sugar beet (Beta vulgaris); the basics of the industrial manufacture of sucrose are outlined in this chapter. Commercial sucrose has a very high purity (> 99.9%) making it one of the purest organic substances produced on an industrial scale. Value-addition to sucrose via chemical and biotechnological reactions is becoming more important for the diversification of the sugar industry to maintain the industries' competitiveness in a world increasingly turning to a bio-based economy. The basis for the chemical reactivity of sucrose is the eight hydroxyl groups present on the molecule, although, sucrose chemical reactivity is regarded as difficult. Increasing use of enzymatic biotechnological techniques to derivatize sucrose is expected, to add special functionalities to sucrose products like biodegradability, biocompatibility, and non-toxicity. Analysis of sucrose by colorimetric, enzymatic, oxidation-reduction and chromatography methods are discussed. Oligosaccharides related to sucrose are outlined in detail and include sucrose-based plant, honey and in vitro oligosaccharides.

  2. High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic receptors

    SciTech Connect

    Kellar, K.J.; Martino, A.M.; Hall, D.P. Jr.; Schwartz, R.D.; Taylor, R.L.

    1985-06-01

    High-affinity binding of (/sup 3/H)acetylcholine to muscarinic cholinergic sites in rat CNS and peripheral tissues was measured in the presence of cytisin, which occupies nicotinic cholinergic receptors. The muscarinic sites were characterized with regard to binding kinetics, pharmacology, anatomical distribution, and regulation by guanyl nucleotides. These binding sites have characteristics of high-affinity muscarinic cholinergic receptors with a Kd of approximately 30 nM. Most of the muscarinic agonist and antagonist drugs tested have high affinity for the (/sup 3/H)acetylcholine binding site, but pirenzepine, an antagonist which is selective for M-1 receptors, has relatively low affinity. The ratio of high-affinity (/sup 3/H)acetylcholine binding sites to total muscarinic binding sites labeled by (/sup 3/H)quinuclidinyl benzilate varies from 9 to 90% in different tissues, with the highest ratios in the pons, medulla, and heart atrium. In the presence of guanyl nucleotides, (/sup 3/H) acetylcholine binding is decreased, but the extent of decrease varies from 40 to 90% in different tissues, with the largest decreases being found in the pons, medulla, cerebellum, and heart atrium. The results indicate that (/sup 3/H)acetylcholine binds to high-affinity M-1 and M-2 muscarinic receptors, and they suggest that most M-2 sites have high affinity for acetylcholine but that only a small fraction of M-1 sites have such high affinity.

  3. In vivo gene transfer to dopamine neurons of rat substantia nigra via the high-affinity neurotensin receptor.

    PubMed Central

    Alvarez-Maya, I.; Navarro-Quiroga, I.; Meraz-Ríos, M. A.; Aceves, J.; Martinez-Fong, D.

    2001-01-01

    BACKGROUND: Recently, we synthesized a nonviral gene vector capable of transfecting cell lines taking advantage of neurotensin (NT) internalization. The vector is NT cross-linked with poly-L-lysine, to which a plasmid DNA was bound to form a complex (NT-polyplex). Nigral dopamine neurons are able to internalize NT, thus representing a target for gene transfer via NT-polyplex. This hypothesis was tested here using reporter genes encoding green fluorescent protein or chloramphenicol acetyl transferase. MATERIALS AND METHODS: NT-polyplex was injected into the substantia nigra. Double immunofluorescence labeling was used to reveal the cell type involved in the propidium iodide-labeled polyplex internalization and reporter gene expression. RESULTS: Polyplex internalization was observed within dopamine neurons but not within glial cells, and was prevented by both hypertonic sucrose solution and SR-48692, a selective nonpeptide antagonist of NT receptors. Reporter gene expression was observed in dopamine neurons from 48 hr up to 15 days after NT-polyplex injection, and was prevented by SR-48692. However, no expression was seen when the NT-polyplex was injected into the ansiform lobule of the cerebellum, which contains low- but not high-affinity NT receptors. Neither internalization nor expression was observed in cultured glial cells, despite the NT-polyplex binding to those cells that was prevented by levocabastine, a low-affinity NT receptor antagonist. CONCLUSIONS: These results suggest that high-affinity NT receptors mediate the uptake of NT-polyplex with the subsequent reporter gene expression in vivo. NT polyfection may be used to transfer genes of physiologic interest to nigrostriatal dopamine neurons, and to produce transgenic animal models of dopamine-related diseases. PMID:11471555

  4. Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

    PubMed Central

    Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria

    2014-01-01

    Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575

  5. The high affinity iron permease is a key virulence factor required for Rhizopus oryzae pathogenesis

    PubMed Central

    Ibrahim, Ashraf S.; Gebremariam, Teclegiorgis; Lin, Lin; Luo, Guanpingsheng; Husseiny, Mohamed I.; Skory, Christopher D.; Fu, Yue; French, Samuel W.; Edwards, John E.; Spellberg, Brad

    2010-01-01

    SUMMARY Rhizopus oryzaeis the most common cause of mucormycosis, an angioinvasive fungal infection that causes more then 50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. The high affinity iron permease gene (FTR1) is required for R. oryzae iron transport in iron-depleted environments. Here we demonstrate that FTR1 is required for full virulence of R. oryzae in mice. We show that FTR1 is expressed during infection in diabetic ketoacidotic (DKA) mice. In addition, we disrupted FTR1 by double cross-over homologous recombination, but multinucleated R. oryzae could not be forced to segregate to a homokaryotic null allele. Nevertheless, a reduction of the relative copy number of FTR1 and inhibition of FTR1 expression by RNAi compromised the ability of R. oryzae to acquire iron in vitro and reduced its virulence in DKA mice. Importantly, passive immunization with anti-Ftr1p immune sera protected DKA mice from infection with R. oryzae. Thus FTR1 is a virulence factor for R. oryzae, and anti-Ftr1p passive immunotherapy deserves further evaluation as a strategy to improve outcomes of deadly mucormycosis. PMID:20545847

  6. Isolation and Characterization of a High Affinity Peptide Inhibitor of ClC-2 Chloride Channels*

    PubMed Central

    Thompson, Christopher H.; Olivetti, Pedro R.; Fuller, Matthew D.; Freeman, Cody S.; McMaster, Denis; French, Robert J.; Pohl, Jan; Kubanek, Julia; McCarty, Nael A.

    2009-01-01

    The ClC protein family includes voltage-gated chloride channels and chloride/proton exchangers. In eukaryotes, ClC proteins regulate membrane potential of excitable cells, contribute to epithelial transport, and aid in lysosomal acidification. Although structure/function studies of ClC proteins have been aided greatly by the available crystal structures of a bacterial ClC chloride/proton exchanger, the availability of useful pharmacological tools, such as peptide toxin inhibitors, has lagged far behind that of their cation channel counterparts. Here we report the isolation, from Leiurus quinquestriatus hebraeus venom, of a peptide toxin inhibitor of the ClC-2 chloride channel. This toxin, GaTx2, inhibits ClC-2 channels with a voltage-dependent apparent KD of ∼20 pm, making it the highest affinity inhibitor of any chloride channel. GaTx2 slows ClC-2 activation by increasing the latency to first opening by nearly 8-fold but is unable to inhibit open channels, suggesting that this toxin inhibits channel activation gating. Finally, GaTx2 specifically inhibits ClC-2 channels, showing no inhibitory effect on a battery of other major classes of chloride channels and voltage-gated potassium channels. GaTx2 is the first peptide toxin inhibitor of any ClC protein. The high affinity and specificity displayed by this toxin will make it a very powerful pharmacological tool to probe ClC-2 structure/function. PMID:19574231

  7. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  8. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes.

  9. Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae. Role of dioxygen and Fe.

    PubMed

    Hassett, R F; Romeo, A M; Kosman, D J

    1998-03-27

    High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease. Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T. V. (1996) Science 271, 1510-1512). We show here that dioxygen also plays an essential role in the expression of these iron uptake activities. Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air. Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen. The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins. Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells. In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2, 2'-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions. An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2'-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities. The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein. The data suggest a model in which dioxygen directly or indirectly modulates the Fe

  10. Contributions of sucrose synthase and invertase to the metabolism of sucrose in developing leaves: estimation by alternate substrate utilization

    SciTech Connect

    Schmalstig, J.G.; Hitz, W.D.

    1987-10-01

    The relative contributions of invertase and sucrose synthase to initial cleavage of phloem-imported sucrose was calculated for sink leaves of soybean (Glycine max L. Merr cv Wye) and sugar beet (Beta vulgaris L. monohybrid). Invertase from yeast hydrolyzed sucrose 4200 times faster than 1'-deoxy-1'-fluorosucrose (FS) while sucrose cleavage by sucrose synthase from developing soybean leaves proceeded only 3.6 times faster than cleavage of FS.(/sup 14/C)Sucrose and (/sup 14/C)FS, used as tracers of sucrose, were transported at identical rates to developing leaves through the phloem. The rate of label incorporation into insoluble products varied with leaf age from 3.4 to 8.0 times faster when (/sup 14/C)sucrose was supplied than when (/sup 14/C)FS was supplied. The discrimination in metabolism was related to enzymatic discriminations against FS to calculate the relative contributions of invertase and sucrose synthase to sucrose cleavage. In the youngest soybean leaves measured, 4% of final laminar length (FLL), all cleavage was by sucrose synthase. Invertase contribution to sucrose metabolism was 47% by 7.6% FLL, increased to 54% by 11% FLL, then declined to 42% for the remainder of the import phase. In sugar beet sink leaves at 30% FLL invertase contribution to sucrose metabolism was 58%.

  11. Sucrose ingestion induces rapid AMPA receptor trafficking.

    PubMed

    Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B

    2013-04-03

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

  12. Sucrose signaling in plants

    PubMed Central

    Tognetti, Jorge A.; Pontis, Horacio G.; Martínez-Noël, Giselle M.A.

    2013-01-01

    The role of sucrose as a signaling molecule in plants was originally proposed several decades ago. However, recognition of sucrose as a true signal has been largely debated and only recently this role has been fully accepted. The best-studied cases of sucrose signaling involve metabolic processes, such as the induction of fructan or anthocyanin synthesis, but a large volume of scattered information suggests that sucrose signals may control a vast array of developmental processes along the whole life cycle of the plant. Also, wide gaps exist in our current understanding of the intracellular steps that mediate sucrose action. Sucrose concentration in plant tissues tends to be directly related to light intensity, and inversely related to temperature, and accordingly, exogenous sucrose supply often mimics the effect of high light and cold. However, many exceptions to this rule seem to occur due to interactions with other signaling pathways. In conclusion, the sucrose role as a signal molecule in plants is starting to be unveiled and much research is still needed to have a complete map of its significance in plant function. PMID:23333971

  13. MKC-231, a choline uptake enhancer: (3) Mode of action of MKC-231 in the enhancement of high-affinity choline uptake.

    PubMed

    Takashina, Ken; Bessho, Tomoko; Mori, Reiko; Kawai, Kunji; Eguchi, Junichi; Saito, Ken-Ichi

    2008-07-01

    MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.

  14. Identification of a high-affinity Ca sup 2+ pump associated with endocytotic vesicles in Dictyostelium discoideum

    SciTech Connect

    Milne, J.L.; Coukell, M.B. )

    1989-11-01

    In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca{sup 2+} are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca{sup 2+} levels in this organism, the authors previously isolated and characterized an ATP/Mg{sup 2+}-dependent, high-affinity Ca{sup 2+} pump which appeared to be a component of inside-out plasma membrane vesicles. In this report, they demonstrate that a high-affinity Ca{sup 2+} pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca{sup 2+}-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca{sup 2+} transporter is associated with a high-capacity non-IP{sub 3}-releasable Ca{sup 2+} store which is generated by endocytosis. A possible role for this store in maintaining Ca{sup 2+} homeostasis in Dictyostelium is discussed.

  15. The shoot is important for high-affinity nitrate uptake in Egeria densa, a submerged vascular plant.

    PubMed

    Takayanagi, Shu; Takagi, Yuma; Shimizu, Akifumi; Hasegawa, Hiroshi

    2012-09-01

    To understand the mechanisms of nitrate uptake by submerged vascular plants, a cDNA for a high-affinity nitrate transporter, NRT2, was isolated from Egeria densa, a submerged monocot. The deduced EdNRT2 protein was similar to the proteins of a conserved NRT2 group in higher plants. Real-time reverse transcription-PCR analysis revealed that after feeding whole plants with 0.2 mM nitrate, the EdNRT2 transcripts were induced in both shoots and roots within 0.5 h, reached the maximum by 1-3 h and then decreased. The EdNRT2 transcript levels in shoots were comparable to those in roots. When nitrate was applied separately to shoots and roots, the EdNRT2 transcripts were induced only in nitrate-treated organs and reached the maximum levels comparable to those in organs when nitrate was applied to whole plants. (15)N-nitrate feeding experiments demonstrated that both shoots and roots are responsible for nitrate uptake and that biomass and (15)N content in shoots was even higher than that in roots. We concluded that EdNRT2 is involved in high-affinity nitrate uptake by shoots and roots of E. densa, that nitrate is taken up independently by shoots and roots and that shoots play an important role in nitrate uptake from aquatic ecosystem.

  16. GHB receptor targets in the CNS: focus on high-affinity binding sites.

    PubMed

    Bay, Tina; Eghorn, Laura F; Klein, Anders B; Wellendorph, Petrine

    2014-01-15

    γ-Hydroxybutyric acid (GHB) is an endogenous compound in the mammalian brain with both low- and high-affinity receptor targets. GHB is used clinically in the treatment of symptoms of narcolepsy and alcoholism, but also illicitly abused as the recreational drug Fantasy. Major pharmacological effects of exogenous GHB are mediated by GABA subtype B (GABAB) receptors that bind GHB with low affinity. The existence of GHB high-affinity binding sites has been known for more than three decades, but the uncovering of their molecular identity has only recently begun. This has been prompted by the generation of molecular tools to selectively study high-affinity sites. These include both genetically modified GABAB knock-out mice and engineered selective GHB ligands. Recently, certain GABA subtype A (GABAA) receptor subtypes emerged as high-affinity GHB binding sites and potential physiological mediators of GHB effects. In this research update, a description of the various reported receptors for GHB is provided, including GABAB receptors, certain GABAA receptor subtypes and other reported GHB receptors. The main focus will thus be on the high-affinity binding targets for GHB and their potential functional roles in the mammalian brain.

  17. ( sup 14 C)-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

    SciTech Connect

    Rohrig, K.; Raschke, K. )

    1991-05-01

    Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated ({sup 14}C)-sucrose. Uptake rates were corrected after measurement of {sup 14}C-sorbitol and {sup 3}H{sub 2}O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent K{sub m} 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related to an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours.

  18. Synthesis and biological activity of a novel series of 6-substituted thieno[2,3-d]pyrimidine antifolate inhibitors of purine biosynthesis with selectivity for high affinity folate receptors over the reduced folate carrier and proton-coupled folate transporter for cellular entry

    PubMed Central

    Deng, Yijun; Zhou, Xilin; Desmoulin, Sita Kugel; Wu, Jianmei; Cherian, Christina; Hou, Zhanjun; Matherly, Larry H.; Gangjee, Aleem

    2009-01-01

    A series of seven 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines, with bridge length variations (from 2-8 carbon atoms) were synthesized as selective folate receptor (FR) α and β substrates and as antitumor agents. The syntheses were accomplished from appropriate allylalcohols and 4-iodobenzoate to afford the aldehydes which were converted to the appropriate 2-amino-4-carbethoxy-5-substituted thiophenes 23-29. Cyclization with chlorformamidine afforded the thieno[2,3-d]pyrimidines 30-36 which were hydrolyzed and coupled with diethyl-L-glutamate, followed by saponification to give the target compounds 2-8. Compounds 3-6 were potent growth inhibitors (IC50 4.7 to 334 nM) of human tumor cells (KB and IGROV1) that express FRs. In addition, compounds 3-6 inhibited the growth of Chinese hamster ovary (CHO) cells that expressed FRs but not the reduced folate carrier (RFC) or proton-coupled folate transporter (PCFT). However, the compounds were inactive toward CHO cells that lacked FRs but contained either the RFC or PCFT. By nucleoside and 5-amino-4-imidazole carboxamide (AICA) protection studies, along with in vitro and in situ enzyme activity assays, the mechanism of antitumor activity was identified as the dual inhibition of glycinamide ribonucleotide formyltransferase and, likely, AICA ribonucleotide formyltransferase. The dual inhibitory activity of the active thieno[2,3-d]pyrimidine antifolates and the FR specificity represent unique mechanistic features for these compounds distinct from all other known antifolates. The potent inhibitory effects of compounds 3-6 toward cells expressing FRs but not PCFT provide direct evidence that cellular uptake of this series of compounds by FRs does not depend on the presence of PCFT and argues that direct coupling between these transporters is not obligatory. PMID:19371039

  19. Sucrose and behavioral problems.

    PubMed

    Benton, David

    2008-05-01

    Various mechanisms by which sucrose could influence behavior are reviewed. Firstly there is food intolerance. There are dozens of foods to which an adverse reaction has been demonstrated, although a reaction to sucrose is less frequent than many other foods. A second possible mechanism is hypoglycemia. There is evidence that a tendency to develop low blood glucose levels, but higher than those that can be described clinically as hypoglycemic, is associated with irritability and violence. However, sucrose is not the predominant cause of swings in blood glucose levels. Thirdly, the role of sucrose intake on micro-nutrient status has been considered as studies have found that micro-nutrient supplementation decreased anti-social behavior. Micro-nutrient intake is more closely associated with the total energy rather than sucrose intake; typically the amount of sucrose in the diet does not lead to micro-nutrient deficiency. In fact meta-analysis of well designed studies that have examined the impact of sucrose on the behavior of children produced no evidence that it has an adverse influence.

  20. Sertraline and its metabolite desmethylsertraline, but not bupropion or its three major metabolites, have high affinity for P-glycoprotein.

    PubMed

    Wang, Jun-Sheng; Zhu, Hao-Jie; Gibson, Bryan Bradford; Markowitz, John Seth; Donovan, Jennifer Lyn; DeVane, Carl Lindsay

    2008-02-01

    The ATP-binding cassette (ABC) transporter protein subfamily B1 line (ABCB1) transporter P-glycoprotein (P-gp) plays an important role in the blood-brain barrier limiting a broad spectrum of substrates from entering the central nervous system. In the present study, the transport activity of P-gp for sertraline, desmethylsertraline, bupropion, and the major metabolites of bupropion, threo-amino alcohol (TB), erythro-amino alcohol (EB), and hydroxy metabolite (HB) was studied using an ATPase assay in expressed human P-gp membranes by measuring concentrations of inorganic P(i) in expressed human P-gp membranes. Verapamil was included as a positive control. The Michaelis-Menten equation was used for characterizing the kinetic data. Sertraline and desmethylsertraline showed high affinity for P-gp. The V(max)/K(m) values of sertraline (1.6 min(-1) x 10(-3)) and desmethylsertraline (1.4 min(-1) x 10(-3)) were comparable with that of verapamil (1.7 min(-1) x 10(-3)). Bupropion and its three metabolites showed very weak affinity for P-gp, with V(max)/K(m) values lower than 0.01 min(-1) x 10(-3). The results of the present study indicate that sertraline and desmethylsertraline have high affinity for P-gp, whereas bupropion and its three major metabolites TB, EB, and HB have very weak affinity for P-gp. These findings may help to explain observed drug-drug interactions among antidepressants.

  1. Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

    PubMed

    Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

    2017-02-01

    To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  2. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  3. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  4. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  5. SORPTION OF LEAD ON A HIGH AFFINITY OXIDE: MACROSCOPIC AND MICROSCOPIC STUDIES (ABSTRACT)

    EPA Science Inventory

    Sorption of lead (Pb) was investigated on an innovative metal oxide compound using macroscopic and microscopic techniques. The objective of this study was to elucidate the sorption mechanism of Pb on the high-affinity engineered oxide with time at pH 6 employing batch methods an...

  6. Two distinct functional high affinity receptors for mouse interleukin-3 (IL-3).

    PubMed Central

    Hara, T; Miyajima, A

    1992-01-01

    The human interleukin-3 receptor (IL-3R) is composed of an IL-3 specific alpha subunit (IL-3R alpha) and a common beta subunit (beta c) that is shared by IL-3, granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-5 receptors. In contrast to the human, the mouse has two distinct but related genes, AIC2A and AIC2B, both of which are homologous to the human beta c gene. AIC2B has proved to encode a common beta subunit between mouse GM-CSF and IL-5 receptors. AIC2A is unique to the mouse and encodes a low affinity IL-3 binding protein. Based on the observation that the AIC2A protein is a component of a high affinity IL-3R, we searched for a cDNA encoding a protein which conferred high affinity IL-3 binding when coexpressed with the AIC2A protein in COS7 cells. We obtained such a cDNA (SUT-1) encoding a mature protein of 70 kDa that has weak homology to the human IL-3R alpha. The SUT-1 protein bound IL-3 with low affinity and formed high affinity receptors not only with the AIC2A protein but also with the AIC2B protein. Both high affinity IL-3Rs expressed on a mouse T cell line, CTLL-2, showed similar IL-3 binding properties and transmitted a growth signal in response to IL-3. Thus, the mouse has two distinct functional high affinity IL-3Rs, providing a molecular explanation for the differences observed between mouse and human IL-3Rs. Images PMID:1582416

  7. ZrFsy1, a High-Affinity Fructose/H+ Symporter from Fructophilic Yeast Zygosaccharomyces rouxii

    PubMed Central

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C.

    2013-01-01

    Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H+ symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H+ symporter, with Km 0.45±0.07 mM and Vmax 0.57±0.02 mmol h−1 (gdw) −1. We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H+ symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system. PMID:23844167

  8. ZrFsy1, a high-affinity fructose/H+ symporter from fructophilic yeast Zygosaccharomyces rouxii.

    PubMed

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C

    2013-01-01

    Zygosaccharomyces rouxii is a fructophilic yeast than can grow at very high sugar concentrations. We have identified an ORF encoding a putative fructose/H(+) symporter in the Z. rouxii CBS 732 genome database. Heterologous expression of this ORF in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic characterization of its sugar transport activity showed it is a high-affinity low-capacity fructose/H(+) symporter, with Km 0.45 ± 0.07 mM and Vmax 0.57 ± 0.02 mmol h(-1) (gdw)(-1). We named it ZrFsy1. This protein also weakly transports xylitol and sorbose, but not glucose or other hexoses. The expression of ZrFSY1 in Z. rouxii is higher when the cells are cultivated at extremely low fructose concentrations (<0.2%) and on non-fermentable carbon sources such as mannitol and xylitol, where the cells have a prolonged lag phase, longer duplication times and change their microscopic morphology. A clear phenotype was determined for the first time for the deletion of a fructose/H(+) symporter in the genome where it occurs naturally. The effect of the deletion of ZrFSY1 in Z. rouxii cells is only evident when the cells are cultivated at very low fructose concentrations, when the ZrFsy1 fructose symporter is the main active fructose transporter system.

  9. Sucrose secreted by the engineered cyanobacterium and its fermentability

    NASA Astrophysics Data System (ADS)

    Duan, Yangkai; Luo, Quan; Liang, Feiyan; Lu, Xuefeng

    2016-10-01

    The unicellular cyanobacterium, Synechococcus elongatus PCC 7942 (Syn7942), synthesizes sucrose as the only compatible solute under salt stress. A series of engineered Syn7942 strains for sucrose production were constructed. The overexpression of the native sps (encoding a natively fused protein of sucrose phosphate synthase SPS and sucrose phosphate phosphatase SPP) in Syn7942 wild type caused a 93% improvement of sucrose productivity. The strain FL130 co-overexpressing sps and cscB (encoding a sucrose transporter) exhibited a 74% higher extracellular sucrose production than that overexpressing cscB only. Both results showed the significant improvement of sucrose productivity by the double functional protein SPS-SPP. Afterwards, FL130 was cultivated under a modified condition, and the cell-free culture medium containing 1.5 g L-1 sucrose was pre-treated with an acid hydrolysis technique. Cultivated with the neutralized hydrolysates as the starting media, two widely used microorganisms, Escherichia coli and Saccharomyces cerevisiae, showed a comparable growth with that in the control media supplemented with glucose. These results clearly demonstrated that the cell-free culture of sucrose-secreting cyanobacteria can be applied as starting media in microbial cultivation.

  10. Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

    PubMed Central

    Echenique, J R; Arienti, H; Tolmasky, M E; Read, R R; Staneloni, R J; Crosa, J H; Actis, L A

    1992-01-01

    Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Images PMID:1447137

  11. Sucrose-mediated transcriptional regulation of sucrose symporter activity in the phloem.

    PubMed

    Vaughn, Matthew W; Harrington, Gregory N; Bush, Daniel R

    2002-08-06

    A proton-sucrose symporter mediates the key step in carbon export from leaves of most plants. Sucrose transport activity and steady-state mRNA levels of BvSUT1, a sugar beet leaf sucrose symporter, are negatively regulated specifically by sucrose. Results reported here show that BvSUT1 mRNA was localized to companion cells of the leaf's vascular system, which supports its role in the systemic distribution of photoassimilate. Immunoblot analysis showed that decreased transport activity was caused by a reduction in the abundance of symporter protein. RNA gel blot analysis of the leaf symporter revealed that message levels also declined, and nuclear run-on experiments demonstrated that this was the result of decreased transcription. Further analysis showed that symporter protein and message are both degraded rapidly. Taken together, these data show that phloem loading is regulated by means of sucrose-mediated changes in transcription of a phloem-specific sucrose symporter gene in a regulatory system that may play a pivotal role in balancing photosynthetic activity with resource utilization.

  12. Upregulation of high affinity GABAA receptors in cultured rat dorsal root ganglion neurons

    PubMed Central

    Lee, Kwan Yeop; Charbonnet, Marcel; Gold, Michael S.

    2012-01-01

    Despite evidence that high-affinity GABAA receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABAA currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 hrs (acute) and 24 hrs (cultured) after plating with whole cell patch clamp techniques, western blot and qRT-PCR analysis. GABAA current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50’s of 0.25 ± 0.01 μM and 3.2 ± 0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16 ± 10.1 μM to 9.56 ± 1.3 μM with time in culture. A fraction of the high affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute but the membrane fraction in cultured DRG neurons with no detectable change in δ-subunit mRNA. However, the emergence of a high affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2 and 3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit containing GABAA receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution. PMID:22366297

  13. Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals.

    PubMed

    Vert, Grégory A; Briat, Jean-François; Curie, Catherine

    2003-06-01

    Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.

  14. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

  15. Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations.

    PubMed

    Rubio, Francisco; Nieves-Cordones, Manuel; Alemán, Fernando; Martínez, Vicente

    2008-12-01

    The relative contribution of the high-affinity K(+) transporter AtHAK5 and the inward rectifier K(+) channel AtAKT1 to K(+) uptake in the high-affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col-0). The results obtained with wild-type lines, with T-DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the NH4+-sensitive and the Ba(2+)-sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high-affinity range of Rb(+) concentrations. Using Rb(+) as a K(+) analogue, it was shown that AtHAK5 mediates absorption at lower Rb(+) concentrations than AtAKT1 and depletes external Rb(+) to values around 1 muM. Factors such as the presence of K(+) or NH4+ during plant growth determine the relative contribution of each system. The presence of NH4+ in the growth solution inhibits the induction of AtHAK5 by K(+) starvation. In K(+)-starved plants grown without NH4+, both systems are operative, but when NH4+ is present in the growth solution, AtAKT1 is probably the only system mediating Rb(+) absorption, and the capacity of the roots to deplete Rb(+) is reduced.

  16. Sucrose Ingestion Induces Rapid AMPA Receptor Trafficking

    PubMed Central

    Tukey, David S.; Ferreira, Jainne M.; Antoine, Shannon O.; D’amour, James A.; Ninan, Ipe; de Vaca, Soledad Cabeza; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K.; Hartner, Diana T.; Guarini, Carlo B.; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F.; Khatri, Megna; Marzan, Dave S.; Mahajan, Shahana S.; Wang, Jing; Froemke, Robert C.; Carr, Kenneth D.; Aoki, Chiye; Ziff, Edward B.

    2013-01-01

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca2+-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose. PMID:23554493

  17. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity

    PubMed Central

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K.; Corey, David P.

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications. PMID:26943906

  18. The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

    PubMed

    Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

    2012-09-01

    Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses.

  19. Three Recombinant Engineered Antibodies against Recombinant Tags with High Affinity and Specificity.

    PubMed

    Zhao, Hongyu; Shen, Ao; Xiang, Yang K; Corey, David P

    2016-01-01

    We describe three recombinant engineered antibodies against three recombinant epitope tags, constructed with divalent binding arms to recognize divalent epitopes and so achieve high affinity and specificity. In two versions, an epitope is inserted in tandem into a protein of interest, and a homodimeric antibody is constructed by fusing a high-affinity epitope-binding domain to a human or mouse Fc domain. In a third, a heterodimeric antibody is constructed by fusing two different epitope-binding domains which target two different binding sites in GFP, to polarized Fc fragments. These antibody/epitope pairs have affinities in the low picomolar range and are useful tools for many antibody-based applications.

  20. Selection of short peptides with high affinity for various metal ions: Strategies and applications

    SciTech Connect

    Ataai, M.M.; Whiteford, W.K.

    1995-12-01

    The authors have used a random combinatorial library to generate an immensely diverse population of candidate peptides and have screened the library, using novel strategies, for selection of peptides of displaying high affinity towards a target metal. As the pool of available binding candidates, we have employed a hexapeptide library, containing essentially all the possible combination of sequences for a hexapeptide, expressed on surface of phage. Our experiments with copper demonstrated that our amplification and selection procedure is very effective. Out of 20 clones sequenced, after four rounds of amplification and selection 18 shared the same sequence. The corresponding peptide which contain two histidines was then synthesized and its binding affinity for copper was assessed. The results revealed that the selected peptide has a very high affinity for copper; its affinity is significantly higher than any hexapeptide which we are aware of including six adjacent histidine, (his)6, frequently used as an affinity tag in bioseparation. Selection of short peptides with high affinity and selectivity for arsenic, mercury, and lead is also underway. We envision important application of this work including discovery of highly effective drugs for treating metal toxicity replacing the chelating agents currently in use, and development of inexpensive whole cell adsorbents for toxicity replacing the chelating agents currently in use, and development of inexpensive whole cell adsorbents for the removal of toxic metals from dilute aqueous streams because dead cells or isolated cell wall, covered by the desired metal-binding domain may serve as high affinity and high capacity metal adsorbents. Other potential application will also be discussed in this presentation.

  1. Quantifying high-affinity binding of hydrophobic ligands by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Broecker, Jana; Vargas, Carolyn; Fanghänel, Jörg; Keller, Sandro

    2012-12-18

    A fast and reliable quantification of the binding thermodynamics of hydrophobic high-affinity ligands employing a new calorimetric competition experiment is described. Although isothermal titration calorimetry is the method of choice for a quantitative characterization of intermolecular interactions in solution, a reliable determination of a dissociation constant (K(D)) is typically limited to the range 100 μM > K(D) > 1 nM. Interactions displaying higher or lower K(D) values can be assessed indirectly, provided that a suitable competing ligand is available whose K(D) falls within the directly accessible affinity window. This established displacement assay, however, requires the high-affinity ligand to be soluble at high concentrations in aqueous buffer and, consequently, poses serious problems in the study of protein binding involving small-molecule ligands dissolved in organic solvents--a familiar case in many drug-discovery projects relying on compound libraries. The calorimetric competition assay introduced here overcomes this limitation, thus allowing for a detailed thermodynamic description of high-affinity receptor-ligand interactions involving poorly water-soluble compounds. Based on a single titration of receptor into a dilute mixture of the two competing ligands, this competition assay provides accurate and precise values for the dissociation constants and binding enthalpies of both high- and moderate-affinity ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation and high-affinity protein-inhibitor interactions, and explore its potential and limitations with the aid of simulations and statistical analyses.

  2. Amyloid-beta binds catalase with high affinity and inhibits hydrogen peroxide breakdown.

    PubMed Central

    Milton, N G

    1999-01-01

    Amyloid-beta (Abeta) specifically bound purified catalase with high affinity and inhibited catalase breakdown of H(2)O(2). The Abeta-induced catalase inhibition involved formation of the inactive catalase Compound II and was reversible. Catalase<-->Abeta interactions provide rapid functional assays for the cytotoxic domain of Abeta and suggest a mechanism for some of the observed actions of Abeta plus catalase in vitro. PMID:10567208

  3. Selective high-affinity polydentate ligands and methods of making such

    DOEpatents

    DeNardo, Sally; DeNardo, Gerald; Balhorn, Rodney

    2013-09-17

    This invention provides polydentate selective high affinity ligands (SHALs) that can be used in a variety of applications in a manner analogous to the use of antibodies. SHALs typically comprise a multiplicity of ligands that each binds different regions on the target molecule. The ligands are joined directly or through a linker thereby forming a polydentate moiety that typically binds the target molecule with high selectivity and avidity.

  4. The Structure of Sucrose Synthase-1 from Arabidopsis thaliana and Its Functional Implications

    SciTech Connect

    Zheng, Yi; Anderson, Spencer; Zhang, Yanfeng; Garavito, R. Michael

    2014-10-02

    Sucrose transport is the central system for the allocation of carbon resources in vascular plants. During growth and development, plants control carbon distribution by coordinating sites of sucrose synthesis and cleavage in different plant organs and different cellular locations. Sucrose synthase, which reversibly catalyzes sucrose synthesis and cleavage, provides a direct and reversible means to regulate sucrose flux. Depending on the metabolic environment, sucrose synthase alters its cellular location to participate in cellulose, callose, and starch biosynthesis through its interactions with membranes, organelles, and cytoskeletal actin. The x-ray crystal structure of sucrose synthase isoform 1 from Arabidopsis thaliana (AtSus1) has been determined as a complex with UDP-glucose and as a complex with UDP and fructose, at 2.8- and 2.85-{angstrom} resolutions, respectively. The AtSus1 structure provides insights into sucrose catalysis and cleavage, as well as the regulation of sucrose synthase and its interactions with cellular targets.

  5. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

    PubMed Central

    Ebisawa, T; Karne, S; Lerner, M R; Reppert, S M

    1994-01-01

    Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family. Images PMID:7517042

  6. Reconstitution of high-affinity opioid agonist binding in brain membranes

    SciTech Connect

    Remmers, A.E.; Medzihradsky, F. )

    1991-03-15

    In synaptosomal membranes from rat brain cortex, the {mu} selective agonist ({sup 3}H)dihydromorphine in the absence of sodium, and the nonselective antagonist ({sup 3}H)naltrexone in the presence of sodium, bound to two populations of opioid receptor sites with K{sub d} values of 0.69 and 8.7 nM for dihydromorphine, and 0.34 and 5.5 nM for naltrexone. The addition of 5 {mu}M guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)) strongly reduced high-affinity agonist but not antagonist binding. Exposure of the membranes to high pH reduced the number of GTP({gamma}-{sup 35}S) binding sites by 90% and low K{sub m}, opioid-sensitive GTPase activity by 95%. In these membranes, high-affinity agonist binding was abolished and modulation of residual binding by GTP({gamma}S) was diminished. Alkali treatment of the glioma cell membranes prior to fusion inhibited most of the low K{sub m} GTPase activity and prevented the reconstitution of agonist binding. The results show that high-affinity opioid agonist binding reflects the ligand-occupied receptor - guanine nucleotide binding protein complex.

  7. Structural Basis for High-Affinity Peptide Inhibition of Human Pin1

    PubMed Central

    Zhang, Yan; Daum, Sebastian; Wildemann, Dirk; Zhou, Xiao Zhen; Verdecia, Mark A.; Bowman, Marianne E.; Lücke, Christian; Hunter, Tony; Lu, Kun-Ping; Fischer, Gunter; Noel, Joseph P.

    2009-01-01

    Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I β-turn conformation for Pin1 prolyl peptide isomerase domain–peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors. PMID:17518432

  8. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates

    PubMed Central

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-01-01

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (koff < 1 × 10−7 s−1) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. PMID:26950154

  9. Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors.

    PubMed

    Hwang, Dah-Ren; Narendran, Raj; Laruelle, Marc

    2005-01-01

    It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3''). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3'' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.

  10. Solution assembly of the pseudo-high affinity and intermediate affinity interleukin-2 receptor complexes.

    PubMed Central

    Wu, Z.; Goldstein, B.; Laue, T. M.; Liparoto, S. F.; Nemeth, M. J.; Ciardelli, T. L.

    1999-01-01

    The high affinity interleukin-2 receptor is composed of three cell surface subunits, IL-2Ralpha, IL-2Rbeta, and IL-2Rgamma. Functional forms of the IL-2 receptor exist, however, that enlist only two of the three subunits. On activated T-cells, the alpha- and beta-subunits combine as a preformed heterodimer (the pseudo-high affinity receptor) that serves to capture IL-2. On a subpopulation of natural killer cells, the beta- and gamma-subunits interact in a ligand-dependent manner to form the intermediate affinity receptor site. Previously, we have demonstrated the feasibility of employing coiled-coil molecular recognition for the solution assembly of a heteromeric IL-2 receptor complex. In that study, although the receptor was functional, the coiled-coil complex was a trimer rather than the desired heterodimer. We have now redesigned the hydrophobic heptad sequences of the coiled-coils to generate soluble forms of both the pseudo-high affinity and the intermediate affinity heterodimeric IL-2 receptors. The properties of these complexes were examined and their relevance to the physiological IL-2 receptor mechanism is discussed. PMID:10091650

  11. Single-experiment displacement assay for quantifying high-affinity binding by isothermal titration calorimetry.

    PubMed

    Krainer, Georg; Keller, Sandro

    2015-04-01

    Isothermal titration calorimetry (ITC) is the gold standard for dissecting the thermodynamics of a biomolecular binding process within a single experiment. However, reliable determination of the dissociation constant (KD) from a single titration is typically limited to the range 100 μM>KD>1 nM. Interactions characterized by a lower KD can be assessed indirectly by so-called competition or displacement assays, provided that a suitable competitive ligand is available whose KD falls within the directly accessible window. However, this protocol is limited by the fact that it necessitates at least two titrations to characterize one high-affinity inhibitor, resulting in considerable consumption of both sample material and time. Here, we introduce a fast and efficient ITC displacement assay that allows for the simultaneous characterization of both a high-affinity ligand and a moderate-affinity ligand competing for the same binding site on a receptor within a single experiment. The protocol is based on a titration of the high-affinity ligand into a solution containing the moderate-affinity ligand bound to the receptor present in excess. The resulting biphasic binding isotherm enables accurate and precise determination of KD values and binding enthalpies (ΔH) of both ligands. We discuss the theoretical background underlying the approach, demonstrate its practical application to metal ion chelation, explore its potential and limitations with the aid of simulations and statistical analyses, and elaborate on potential applications to protein-inhibitor interactions.

  12. Isolation of Anti-Ricin Protective Antibodies Exhibiting High Affinity from Immunized Non-Human Primates.

    PubMed

    Noy-Porat, Tal; Rosenfeld, Ronit; Ariel, Naomi; Epstein, Eyal; Alcalay, Ron; Zvi, Anat; Kronman, Chanoch; Ordentlich, Arie; Mazor, Ohad

    2016-03-03

    Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with KD values below pM (k(off )< 1 × 10(-7) s(-1)) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication.

  13. High affinity binding of (/sup 3/H)neurotensin of rat uterus

    SciTech Connect

    Pettibone, D.J.; Totaro, J.A.

    1987-11-01

    (/sup 3/H)Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that (/sup 3/H)NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited (/sup 3/H)NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

  14. Induction of Sucrose Utilization Genes from Bifidobacterium lactis by Sucrose and Raffinose

    PubMed Central

    Trindade, Marla I.; Abratt, Valerie R.; Reid, Sharon J.

    2003-01-01

    The probiotic organism Bifidobacterium lactis was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of B. lactis was identified by complementation of a gene library in Escherichia coli. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The scrP gene was expressed constitutively from its own promoter in E. coli grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with scrP performed by using RNA isolated from B. lactis identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In B. lactis, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the scrP, scrR, and scrT genes in B. lactis indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous E. coli indicated that ScrR functions as a positive regulator. PMID:12513973

  15. Sucrose phloem unloading follows an apoplastic pathway with high sucrose synthase in Actinidia fruit.

    PubMed

    Chen, Cheng; Yuan, Yulin; Zhang, Chen; Li, Huixia; Ma, Fengwang; Li, Mingjun

    2017-02-01

    Phloem unloading plays a pivotal role in photoassimilate partitioning and the accumulation of sugars in sink organs, e.g. fruit. Here, we investigated the pathway of sucrose unloading in kiwifruit (Actinidia deliciasa cv. Qinmei) using a combination of electron microscopy, transport of the phloem-mobile symplastic tracer carboxyfluorescein and enzyme activity and gene expression assays. Our structural investigation revealed that the sieve element-companion cell complex of bundles feeding the fruit flesh was symplastically isolated from its surrounding parenchyma cells throughout fruit development, whereas numerous plasmodesmata were present between the phloem parenchyma cells. Consistent with this, carboxyfluorescein unloading showed that the dye remained confined in the phloem strands during fruit development. The activities and expression of cell wall acid invertase in fruit flesh were lower than those of other enzymes that catalyze sucrose dissociation. However, sucrose synthase showed higher enzyme activities and mRNA expression in fruit flesh compared with other detected enzymes. These results imply that, in kiwifruit flesh, phloem unloading of sucrose is predominantly an apoplastic pathway during fruit development, and that sucrose synthase is a key enzyme for sucrose post-unloading pathways. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  16. Monitoring of the velocity of high-affinity glutamate uptake by isolated brain nerve terminals using amperometric glutamate biosensor.

    PubMed

    Soldatkin, O; Nazarova, A; Krisanova, N; Borуsov, A; Kucherenko, D; Kucherenko, I; Pozdnyakova, N; Soldatkin, A; Borisova, T

    2015-04-01

    Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na(+)-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Sucrose Synthase in Wild Tomato, Lycopersicon chmielewskii, and Tomato Fruit Sink Strength.

    PubMed

    Sun, J; Loboda, T; Sung, S J; Black, C C

    1992-03-01

    Here it is reported that sucrose synthase can be readily measured in growing wild tomato fruits (Lycopersicon chmielewskii) when suitable methods are adopted during fruit extraction. The enzyme also was present in fruit pericarp tissues, in seeds, and in flowers. To check for novel characteristics, the wild tomato fruit sucrose synthase was purified, by (NH(4))(2)SO(4) fraction and chromatography with DE-32, Sephadex G-200, and PBA-60, to one major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The following characteristics were obtained: native protein relative molecular weight 380,000; subunit relative molecular weight 89,000; K(m) values with: sucrose 53 millimolar, UDP 18.9 micromolar, UDP-glucose 88 micromolar, fructose 8.4 millimolar; pH optima between 6.2 to 7.3 for sucrose breakdown and 7 to 9 for synthesis; and temperature optima near 50 degrees C. The enzyme exhibited a high affinity and a preference for uridylates. The enzyme showed more sensitivity to divalent cations in the synthesis of sucrose than in its breakdown. Sink strength in tomato fruits also was investigated in regard to sucrose breakdown enzyme activities versus fruit weight gain. Sucrose synthase activity was consistently related to increases in fruit weight (sink strength) in both wild and commercial tomatoes. Acid and neutral invertases were not, because the published invertase activity values were too variable for quantitative analyses regarding the roles of invertases in tomato fruit development. In rapidly growing fruits of both wild and commercially developed tomato plants, the activity of sucrose synthase per growing fruit, i.e. sucrose synthase peak activity X fruit size, was linearly related to final fruit size; and the activity exceeded fruit growth and carbon import rates by at least 10-fold. In mature, nongrowing fruits, sucrose synthase activities approached nil values. Therefore, sucrose synthase can serve as an indicator of sink strength in growing tomato

  18. Sucrose Synthase: Expanding Protein Function

    USDA-ARS?s Scientific Manuscript database

    Sucrose synthase (SUS: EC 2.4.1.13), a key enzyme in plant sucrose catabolism, is uniquely able to mobilize sucrose into multiple pathways involved in metabolic, structural, and storage functions. Our research indicates that the biological function of SUS may extend beyond its catalytic activity. Th...

  19. Assessment of Sugar Components and Genes Involved in the Regulation of Sucrose Accumulation in Peach Fruit.

    PubMed

    Vimolmangkang, Sornkanok; Zheng, Hongyu; Peng, Qian; Jiang, Quan; Wang, Huiliang; Fang, Ting; Liao, Liao; Wang, Lu; He, Huaping; Han, Yuepeng

    2016-09-07

    Soluble sugar contents in mature fruits of 45 peach accessions were quantified using gas chromatography analysis. Sucrose is the predominant sugar in mature fruit, followed by glucose and fructose, which have similar concentrations. Overall, sucrose metabolism and accumulation are crucial determinants of sugar content in peach fruit, and there is a wide range of sucrose concentrations among peach genotypes. To understand the mechanisms regulating sucrose accumulation in peach fruit, expression profiles of genes involved in sucrose metabolism and transport were compared among four genotypes. Two sucrose-cleaving enzyme genes (SUS4 and NINV8), one gene involved in sucrose resynthesis (SPS3), and three sugar transporter genes (SUT2, SUT4, and TMT2) were prevalently expressed in peach fruit, and their expression levels are significantly correlated with sucrose accumulation. In contrast, the VAINV genes responsible for sucrose cleavage in the vacuole were weakly expressed in mature fruit, suggesting that the sucrose-cleaving reaction is not active in the vacuole of sink cells of mature peach fruit. This study suggests that sucrose accumulation in peach fruit involves the coordinated interaction of genes related to sucrose cleavage, resynthesis, and transport, which could be helpful for future peach breeding.

  20. The thuEFGKAB Operon of Rhizobia and Agrobacterium tumefaciens Codes for Transport of Trehalose, Maltitol, and Isomers of Sucrose and Their Assimilation through the Formation of Their 3-Keto Derivatives

    PubMed Central

    Ampomah, Osei Yaw; Avetisyan, Anna; Hansen, Espen; Svenson, Johan; Huser, Thomas; Bhuvaneswari, T. V.

    2013-01-01

    The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism. PMID:23772075

  1. Oligosaccharides Derived from Sucrose

    NASA Astrophysics Data System (ADS)

    Monsan, Pierre F.; Ouarné, Francois

    Sucrose is a non-reducing disaccharide, consisting of an α-D-glucopyranosyl residue and a β-D-fructofuranosyl residue linked covalently by their respective anomeric carbons (α-D-glucopyranosyl-1,2-β-D-fructofuranoside). It is not just a simple disaccharide, among others: in fact, the energy of its glycosidic bond is higher than that of a usual glycosidic bond. It is equal to 27.6 kJ/mol, which is similar to the energy of a nucleotide-sugar bond as in UDP-glucose or ADP-glucose. This means that sucrose is a protected and activated form of D-glucose (as well as of D-fructose), which plays a key role in the metabolism of plants, for a wide variety of synthesis reactions.

  2. Sugar (sucrose) holograms

    NASA Astrophysics Data System (ADS)

    Ponce-Lee, E. L.; Olivares-Pérez, A.; Fuentes-Tapia, I.

    2004-06-01

    Computer holograms made with sugar crystals are reported. This material is well known as a good sweetener; the sugar from sugar cane or sugar beet (sucrose). These sweetener can be applied as honey "water and diluted sugar" easily on any substrate such as plastics or glasses without critical conditions for developed process. This step corresponds only to the cured sucrose as a photopolymer process. The maximum absorption spectra is localized at UV region λ=240 nm. We record with lithographic techniques some gratings, showing a good diffraction efficiency around 45%. This material has good resolution to make diffraction gratings. These properties are attractive because they open the possibility to make phase holograms on candies. Mainly the phase modulation is by refraction index.

  3. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display.

    PubMed

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C; Van Bergen En Henegouwen, Paul M P; Fernández, Luis Ángel

    2016-10-01

    Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.

  4. Binding and structural diversity among high-affinity monoclonal anti-digoxin antibodies.

    PubMed

    Mudgett-Hunter, M; Anderson, W; Haber, E; Margolies, M N

    1985-04-01

    High-affinity monoclonal antibodies specific for the cardiac glycoside digoxin provide a useful system for the study of structure-function relationships between antibody combining site and specific antigenic determinants. Fifteen high-affinity monoclonal anti-digoxin antibodies were produced when spleen cells from A/J mice immunized with digoxin coupled to human serum albumin (Dig-HSA) were fused with the non-secreting murine myeloma Sp2/0 cell line. Each subcloned hybridoma antibody was analyzed for affinity and specificity for structurally related cardiac glycosides by a radioimmunoassay based on the adsorption of free [3H]digoxin to dextran-coated charcoal. All of the anti-digoxin hybridoma proteins demonstrated high affinity constants ranging from 10(9) to 10(12) M-1. Using seven different analogs of digoxin, binding specificities of the monoclonal antibodies were assessed by inhibition radioimmunoassay. The 15 hybridomas produced from fusions involving five mice could be divided into eight sets on the basis of these binding specificities. Certain antibodies exhibit a preference for the aglycone portion of digoxin, while others are more specific for the tridigitoxose sugar moiety of digoxin. Monoclonal antibody H- and L-chains were subjected to N-terminal amino acid sequence analysis. The antibodies may be divided into several sequence homology sets for both H- and L-chains. In most instances, homologous heavy chains are associated with a set of homologous light chains. Homologous partial sequences, however, do not correlate with similar antigenic specificities and affinities for digoxin. Thus the fine specificity for antigen is not dependent on VH- and VL-encoded sequences alone. These data illustrate the broad diversity of the elicited response to a single hapten, even in inbred mice.

  5. Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

    PubMed Central

    Kragh-Hansen, U

    1985-01-01

    Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made. PMID:3977850

  6. Rational development of high-affinity T-cell receptor-like antibodies

    PubMed Central

    Stewart-Jones, Guillaume; Wadle, Andreas; Hombach, Anja; Shenderov, Eugene; Held, Gerhard; Fischer, Eliane; Kleber, Sascha; Nuber, Natko; Stenner-Liewen, Frank; Bauer, Stefan; McMichael, Andrew; Knuth, Alexander; Abken, Hinrich; Hombach, Andreas A.; Cerundolo, Vincenzo; Jones, E. Yvonne; Renner, Christoph

    2009-01-01

    T-cell interaction with a target cell is a key event in the adaptive immune response and primarily driven by T-cell receptor (TCR) recognition of peptide-MHC (pMHC) complexes. TCR avidity for a given pMHC is determined by number of MHC molecules, availability of coreceptors, and TCR affinity for MHC or peptide, respectively, with peptide recognition being the most important factor to confer target specificity. Here we present high-resolution crystal structures of 2 Fab antibodies in complex with the immunodominant NY-ESO-1157–165 peptide analogue (SLLMWITQV) presented by HLA-A*0201 and compare them with a TCR recognizing the same pMHC. Binding to the central methionine-tryptophan peptide motif and orientation of binding were almost identical for Fabs and TCR. As the MW “peg” dominates the contacts between Fab and peptide, we estimated the contributions of individual amino acids between the Fab and peptide to provide the rational basis for a peptide-focused second-generation, high-affinity antibody library. The final Fab candidate achieved better peptide binding by 2 light-chain mutations, giving a 20-fold affinity improvement to 2–4 nM, exceeding the affinity of the TCR by 1,000-fold. The high-affinity Fab when grafted as recombinant TCR on T cells conferred specific killing of HLA-A*0201/NY-ESO-1157–165 target cells. In summary, we prove that affinity maturation of antibodies mimicking a TCR is possible and provide a strategy for engineering high-affinity antibodies that can be used in targeting specific pMHC complexes for diagnostic and therapeutic purposes. PMID:19307587

  7. High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

    PubMed Central

    Salema, Valencio; Mañas, Carmen; Cerdán, Lidia; Piñero-Lambea, Carlos; Marín, Elvira; Roovers, Rob C.; Van Bergen en Henegouwen, Paul M.P.; Fernández, Luis Ángel

    2016-01-01

    ABSTRACT Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens. PMID:27472381

  8. High-affinity antibodies to the 1,4-dihydropyridine Ca2+-channel blockers

    SciTech Connect

    Campbell, K.P.; Sharp, A.; Strom, M.; Kahl, S.D.

    1986-05-01

    Antibodies with high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers have been produced in rabbits by immunization with dihydropyridine-protein conjugates. Anti-dihydropyridine antibodies were found to specifically bind (/sup 3/H)nitrendipine, (/sup 3/H)-nimodipine, (/sup 3/H)nisoldipine, and (/sup 3/H)PN 200-110 (all 1,4-dihydropyridine Ca2+-channel blockers) with high affinity, while (/sup 3/H)verapamil, (/sup 3/H)diltiazem, and (/sup 3/H)trifluoperazine were not recognized. The average dissociation constant of the (/sup 3/H)nitrendipine-antibody complex was 0.06 (+/- 0.02) X 10(-9) M for an antiserum studied in detail and ranged from 0.01 to 0.24 X 10(-9) M for all antisera. Inhibition of (/sup 3/H)nitrendipine binding was specific for the 1,4-dihydropyridine Ca2+-channel modifiers and the concentrations required for half-maximal inhibition ranged between 0.25 and 0.90 nM. Structurally unrelated Ca2+-channel blockers, calmodulin antagonists, inactive metabolites of nitrendipine, and UV-inactivated nisoldipine did not modify (/sup 3/H)nitrendipine binding to the anti-dihydropyridine antibodies. Dihydropyridines without a bulky substituent in the 4-position of the heterocycle were able to displace (/sup 3/H)nitrendipine binding, but the concentrations required for half-maximal inhibition were greater than 800 nM. In summary, anti-dihydropyridine antibodies have been shown to have high affinity and specificity for the 1,4-dihydropyridine Ca2+-channel blockers and to exhibit dihydropyridine binding properties similar to the membrane receptor for the 1,4-dihydropyridine Ca2+-channel blockers.

  9. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  10. Aminopyrazoles with high affinity for the human neuropeptide Y5 receptor.

    PubMed

    Kordik, C P; Luo, C; Zanoni, B C; Dax, S L; McNally, J J; Lovenberg, T W; Wilson, S J; Reitz, A B

    2001-09-03

    1,3-Disubstituted-5-aminopyrazoles were prepared based on a lead compound found through high-throughput screening of our corporate compound library in an assay measuring affinity for the human neuropeptide Y5 receptor. The target compounds were prepared by cyclization of alpha-cyanoketones with appropriate hydrazines, followed by reduction and coupling to various sulfonamido-carboxylic acids. Several of these arylpyrazoles (e.g., 19 and 45) displayed high affinity for the human NPY Y5 receptor (<20nM IC(50)s).

  11. Valproate is neuroprotective against malonate toxicity in rat striatum: an association with augmentation of high-affinity glutamate uptake.

    PubMed

    Morland, Cecilie; Boldingh, Karen Astrid; Iversen, Evy Grini; Hassel, Bjørnar

    2004-11-01

    The antiepileptic drug valproate (VPA) may be neuroprotective. We treated rats with VPA for 14 days (300 mg/kg twice daily) before intrastriatal injection of 1.5 micromol (1 M) of the succinate dehydrogenase inhibitor malonate. VPA-treated animals developed smaller lesions than control animals: 10 +/- 2 mm(3) versus 26 +/- 8 mm(3) (means +/- SD; P = 10(-4). Injection of NaCl that was equiosmolar with 1 M malonate caused lesions of only 1.2 +/- 0.4 mm(3) in control animals, whereas physiologic saline produced no lesion. VPA pretreatment reduced the malonate-induced extracellular accumulation of glutamate. This effect paralleled an increase in the striatal level of the glutamate transporter GLT, which augmented high-affinity glutamate uptake by 25%, as determined from the uptake of [(3)H] glutamate into striatal proteoliposomes. Malonate caused a 76% reduction in striatal adenosine triphosphate (ATP) content, but the glial, ATP-dependent formation of glutamine from radiolabeled glucose or glutamate was intact, indicating that glial ATP production supported uptake of glutamate. Striatal levels of HSP-70 and fos were reduced, and the levels of bcl-2 and phosphorylated extracellular signal-regulated kinase remained unaffected, but histone acetylation was increased by VPA treatment. The results suggest that augmentation of glutamate uptake may contribute importantly to VPA-mediated neuroprotection in striatum.

  12. Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

    PubMed

    Willmann, Anita; Weiss, Michael; Nehls, Uwe

    2007-02-01

    A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

  13. Phloem-localized, proton-coupled sucrose carrier ZmSUT1 mediates sucrose efflux under the control of the sucrose gradient and the proton motive force.

    PubMed

    Carpaneto, Armando; Geiger, Dietmar; Bamberg, Ernst; Sauer, Norbert; Fromm, Jörg; Hedrich, Rainer

    2005-06-03

    The phloem network is as essential for plants as the vascular system is for humans. This network, assembled by nucleus- and vacuole-free interconnected living cells, represents a long distance transport pathway for nutrients and information. According to the Münch hypothesis, osmolytes such as sucrose generate the hydrostatic pressure that drives nutrient and water flow between the source and the sink phloem (Münch, E. (1930) Die Stoffbewegungen in der Pflanze, Gustav Fischer, Jena, Germany). Although proton-coupled sucrose carriers have been localized to the sieve tube and the companion cell plasma membrane of both source and sink tissues, knowledge of the molecular representatives and the mechanism of the sucrose phloem efflux is still scant. We expressed ZmSUT1, a maize sucrose/proton symporter, in Xenopus oocytes and studied the transport characteristics of the carrier by electrophysiological methods. Using the patch clamp techniques in the giant inside-out patch mode, we altered the chemical and electrochemical gradient across the sucrose carrier and analyzed the currents generated by the proton flux. Thereby we could show that ZmSUT1 is capable of mediating both the sucrose uptake into the phloem in mature leaves (source) as well as the desorption of sugar from the phloem vessels into heterotrophic tissues (sink). As predicted from a perfect molecular machine, the ZmSUT1-mediated sucrose-coupled proton current was reversible and depended on the direction of the sucrose and pH gradient as well as the membrane potential across the transporter.

  14. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  15. Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

    NASA Astrophysics Data System (ADS)

    Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

    2017-07-01

    The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

  16. High affinity binding site-mediated prevention of chemical absorption across the gastrointestinal tract.

    PubMed

    Rasmussen, M V; Barker, T T; Silbart, L K

    2001-12-15

    Preventing mucosal absorption of low-molecular weight compounds such as carcinogens, toxins and drugs could help prevent many diseases. To characterize the effects of dose and timing on high-affinity binding site mediated sequestration of specific chemical ligands in the gastrointestinal tract, avidin was perorally-administered to mice either prior to or mixed with 3H-biotin. Avidin enhanced fecal 3H-biotin excretion in a dose-dependent manner, consistent with the accepted mechanism of egg white-induced biotin deficiency syndrome. Avidin administration up to 4 h before 3H-biotin administration also enhanced fecal 3H-biotin excretion. Activated charcoal (AC) reduced 3H-biotin absorption when mixed with 3H-biotin before ingestion, but was ineffective when ingested prior to 3H-biotin. These studies suggest that ingestion of high-affinity protein binding sites can establish an absorptive barrier at the gastrointestinal mucosa to prevent the uptake of unwanted low molecular-weight chemicals.

  17. Rac recruits high-affinity integrin alphavbeta3 to lamellipodia in endothelial cell migration.

    PubMed

    Kiosses, W B; Shattil, S J; Pampori, N; Schwartz, M A

    2001-03-01

    Integrin alphavbeta3 has an important role in the proliferation, survival, invasion and migration of vascular endothelial cells. Like other integrins, alphavbeta3 can exist in different functional states with respect to ligand binding. These changes involve both affinity modulation, by which conformational changes in the integrin heterodimer govern affinity for individual extracellular matrix proteins, and avidity modulation, by which changes in lateral mobility and integrin clustering affect the binding of cells to multivalent matrices. Here we have used an engineered monoclonal antibody Fab (antigen-binding fragment) named WOW-1, which binds to activated integrins alphavbeta3 and alphavbeta5 from several species, to investigate the role of alphavbeta3 activation in endothelial cell behaviour. Because WOW-1 is monovalent, it is insensitive to changes in integrin clustering and therefore reports only changes in affinity. WOW-1 contains an RGD tract in its variable region and binds only to unoccupied, high-affinity integrins. By using WOW-1, we have identified the selective recruitment of high-affinity integrins as a mechanism by which lamellipodia promote formation of new adhesions at the leading edge in cell migration.

  18. A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

    PubMed

    Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

    2012-12-01

    Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

  19. The AVR4 elicitor protein of Cladosporium fulvum binds to fungal components with high affinity.

    PubMed

    Westerink, Nienke; Roth, Ronelle; Van den Burg, Harrold A; De Wit, Pierre J G M; Joosten, Matthieu H A J

    2002-12-01

    The interaction between tomato and the fungal pathogen Cladosporium fulvum complies with the gene-for-gene system. Strains of C. fulvum that produce race-specific elicitor AVR4 induce a hypersensitive response, leading to resistance, in tomato plants that carry the Cf-4 resistance gene. The mechanism of AVR4 perception was examined by performing binding studies with 125I-AVR4 on microsomal membranes of tomato plants. We identified an AVR4 high-affinity binding site (KD = 0.05 nM) which exhibited all the characteristics expected for ligand-receptor interactions, such as saturability, reversibility, and specificity. Surprisingly, the AVR4 high-affinity binding site appeared to originate from fungi present on infected tomato plants rather than from the tomato plants themselves. Detailed analysis showed that this fungus-derived, AVR4-specific binding site is heat- and proteinase K-resistant. Affinity crosslinking demonstrated that AVR4 specifically binds to a component of approximately 75 kDa that is of fungal origin. Our data suggest that binding of AVR4 to a fungal component or components is related to the intrinsic virulence function of AVR4 for C. fulvum.

  20. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding.

    PubMed

    Rosilo, Henna; McKee, Jason R; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P; Ikkala, Olli; Kostiainen, Mauri A

    2014-10-21

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.

  1. Purification of high affinity benzodiazepine receptor binding site fragments from rat brain

    SciTech Connect

    Klotz, K.L.

    1984-01-01

    In central nervous system benzodiazepine recognition sites occur on neuronal cell surfaces as one member of a multireceptor complex, including recognition sites for benzodiazepines, gamma aminobutyric acid (GABA), barbiturates and a chloride ionophore. During photoaffinity labelling, the benzodiazepine agonist, /sup 3/H-flunitrazepam, is irreversibly bound to central benzodiazepine high affinity recognition sites in the presence of ultraviolet light. In these studies a /sup 3/H-flunitrazepam radiolabel was used to track the isolation and purification of high affinity agonist binding site fragments from membrane-bound benzodiazepine receptor in rat brain. The authors present a method for limited proteolysis of /sup 3/H-flunitrazepam photoaffinity labeled rat brain membranes, generating photolabeled benzodiazepine receptor fragments containing the agonist binding site. Using trypsin chymotrypsin A/sub 4/, or a combination of these two proteases, they have demonstrated the extent and time course for partial digestion of benzodiazepine receptor, yielding photolabeled receptor binding site fragments. These photolabeled receptor fragments have been further purified on the basis of size, using ultrafiltration, gel permeation chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as on the basis of hydrophobicity, using a high performance liquid chromatography (HPLC) precolumn, several HPLC elution schemes, and two different HPLC column types. Using these procedures, they have purified three photolabeled benzodiazepine receptor fragments containing the agonist binding site which appear to have a molecular weight of less than 2000 daltons each.

  2. Ontogeny of high-affinity GABA and benzodiazepine receptors in the rat cerebellum: an autoradiographic study.

    PubMed

    Palacios, J M; Kuhar, M J

    1981-11-01

    High-affinity GABA and benzodiazepine receptors were localized by light microscopic autoradiography in the developing rat cerebellum. [3H]muscimol was used for the labeling of GABA receptors and [3H]flunitrazepam for benzodiazepine receptors. Very low densities of GABA sites were found during the first postnatal week. GABA receptors start increasing linearly at the end of the second week up to adult levels around the fourth postnatal week. The increase in receptor density is concentrated in the developing granule cell layer. Benzodiazepine receptors are present at birth and increases in the density of receptors were observed already during the first postnatal week. Receptor concentrations reached adult values around the third to fourth weeks postnatally. The increase in benzodiazepine receptors in concentrated in the growing molecular layer with little change in the granule cell layer. The immature cell of the external granule layer were characterized by the absence of receptor sites. At least partial association of high-affinity GABA receptors with granule cells and benzodiazepine receptor with Purkinje cell dendrites is suggested by these developmental profiles.

  3. Competition STD NMR for the detection of high-affinity ligands and NMR-based screening.

    PubMed

    Wang, Yu-Sen; Liu, Dingjiang; Wyss, Daniel F

    2004-06-01

    The reported competition STD NMR method combines saturation transfer difference (STD) NMR with competition binding experiments to allow the detection of high-affinity ligands that undergo slow chemical exchange on the NMR time-scale. With this technique, the presence of a competing high-affinity ligand in the compound mixture can be detected by the disappearance or reduction of the STD signals of a low-affinity indicator ligand. This is demonstrated on a BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) protein-inhibitor system. This method can also be used to derive an approximate value, or a lower limit, for the dissociation constant of the potential ligand based on the reduction of the signal intensity of the STD indicator, which is illustrated on an HSA (human serum albumin) model system. This leads to important applications of the competition STD NMR method for lead discovery: it can be used (i) for compound library screening against a broad range of drug targets to identify both high- and low-affinity ligands and (ii) to rank order analogs rapidly and derive structure-activity relationships, which are used to optimize these NMR hits into viable drug leads. Copyright 2004 John Wiley & Sons, Ltd.

  4. Selecting highly affine and well-expressed TCRs for gene therapy of melanoma.

    PubMed

    Jorritsma, Annelies; Gomez-Eerland, Raquel; Dokter, Maarten; van de Kasteele, Willeke; Zoet, Yvonne M; Doxiadis, Ilias I N; Rufer, Nathalie; Romero, Pedro; Morgan, Richard A; Schumacher, Ton N M; Haanen, John B A G

    2007-11-15

    A recent phase 1 trial has demonstrated that the generation of tumor-reactive T lymphocytes by transfer of specific T-cell receptor (TCR) genes into autologous lymphocytes is feasible. However, compared with results obtained by infusion of tumor-infiltrating lymphocytes, the response rate observed in this first TCR gene therapy trial is low. One strategy that is likely to enhance the success rate of TCR gene therapy is the use of tumor-reactive TCRs with a higher capacity for tumor cell recognition. We therefore sought to develop standardized procedures for the selection of well-expressed, high-affinity, and safe human TCRs. Here we show that TCR surface expression can be improved by modification of TCR alpha and beta sequences and that such improvement has a marked effect on the in vivo function of TCR gene-modified T cells. From a panel of human, melanoma-reactive TCRs we subsequently selected the TCR with the highest affinity. Furthermore, a generally applicable assay was used to assess the lack of alloreactivity of this TCR against a large series of common human leukocyte antigen alleles. The procedures described in this study should be of general value for the selection of well- and stably expressed, high-affinity, and safe human TCRs for subsequent clinical testing.

  5. High-affinity interactions of ligands at recombinant Guinea pig 5HT7 receptors

    NASA Astrophysics Data System (ADS)

    Wilcox, R. E.; Ragan, J. E.; Pearlman, R. S.; Brusniak, M. Y.-. K.; Eglen, R. M.; Bonhaus, D. W.; Tenner, T. E., Jr.; Miller, J. D.

    2001-10-01

    The serotonin 5HT7 receptor has been implicated in numerous physiological and pathological processes from circadian rhythms [1] to depression and schizophrenia. Clonal cell lines heterologously expressing recombinant receptors offer good models for understanding drug-receptor interactions and development of quantitative structure-activity relationships (QSAR). Comparative Molecular Field Analysis (CoMFA) is an important modern QSAR procedure that relates the steric and electrostatic fields of a set of aligned compounds to affinity. Here, we utilized CoMFA to predict affinity for a number of high-affinity ligands at the recombinant guinea pig 5HT7 receptor. Using R-lisuride as the template, a final CoMFA model was derived using procedures similar to those of our recent papers [2, 3, 4] The final cross-validated model accounted for >85% of the variance in the compound affinity data, while the final non-cross validated model accounted for >99% of the variance. Model evaluation was done using cross-validation methods with groups of 5 ligands. Twenty cross-validation runs yielded an average predictive r2(q2) of 0.779 ± 0.015 (range: 0.669-0.867). Furthermore, 3D-chemical database search queries derived from the model yielded hit lists of promising agents with high structural similarity to the template. Together, these results suggest a possible basis for high-affinity drug action at 5HT7 receptors.

  6. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  7. Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma

    PubMed Central

    Zhang, Yi-Fan; Ho, Mitchell

    2016-01-01

    Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer. PMID:27667400

  8. Evidence for the presence of a sucrose carrier in immature sugar beet tap roots. [Beta vulgaris L

    SciTech Connect

    Lemoine, R.; Daie, J.; Wyse, R. )

    1988-02-01

    The objectives of this work were to determine the path of phloem unloading and if a sucrose carrier was present in young sugar beet (Beta vulgaris L.) taproots. The approach was to exploit the characteristics of the sucrose analog, 1{prime}-fluorosucrose (F-sucrose) which is a poor substrate for acid invertase but is a substrate for sucrose synthase. Ten millimolar each of ({sup 3}H) sucrose and ({sup 14}C)F-sucrose were applied in a 1:1 ratio to an abraded region of an attached leaf for 6 hours. ({sup 14}C)F-sucrose was translocated and accumulated in the roots at a higher rate than ({sup 3}H)sucrose. This was due to ({sup 3}H)sucrose hydrolysis along the translocation path. Presence of ({sup 3}H)hexose and ({sup 14}C)F-sucrose in the root apoplast suggested apoplastic sucrose unloading with its subsequent hydrolysis. Labeled F-sucrose uptake by root tissue discs exhibited biphasic kinetics and was inhibited by unlabeled sucrose, indicating that immature roots have the ability for carrier-mediated sucrose transport from the apoplast. Collectively, in vivo and in vitro data indicate that despite sucrose hydrolysis by the wall-bound invertase, sucrose hydrolysis is not entirely essential for sugar accumulation in this tissue.

  9. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

  10. Extraction of sucrose from molasses

    SciTech Connect

    Landis, A.M.

    1982-01-26

    Sucrose is extracted from molasses by passing an aqueous molasses solution over an adsorbent, e.g., calcined Al/sub 2/O/sub 3/-supported pyrolyzed C/sub 6/H/sub 6/. Thus, 10 mL molasses (approximately 46% solids) was run through a column containing 70 cubic centimetres above adsorbent with sucrose retention volume 21.4 and selectivity for sucrose - betaine 23.8.

  11. Putative M2 muscarinic receptors of rat heart have high affinity for organophosphorus anticholinesterases

    SciTech Connect

    Silveira, C.L.; Eldefrawi, A.T.; Eldefrawi, M.E. )

    1990-05-01

    The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-(3H)cis-methyldioxolane ((3H)CD), which has been used to label a high affinity population of M2 receptors. A single population of sites was detected and (3H)CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with (3H)CD and considered to be of the M2 subtype. Up to 70% of the (3H)CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. (3H)CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of (3H)CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM (3H)CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting (3H)CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration.

  12. Location of high-affinity metal binding sites in the profile structure of the Ca+2-ATPase in the sarcoplasmic reticulum by resonance x-ray diffraction.

    PubMed Central

    Asturias, F J; Blasie, J K

    1991-01-01

    Resonance x-ray diffraction measurements on the lamellar diffraction from oriented multilayers of isolated sarcoplasmic reticulum (SR) membranes containing a small concentration of lanthanide (III) ions (lanthanide/protein molar ratio approximately 4) have allowed us to calculate both the electron density profile of the SR membrane and the separate electron density profile of the resonant lanthanide atoms bound to the membrane to a relatively low spatial resolution of approximately 40 A. Analysis of the membrane electron density profile and modeling of the separate low resolution lanthanide atom profile, using step-function electron density models based on the assumption that metal binding sites in the membrane profile are discrete and localized, resulted in the identification of a minimum of three such binding sites in the membrane profile. Two of these sites are low-affinity, low-occupancy sites identified with the two phospholipid polar headgroup regions of the lipid bilayer within the membrane profile. Up to 20% of the total lanthanide (III) ions bind to these low-affinity sites. The third site has relatively high affinity for lanthanide ion binding; its Ka is roughly an order of magnitude larger than that for the lower affinity polar headgroup sites. Approximately 80% of the total lanthanide ions present in the sample are bound to this high-affinity site, which is located in the "stalk" portion of the "headpiece" within the profile structure of the Ca+2 ATPase protein, approximately 12 A outside of the phospholipid polar headgroups on the extravesicular side of the membrane profile. Based on the nature of our results and on previous reports in the literature concerning the ability of lanthanide (III) ions to function as Ca+2 analogues for the Ca+2 ATPase we suggest that we have located a high-affinity metal binding site in the membrane profile which is involved in the active transport of Ca+2 ions across the SR membrane by the Ca+2 ATPase. PMID:1826221

  13. Pinealectomy increases ouabain high-affinity binding sites and dissociation constant in rat cerebral cortex.

    PubMed

    Acuña Castroviejo, D; del Aguila, C M; Fernández, B; Gomar, M D; Castillo, J L

    1991-06-24

    The effect of the pineal gland on the ouabain high-affinity binding sites (Kd = 3.1 +/- 0.4 nM, Bmax = 246.4 +/- 18.4 fmol/mg protein) in rat cerebral cortex was studied. Pinealectomy increased Bmax (940.7 +/- 42.8 fmol/mg protein) and Kd (7.6 +/- 1.5 nM) while melatonin injection (100 micrograms/kg b.wt.) counteracted these effects, restoring kinetic parameters (Kd = 1.9 +/- 0.05 nM; Bmax = 262.2 +/- 29.6 fmol/mg prot) to control values. Melatonin activity on ouabain binding in vitro did not depend upon a direct effect on the binding sites themselves. However, in competition experiments, melatonin increased binding affinity of ouabain as shown by the decreased IC50 values.

  14. Quantitation of the P2Y1 Receptor with a High Affinity Radiolabeled Antagonist

    PubMed Central

    Waldo, Gary L.; Corbitt, James; Boyer, José L.; Ravi, Gnana; Kim, Hak Sung; Ji, Xiao-Duo; Lacy, James; Jacobson, Kenneth A.; Harden, T. Kendall

    2015-01-01

    2-Chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y1 receptor (P2Y1-R) antagonist (J Med Chem 43:829–842, 2002; Br J Pharmacol 135:2004–2010, 2002). We have taken advantage of the N6-methyl substitution in the adenine base to incorporate [3H]methylamine into the synthesis of [3H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [3H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y2 or P2Y12 receptors. Equilibrium binding of [3H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (Kd = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R–promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived Kd calculated from independent determinations of the rate constants of association (7.15 × 107 M−1 min−1) and dissociation (0.72 min−1) of [3H]MRS2279 also was in good agreement with the Kd derived from equilibrium binding studies. Competition binding assays with [3H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed Ki values for the P2Y1-R antagonists MRS2279 (Ki = 13 nM), N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179; Ki = 84 nM), adenosine-3′, 5′-bisphosphate (Ki = 900 nM), and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (Ki = 6 µM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [3H]MRS2279 also bound with high affinity (Kd = 4–8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [3H]MRS2279 bound

  15. Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions

    PubMed Central

    Altschuler, Sarah E.; Lewis, Karen A.; Wuttke, Deborah S.

    2014-01-01

    The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization. PMID:25197549

  16. Glycan-based high-affinity ligands for toxins and pathogen receptors.

    PubMed

    Kulkarni, Ashish A; Weiss, Alison A; Iyer, Suri S

    2010-03-01

    Glycans decorate over 95% of the mammalian cell surface in the form of glycolipids and glycoproteins. Several toxins and pathogens bind to these glycans to enter the cells. Understanding the fundamentals of the complex interplay between microbial pathogens and their glycan receptors at the molecular level could lead to the development of novel therapeutics and diagnostics. Using Shiga toxin and influenza virus as examples, we describe the complex biological interface between host glycans and these infectious agents, and recent strategies to develop glycan-based high-affinity ligands. These molecules are expected to ultimately be incorporated into diagnostics and therapeutics, and can be used as probes to study important biological processes. Additionally, by focusing on the specific glycans that microbial pathogens target, we can begin to decipher the "glycocode" and how these glycans participate in normal and aberrant cellular communication.

  17. Preorganized Peptide Scaffolds as Mimics of Phosphorylated Proteins Binding Sites with a High Affinity for Uranyl.

    PubMed

    Starck, Matthieu; Sisommay, Nathalie; Laporte, Fanny A; Oros, Stéphane; Lebrun, Colette; Delangle, Pascale

    2015-12-07

    Cyclic peptides with two phosphoserines and two glutamic acids were developed to mimic high-affinity binding sites for uranyl found in proteins such as osteopontin, which is believed to be a privileged target of this ion in vivo. These peptides adopt a β-sheet structure that allows the coordination of the latter amino acid side chains in the equatorial plane of the dioxo uranyl cation. Complementary spectroscopic and analytical methods revealed that these cyclic peptides are efficient uranyl chelating peptides with a large contribution from the phosphorylated residues. The conditional affinity constants were measured by following fluorescence tryptophan quenching and are larger than 10(10) at physiological pH. These compounds are therefore promising models for understanding uranyl chelation by proteins, which is relevant to this actinide ion toxicity.

  18. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  19. Effects of anticonvulsants in vivo on high affinity choline uptake in vitro in mouse hippocampal synaptosomes.

    PubMed Central

    Miller, J. A.; Richter, J. A.

    1985-01-01

    The effects of several anticonvulsant drugs on sodium-dependent high affinity choline uptake (HACU) in mouse hippocampal synaptosomes was investigated. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by drugs which have in common the ability to facilitate gamma-aminobutyric acid (GABA) transmission, pentobarbitone, phenobarbitone, barbitone, diazepam, chloridiazepoxide, and valproic acid. Dose-response relationships were determined for these drugs and the drugs' potencies at inhibiting HACU correlated well with their anticonvulsant potencies. Clonazepam, ethosuximide, carbamazepine, and barbituric acid had no effect on HACU in the doses used while phenytoin and trimethadione stimulated HACU. These results suggest that certain anticonvulsants may elicit a part of their anticonvulsant activity by modulating cholinergic neurones. This effect may be mediated through a GABA mechanism. PMID:3978310

  20. Identification of Nucleic Acid High Affinity Binding Sequences of Proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2015-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  1. Identification of nucleic acid high-affinity binding sequences of proteins by SELEX.

    PubMed

    Bouvet, Philippe

    2009-01-01

    A technique is described for the identification of nucleic acid sequences bound with high affinity by proteins or by other molecules suitable for a partitioning assay. Here, a histidine-tagged protein is allowed to interact with a pool of nucleic acids and the protein-nucleic acid complexes formed are retained on a Ni-NTA matrix. Nucleic acids with a low level of recognition by the protein are washed away. The pool of recovered nucleic acids is amplified by the polymerase chain reaction and is submitted to further rounds of selection. Each round of selection increases the proportion of sequences that are avidly bound by the protein of interest. The cloning and sequencing of these sequences finally completes their identification.

  2. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  3. A linker peptide with high affinity towards silica-containing materials.

    PubMed

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  4. Mu/sub 1/: A very high affinity subtype of enkephalin binding sites in rat brain

    SciTech Connect

    Lutz, R.A.; Cruciani, R.A.; Munson, P.J.; Rodbard, D.

    1985-06-10

    Displacement studies of (/sup 3/H)-(D-Ala/sup 2/-MePhe/sup 4/-Gly-ol/sup 5/)-enkephalin ((/sup 3/H)-DAGO) and (/sup 3/H)-(D-Ala/sup 2/-D-Leu/sup 5/)-enkephalin ((/sup 3/H)-DADL) by the corresponding unlabeled ligands show that there are at least three classes of sites which bind these enkephalin analogs with high affinity. Using computer modeling, the introduction of the third site significantly improved the goodness of fit in ten consecutive experiments. These sites appear to correspond to the ..mu.., delta, and ..mu../sub 1/ sites, with mean dissociation constants of 11, 1.3 and 0.9 nM for DADL and 2.5, 300 and 0.3 nM for DAGO, respectively. 15 reference, 3 figures, 1 table.

  5. A complex water network contributes to high-affinity binding in an antibody-antigen interface.

    PubMed

    Marino, S F; Olal, D; Daumke, O

    2016-03-01

    This data article presents an analysis of structural water molecules in the high affinity interaction between a potent tumor growth inhibiting antibody (fragment), J22.9-xi, and the tumor marker antigen CD269 (B cell maturation antigen, BCMA). The 1.89 Å X-ray crystal structure shows exquisite details of the binding interface between the two molecules, which comprises relatively few, mostly hydrophobic, direct contacts but many indirect interactions over solvent waters. These are partly or wholly buried in, and therefore part of, the interface. A partial description of the structure is included in an article on the tumor inhibiting effects of the antibody: "Potent anti-tumor response by targeting B cell maturation antigen (BCMA) in a mouse model of multiple myeloma", Mol. Oncol. 9 (7) (2015) pp. 1348-58.

  6. Cationic polymer brush-modified cellulose nanocrystals for high-affinity virus binding

    NASA Astrophysics Data System (ADS)

    Rosilo, Henna; McKee, Jason R.; Kontturi, Eero; Koho, Tiia; Hytönen, Vesa P.; Ikkala, Olli; Kostiainen, Mauri A.

    2014-09-01

    Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface-initiated atom-transfer radical polymerization of poly(N,N-dimethylaminoethyl methacrylate) and subsequent quaternization of the polymer pendant amino groups. The cationic polymer brush-modified CNCs maintained excellent dispersibility and colloidal stability in water and showed a ζ-potential of +38 mV. Dynamic light scattering and electron microscopy showed that the modified CNCs electrostatically bind cowpea chlorotic mottle virus and norovirus-like particles with high affinity. Addition of only a few weight percent of the modified CNCs in water dispersions sufficed to fully bind the virus capsids to form micrometer-sized assemblies. This enabled the concentration and extraction of the virus particles from solution by low-speed centrifugation. These results show the feasibility of the modified CNCs in virus binding and concentrating, and pave the way for their use as transduction enhancers for viral delivery applications.Surfaces capable of high-affinity binding of biomolecules are required in several biotechnological applications, such as purification, transfection, and sensing. Therein, the rod-shaped, colloidal cellulose nanocrystals (CNCs) are appealing due to their large surface area available for functionalization. In order to exploit electrostatic binding, their intrinsically anionic surfaces have to be cationized as biological supramolecules are predominantly anionic. Here we present a facile way to prepare cationic CNCs by surface

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

    PubMed

    Quick, Matthias; Shi, Lei; Zehnpfennig, Britta; Weinstein, Harel; Javitch, Jonathan A

    2012-01-15

    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 homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na(+) ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a 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 points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.

  8. LNP 906, the first high-affinity photoaffinity ligand selective for I1 imidazoline receptors

    PubMed Central

    Dragan, Urosevic; Stephan, Schann; Jean-Daniel, Ehrhardt; Pascal, Bousquet; Hugues, Greney

    2004-01-01

    The hypotensive effect of imidazoline-like drugs, such as clonidine, was attributed both to α2-adrenergic receptors and nonadrenergic imidazoline receptors, which are divided into I1, I2 and I3 subtypes. We have recently synthesized a derivative of (2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), the first high-affinity and selective ligand for I1 receptors (I1R), with a photoactivable function (LNP 906). This work aims to test whether this derivative retained the binding properties of LNP 911 and bound irreversibly to I1R. Binding studies showed that LNP 906 exhibited nanomolar affinity for I1R and was selective for I1R over I2 receptors and α2-adrenergic receptors (α2Ars). Upon exposure to u.v. light, LNP 906 irreversibly blocked the binding of [125I]-paraiodoclonidine (PIC) to I1R, time- and dose-dependently, on PC12 cell membranes and interacted with I1R in a reversible and competitive manner in the absence of light. Pharmacological studies showed that this blockade was prevented by the concomitant presence of rilmenidine (a well-known I1 agonist), but not by rauwolscine (an α2 antagonist). Finally, LNP 906 clearly antagonized the decrease in forskolin-stimulated cAMP level induced by rilmenidine, but not by melatonin. These results indicate that LNP 906 is the first high-affinity and selective photoaffinity ligand for I1R and that it behaves as an I1R antagonist. PMID:15178642

  9. Concurrent low- and high-affinity sulfate reduction kinetics in marine sediment

    NASA Astrophysics Data System (ADS)

    Harder Tarpgaard, Irene; Røy, Hans; Jørgensen, Bo Barker

    Bacterial sulfate reduction in marine sediments generally occurs in the presence of high millimolar concentrations of sulfate. Published data indicate that low sulfate concentrations may limit sulfate reduction rates below 0.2-2 mM. Yet, high sulfate reduction rates occur in the 1-100 μM range in freshwater sediments and at the sulfate-methane transition in marine sediments. Through a combination of 35S-tracer experiments, including initial velocity experiments and time course experiments, we searched for different sulfate affinities in the mixed community of sulfate reducers in a marine sediment. We supported the radiotracer experiments with a highly sensitive ion chromatographic technique for sulfate with a detection limit of 0.15 μM SO 42- in marine pore water. Our results showed that high and low affinities for sulfate co-occur and that the applied experimental approach may determine the observed apparent half saturation constant, Km. Our experimental and model data both show that sulfate reduction in the studied marine sediment could be explained by two dominating affinities for sulfate: a low affinity with a mean half saturation constant, Km, of 430 μM SO 42- and a high affinity with a mean Km of 2.6 μM SO 42-. The high-affinity sulfate reduction was thermodynamically un-constrained down to <1 μM SO 42-, both in our experiments and under in situ conditions. The reduction of radio-labeled sulfate was partly reversible due to concurrent re-oxidation of sulfide by Fe(III) and possibly due to a reversibility of the enzymatic pathway of sulfate reduction. A literature survey of apparent Km values for sediments and pure cultures is presented and discussed.

  10. High affinity ATP/ADP analogues as new tools for studying CFTR gating

    PubMed Central

    Zhou, Zhen; Wang, Xiaohui; Li, Min; Sohma, Yoshiro; Zou, Xiaoqin; Hwang, Tzyh-Chang

    2005-01-01

    Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K½ of 1.6 ± 0.4 μm (>50-fold more potent than ATP). The maximal open probability (Po) in the presence of P-ATP was ∼30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating. PMID:16223764

  11. Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.

    PubMed

    Chaturvedi, Sumit K; Ma, Jia; Zhao, Huaying; Schuck, Peter

    2017-09-01

    Sedimentation velocity (SV) analytical ultracentrifugation (AUC) is a classic technique for the real-time observation of free macromolecular migration in solution driven by centrifugal force. This enables the analysis of macromolecular mass, shape, size distribution, and interactions. Although traditionally limited to determination of the sedimentation coefficient and binding affinity of proteins in the micromolar range, the implementation of modern detection and data analysis techniques has resulted in marked improvements in detection sensitivity and size resolution during the past decades. Fluorescence optical detection now permits the detection of recombinant proteins with fluorescence excitation at 488 or 561 nm at low picomolar concentrations, allowing for the study of high-affinity protein self-association and hetero-association. Compared with other popular techniques for measuring high-affinity protein-protein interactions, such as biosensing or calorimetry, the high size resolution of complexes at picomolar concentrations obtained with SV offers a distinct advantage in sensitivity and flexibility of the application. Here, we present a basic protocol for carrying out fluorescence-detected SV experiments and the determination of the size distribution and affinity of protein-antibody complexes with picomolar KD values. Using an EGFP-nanobody interaction as a model, this protocol describes sample preparation, ultracentrifugation, data acquisition, and data analysis. A variation of the protocol applying traditional absorbance or an interference optical system can be used for protein-protein interactions in the micromolar KD value range. Sedimentation experiments typically take ∼3 h of preparation and 6-12 h of run time, followed by data analysis (typically taking 1-3 h).

  12. Neurotensin decreases high affinity [3H]-ouabain binding to cerebral cortex membranes.

    PubMed

    Rosin, Carina; Ordieres, María Graciela López; Arnaiz, Georgina Rodríguez de Lores

    2011-12-10

    Previous work from this laboratory showed the ability of neurotensin to inhibit synaptosomal membrane Na(+), K(+)-ATPase activity, the effect being blocked by SR 48692, a non-peptidic antagonist for high affinity neurotensin receptor (NTS1) [López Ordieres and Rodríguez de Lores Arnaiz 2000; 2001]. To further study neurotensin interaction with Na(+), K(+)-ATPase, peptide effect on high affinity [(3)H]-ouabain binding was studied in cerebral cortex membranes. It was observed that neurotensin modified