High-affinity Uptake System Research Articles

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

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.

Functional Independence of the Two Cysteine-rich Activation Domains in the Yeast Mac1 Transcription Factor

Mac1 is a transcriptional activator whose activity is inhibited by copper ions. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were identified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich motifs, designated the C1 motif. All but one of the constitutive Mac1 mutations occurred in one of the conserved six residues in the (264)CXC[(X)(4)]CXC[(X)(2)]C[(X)(2)][H(279)]C1 motif. The lone exception was a L260S substitution. Two additional MAC1 mutations exhibiting constitutive activity were in-frame deletions encompassing portions C1. Engineered mutations in the second cysteine-rich motif did not yield a constitutively active Mac1. These results are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 activator was weak relative to the C2 activator. Limited copper metalloregulation of Mac1 was observed with only the C1 activator fused to the N-terminal DNA binding domain. Thus, the two Cys-rich motifs appear to function independently. The C1 motif appears to be a functional copper-regulatory domain.

The establishment of coherent phosphate uptake behaviour by the cyanobacterium Anacystis nidulans

The physiological adaptation of the phosphate uptake behaviour of the cyanobacterium Anacystis nidulans (Synechococcus leopoliensis) has been studied under phosphate-deficient conditions. We show that, during additions of pulses of phosphate, the individual cells of a population potentially develop a coherent behaviour with respect to the kinetic and energetic properties of the high-affinity phosphate uptake system. These adaptive responses are independent of the amount of stored phosphate and the absolute external phosphate concentration, but depend on the exposure time to elevated phosphate concentrations. We demonstrate that the response to additions of pulses of phosphate triggers further metabolic alterations to CO2 fixation, and the flow of fixed carbon into the pool of low-molecular weight-compounds or glycogen. These alterations may also affect subsequent growth. The ecological relevance of the adaptive phosphate uptake behaviour is discussed with respect to the establishment and stability of natural communities.

L-arginine uptake and release by cultured avian retinal cells: differential cellular localization in relation to nitric oxide synthase.

The availability of L-arginine is of pivotal importance for the synthesis of nitric oxide, a signaling molecule in the CNS. Here we show the presence of a high-affinity L-arginine uptake system (Km of 4.4 +/- 0.5 microM and a Vmax of 26.0 +/- 0.9 fmol/well/min) in cultured chick retinal cells. Different compounds, such as N(G)-mono-methyl-L-arginine and L-lysine, were able to inhibit the uptake that was also inhibited 60-70% in the absence of sodium and/or calcium ions. No trans stimulation was observed when cells were preloaded with L-lysine. The data indicate that the L-arginine uptake in cultured retinal cells is partially mediated by the y+ system, but has a great contribution of the B(0,+) system. Autoradiographic studies revealed that the uptake is predominant in glial cells and can also be detected in neurons, whereas immunocytochemistry of nitric oxide synthase and L-citrulline showed that the enzyme is present in neurons and photoreceptors, but not in glial cells. L-[3H]Arginine is released from purified glial cultures incubated with high concentrations of potassium in the extracellular medium. Moreover, the amino acid released from preloaded glial cells was taken up by purified neuronal cultures. These results indicate that L-arginine released from glial cells is taken up by neurons and used as substrate for the synthesis of nitric oxide.

Multiple factors independently regulate hilA and invasion gene expression in Salmonella enterica serovar typhimurium.

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimurium's ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

Pho86p, an endoplasmic reticulum (ER) resident protein in Saccharomyces cerevisiae, is required for ER exit of the high-affinity phosphate transporter Pho84p.

In the budding yeast Saccharomyces cerevisiae, PHO84 and PHO86 are among the genes that are most highly induced in response to phosphate starvation. They are essential for growth when phosphate is limiting, and they function in the high-affinity phosphate uptake system. PHO84 encodes a high-affinity phosphate transporter, and mutations in PHO86 cause many of the same phenotypes as mutations in PHO84, including a phosphate uptake defect and constitutive expression of the secreted acid phosphatase, Pho5p. Here, we show that the subcellular localization of Pho84p is regulated in response to extracellular phosphate levels; it is localized to the plasma membrane in low-phosphate medium but quickly endocytosed and transported to the vacuole upon addition of phosphate to the medium. Moreover, Pho84p is localized to the endoplasmic reticulum (ER) and fails to be targeted to the plasma membrane in the absence of Pho86p. Utilizing an in vitro vesicle budding assay, we demonstrate that Pho86p is required for packaging of Pho84p into COPII vesicles. Pho86p is an ER resident protein, which itself is not transported out of the ER. Interestingly, the requirement of Pho86p for ER exit is specific to Pho84p, because other members of the hexose transporter family to which Pho84 belongs are not mislocalized in the absence of Pho86p.

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures.

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent kS value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h(-1), with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h(-1), but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h(-1), which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.

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

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.

The effect of glutamate uptake inhibitors on hippocampal evoked potentials in vitro

The influence of four inhibitors of the high-affinity glutamate uptake system (DL-aspartic acid beta-hydroxymate, DL-AHM; L-aspartic acid beta-hydroxymate, L-AHM; threo-beta-methylaspartate, DLM; L-transpyrrolidine-2, 4-dicarboxylate, PDC) on potentials recorded from hippocampal slices was investigated. At low concentrations of DL-AHM, L-AHM and DLM (50–150 microM) the population spike was permanently amplified. NMDA receptor antagonists blocked this facilitatory effect of L-AHM, DL-AHM and DLM. At higher concentrations (400–700 microM) DL-AHM and DLM abolished the population spike, while L-AHM did not eliminate the population spike at any concentration tested. None of these uptake inhibitors influenced an antidromic potential recorded in Ca2+-free Ringer solution. PDC at lower concentrations (75 microM) did not affect the population spike and at higher concentrations (150 microM – 500 microM) induced only a transient elevation in population spike. Our data demonstrate that modification of glutamate uptake may be an important factor in the regulation of synaptic efficiency of glutamergic pathways.

Iron acquisition systems in the pathogenic Neisseria.

Pathogenic neisseriae have a repertoire of high-affinity iron uptake systems to facilitate acquisition of this essential element in the human host. They possess surface receptor proteins that directly bind the extracellular host iron-binding proteins transferrin and lactoferrin. Alternatively, they have siderophore receptors capable of scavenging iron when exogenous siderophores are present. Released intracellular haem iron present in the form of haemoglobin, haemoglobin-haptoglobin or free haem can be used directly as a source of iron for growth through direct binding by specific surface receptors. Although these receptors may vary in complexity and composition, the key protein involved in the transport of iron (as iron, haem or iron-siderophore) across the outer membrane is a TonB-dependent receptor with an overall structure presumably similar to that determined recently for Escherichia coli FhuA or FepA. The receptors are potentially ideal vaccine targets in view of their critical role in survival in the host. Preliminary pilot studies indicate that transferrin receptor-based vaccines may be protective in humans.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of gram-negative bacteria.

The Yeast Connection to Friedreich Ataxia

We thank John Ashkenas for his editorial assistance and David Koeller for data prior to publication. A.D. and D.P. are supported by grants DK53953-01 and GM57067-01, respectively, from the National Institutes of Health.

The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake.

The CHL1 (NRT1) gene of Arabidopsis encodes a nitrate-inducible nitrate transporter that is thought to be a component of the low-affinity (mechanism II) nitrate-uptake system in plants. A search was performed to find high-affinity (mechanism I) uptake mutants by using chlorate selections on plants containing Tag1 transposable elements. Chlorate-resistant mutants defective in high-affinity nitrate uptake were identified, and one had a Tag1 insertion in chl1, which was responsible for the phenotype. Further analysis showed that chl1 mutants have reduced high-affinity uptake in induced plants and are missing a saturable component of the constitutive, high-affinity uptake system in addition to reduced low-affinity uptake. The contribution of CHL1 to constitutive high-affinity uptake is higher when plants are grown at more acidic pH, conditions that increase the level of CHL1 mRNA. chl1 mutants show reduced membrane depolarization in root epidermal cells in response to low (250 microM) and high (10 mM) concentrations of nitrate. Low levels of nitrate (100 microM) induce a rapid increase in CHL1 mRNA. These results show that CHL1 is an important component of both the high-affinity and the low-affinity nitrate-uptake systems and indicate that CHL1 may be a dual-affinity nitrate transporter.

Metalloregulation of FRE1 and FRE2Homologs in Saccharomyces cerevisiae

The high affinity uptake systems for iron and copper ions in Saccharomyces cerevisiae involve metal-specific permeases and two known cell surface Cu(II) and Fe(III) metalloreductases, Fre1 and Fre2. Five novel genes found in the S. cerevisiae genome exhibit marked sequence similarity to Fre1 and Fre2, suggesting that the homologs are part of a family of proteins related to Fre1 and Fre2. The homologs are expressed genes in S. cerevisiae, and their expression is metalloregulated as is true with FRE1 and FRE2. Four of the homologs (FRE3-FRE6) are specifically iron-regulated through the Aft1 transcription factor. These genes are expressed either in cells limited for iron ion uptake by treatment with a chelator or in cells lacking the high affinity iron uptake system. Expression of FRE3-FRE6 is elevated in AFT1-1 cells and attenuated in aft1 null cells, showing that iron modulation occurs through the Aft1 transcriptional activator. The fifth homolog FRE7 is specifically copper-metalloregulated. FRE7 is expressed in cells limited in copper ion uptake by a Cu(I)-specific chelator or in cells lacking the high affinity Cu(I) permeases. The constitutive expression of FRE7 in MAC1 cells and the lack of expression in mac1-1 cells are consistent with Mac1 being the critical transcriptional activator of FRE7 expression. The 5' promoter sequence of FRE7 contains three copper-responsive promoter elements. Two elements are critical for Mac1-dependent FRE7 expression. Combinations of either the distal and central elements or the central and proximal elements result in copper-regulated FRE7 expression. Spacing between Mac1-responsive sites is important as shown by the attenuated expression of FRE7 and CTR1 when two elements are separated by over 100 base pairs. From the three Mac1-responsive elements in FRE7, a new consensus sequence for Mac1 binding can be established as TTTGC(T/G)C(A/G).

YIpDCE1 - an integrating plasmid for dual constitutive expression in yeast.

YIpDCE1 (Dual Constitutive Expression), a novel Saccharomyces cerevisiae integrating plasmid, constitutively expresses two genes under the control of separate phosphoglycerol kinase promoters. YIpDCE1 contains the complete ADE2 gene which can be used as a marker for selecting integrants at mutant ade2 loci commonly present in laboratory yeast strains. The YIpDCE1 plasmid can be inserted into the ade2-101 locus of the HF7c strain used in two hybrid screens. Thus it could be useful for analysis of two hybrid interactions that occur in the context of additional protein components (e.g. modifying enzymes such as kinases or phosphatases, or multimeric complexes consisting of three or four distinct protein components). YIpDCE1 has been used to create strains simultaneously overexpressing the permease (FTR1) and oxidase (FET3) components of the yeast high-affinity iron uptake system. This confers constitutive high-affinity iron uptake on the transformed strains, bypassing the normal regulatory mechanisms.

The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli.

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron repletion and repressed under iron depletion. Two fusions were localized in genes that formed an operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 microM Zn2+. A znuA-lacZ operon fusion was repressed by 5 microM Zn2+ and showed a more than 20-fold increase in beta-galactosidase activity when Zn2+ was bound to 1.5 microM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbK of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identity with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.

Extracellular Accumulation of Rhodotorulic Acid in Strains of Microbotryum violaceum

Siderophore production is an integral component of high-affinity iron uptake by many microorganisms. We examined the extracellular production of the siderophore rhodotorulic acid (RA) in 16 field-collected and seven herbarium strains of Microbotryum violaceum. These strains were collected from a variety of caryophyllaceous host plant species, from many geographical locations, over a period of years. All of the tested strains, when starved for iron, produced detectable levels of RA, which is consistent with a critical function of the high-affinity iron uptake system. We found normal patterns of siderophore secretion accompanying iron starvation as well as repression of siderophore secretion when cells were grown in culture containing adequate levels of iron. We detected a fivefold variation in the extracellular siderophore level (0.33 mM-1.71 mM) among the 23 strains. Levels of extracellular siderophore accumulation correlated with cell density; strains that grew to a greater density (cells/mL) in liquid c...

Carrier‐mediated high affinity uptake system for histamine in astroglial and cerebral endothelial cells

Carrier‐mediated high affinity uptake system for histamine in astroglial and cerebral endothelial cells

Effect of methionine sulphoximine on13 N‐ammonium fluxes in the roots of barley and squash seedlings

Abstract13 N‐Iabelled experiments were carried out with barley and squash seedlings to analyse the effect of methionine sulphoximine (MSO), as an inhibitor of the ammonium assimilation, on NH4+ fluxes in the roots at the level (150 μM NH4+) of the high affinity uptake system (HATS). MSO pretreatment in the root medium significantly accelerated NH4+ influx and efflux in NH4+ ‐fed plants of both species. Concomitantly, the level of NH4+ extracted from the root and shoot tissues was strongly elevated whereas the translocation of13 N‐Iabelled organic substances (presumably amino products) into the shoots was markedly reduced. The onset of the increased NH4+ influx did not occur immediately but was indicated about 1 h after the application of MSO to the nutrient solution. Also, MSO did not directly affect the uptake of13 NO3−. The reason why MSO treatment leads to an acceleration of the NH4+ influx against an increasing level in the cell tissue remains obscure. In squash, an intermediate of NO3− metabolism is thought to suppress NH4+ uptake since NH4+ influx was high in N‐free‐ and NH4+ ‐grown seedlings but was reduced about threefold in NO3− ‐fed squash plants; the suppression was not eliminated by MSO treatment.

Amino acid uptake is strongly affected during exponential growth of Saccharomyces cerevisiae in 0.7 M NaCl medium.

Labelling of Saccharomyces cerevisiae grown in 0.7 M NaCl with 35S-methionine revealed a 5-6 fold lowering of the methionine incorporation into protein, which could not be attributed solely to the approximately 50% longer generation time of cells grown in 0.7 M NaCl. Subsequent studies of the high affinity methionine uptake system showed a strongly reduced uptake of methionine during growth in 0.7 M NaCl medium. This reduced uptake was shown to be strain-independent and caused mainly by an approximately 20-fold lowered maximum velocity (Vmax) of the transport system, while the substrate affinity (Km) displayed only a minor change. A salt-instigated reduction of uptake was furthermore demonstrated for the leucine and histidine high affinity uptake systems and also for a mixture of 15 different amino acids. We therefore suggest that the reduced amino acid uptake is a general phenomenon observed in salt-grown cells.