Molecular recognition of siderophores in fungi: role of iron-surrounding N-acyl residues and the peptide backbone during membrane transport in Neurospora crassa.

Recognition of ferric siderophores in Neurospora crassa was found to depend on the number and kind of N-acyl residues that surrounded the iron coordination center. In the coprogen series, uptake decreased in the order of coprogen, neocoprogen I, and neocoprogen II, indicating that gradual replacement of the N-transanhydromevalonyl groups by N-acetyl groups had an adverse effect on uptake. The reverse effect was observed in the ferrichrome series, where uptake decreased in the order of ferrichrysin, asperchrome D1, asperchrome B1, and ferrirubin. Configuration of the anhydromevalonyl group (cis or trans) in ferrichromes was also an important determinant in the recognition process. On the basis of uptake and inhibition studies, it is proposed that in ferrichromes part of the molecule (iron configuration and the N-acyl groups) is responsible for binding, whereas another (cyclic peptide ring) is involved in the subsequent process of transport.     

Siderophore-mediated iron (III) transport in the mycelia of the cultivated fungus, Agaricus bisporus.

Three structurally diverse iron (III) sequestering compounds (siderophores) were isolated from the supernatants of early stationary phase iron-deficient cultures of vegetative mycelia of the cultivated mushroom, Agaricus bisporus (ATCC 36416). The compounds were purified as their ferric chelates to homogeneity by gel permeation, cation exchange, and low-pressure reversed phase C18 chromatographies, and characterized as trihydroxamic acids. The chelates were identified as ferrichrome, ferric fusarinine C, and an unusual compound, des (diserylglycyl) ferrirhodin (DDF) by HPTLC cochromatography and electrophoresis against authentic samples, hydrolysis and amino acid analysis, and FAB-MS and 1H NMR spectroscopy. The iron transport activities of the three compounds (and of some structurally similar exogenous compounds) in young mycelial cells were determined by time- and concentration-dependent kinetic assays and inhibition experiments (CN-, N3-) using 55Fe(3+)-labeled chelates. 55Iron (III) uptake mediated by all three compounds was found to be via high affinity, energy-dependent processes; transport effectiveness was in the order: ferrichrome > DDF > ferric fusarinine C. The relative uptake of iron by lambda-cis ferrichromes was: ferrichrome > ferrirhodin > ferrichrome A; transport activity by the delta-cis fusarinines was: ferric fusarinine C > tris cis-(and trans-) fusarinine iron (III) > ferric N1-triacetylfusarinine C.     

Ferrichrome induces endosome to plasma membrane cycling of the ferrichrome transporter,Arn1p,in Saccharomyces cerevisiae

Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore-iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of ferrichrome-iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.     

Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis

We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1 delta and the hak1 delta TRK1 mutant strains. Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH. At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 microM) where HAK1 is very effective. TRK1 is also involved in the control of the membrane potential.     

GGA2- and ubiquitin-dependent trafficking of Arn1, the ferrichrome transporter of Saccharomyces cerevisiae

The intracellular trafficking of Arn1, a ferrichrome transporter in Saccharomyces cerevisiae, is controlled in part by the binding of ferrichrome to the transporter. In the absence of ferrichrome, Arn1 is sorted directly from the Golgi to endosomes. Ferrichrome binding triggers the redistribution of Arn1 to the plasma membrane, whereas ferrichrome transport is associated with the cycling of Arn1 between the plasma membrane and endosomes. Here, we report that the clathrin adaptor Gga2 and ubiquitination by the Rsp5 ubiquitin ligase are required for trafficking of Arn1. Gga2 was required for Golgi-to-endosomal trafficking of Arn1, which was sorted from endosomes to the vacuole for degradation. Trafficking into the vacuolar lumen was dependent on ubiquitination by Rsp5, but ubiquitination was not required for plasma membrane accumulation of Arn1 in the presence of ferrichrome. Retrograde trafficking via the retromer complex or Snx4 was also not required for plasma membrane accumulation. High concentrations of ferrichrome led to higher levels of ubiquitination of Arn1, but they did not induce degradation. Without this ubiquitination, Arn1 remained on the plasma membrane, where it was active for transport. Arn1 was preferentially modified with polyubiquitin chains on a cluster of lysine residues at the amino terminus of the transporter.     

Substrate recognition by nucleotide sugar transporters: further characterization of substrate recognition regions by analyses of UDP-galactose/CMP-sialic acid transporter chimeras and biochemical analysis of the substrate specificity of parental and chimeric transporters

Human UDP-Gal transporter 1 (hUGT1) and the human CMP-Sia transporter (hCST) are similar in structure, with amino acid sequences that are 43% identical, but they have quite distinct transport substrates. To define their substrate recognition regions, we constructed various chimeras between the two transporters and demonstrated that distinct submolecular regions of the transporter molecules are involved in the specific recognition of UDP-Gal and CMP-Sia (Aoki, K., Ishida, N., and Kawakita, M. (2001) J. Biol. Chem. 276, 21555-21561). In a further attempt to define the minimum submolecular regions required for the recognition of specific substrates, we found that substitution of helix 7 of hCST into the corresponding part of hUGT1 was necessary and sufficient for a chimera to show CST activity. Additional replacement of helix 2 or 3 of hUGT1 with the corresponding hCST sequence markedly increased the efficiency of CMP-Sia transport. For UGT activity, helices 1 and 8 of hUGT1 were necessary (but not sufficient), and helices 9 and 10 or helices 2, 3, and 7 derived from hUGT1 were also required to render the chimera competent for UDP-Gal transport. The in vitro analyses of a chimera with dual specificity indicated that it transported both UMP and CMP and mediated exchange reactions between these nucleotides and nucleotide sugars that are recognized specifically by either of the parental transporters.     

A receptor domain controls the intracellular sorting of the ferrichrome transporter, ARN1

The Saccharomyces cerevisiae transporter Arn1p takes up the ferric-siderophore ferrichrome, and extracellular ferrichrome dramatically influences the intracellular trafficking of Arn1p. In the absence of ferrichrome, Arn1p sorts directly to the endosomal compartment. At low concentrations of ferrichrome, Arn1p stably relocalizes to the plasma membrane, yet little to no uptake of ferrichrome occurs at these low concentrations. At higher concentrations of ferrichrome, Arn1p cycles between the plasma membrane and endosome. Arn1p contains two binding sites for ferrichrome: one site has an affinity similar to the K(T) for transport, but the second site has a much higher affinity. Here we report that this high-affinity binding site lies within a unique extracytosolic, carboxyl-terminal domain. Mutations within this domain lead to loss of ferrichrome binding and uptake activities and missorting of Arn1p, including a failure to relocalize to the plasma membrane in the presence of ferrichrome. Mutation of phenylalanine residues in the cytosolic tail of Arn1p also lead to missorting, but without defects in ferrichrome binding. We propose that the carboxyl terminus of Arn1p contains a receptor domain that controls the intracellular trafficking of the transporter.     

The channel in transporters is formed by residues that are rare in transmembrane helices

Transmembrane transport is an essential component of the cell life. Many genes encoding known or putative transport proteins are found in bacterial genomes. In most cases their substrate specificity is not experimentally determined and only approximately predicted by comparative genomic analysis. Even less is known about the 3D structure of transporters. Nevertheless, the published experimental data demonstrate that channel-forming residues determine the substrate specificity of secondary transporters and analysis of these residues would provide better understanding of the transport mechanism. We developed a simple computational method for identification of channel-forming residues in transporter sequences. It is based on the analysis of amino acids frequencies in bacterial secondary transporters. We applied this method to a variety of transmembrane proteins with resolved 3D structure. The predictions are in sufficiently good agreement with the real protein structure.     

Human and Drosophila UDP-galactose transporters transport UDP-N-acetylgalactosamine in addition to UDP-galactose.

A putative Drosophila nucleotide sugar transporter was characterized and shown to be the Drosophila homologue of the human UDP-Gal transporter (hUGT). When the Drosophila melanogaster UDP-Gal transporter (DmUGT) was expressed in mammalian cells, the transporter protein was localized in the Golgi membranes and complemented the UDP-Gal transport deficiency of Lec8 cells but not the CMP-Sia transport deficiency of Lec2 cells. DmUGT and hUGT were expressed in Saccharomyces cerevisiae cells in functionally active forms. Using microsomal vesicles isolated from Saccharomyces cerevisiae expressing these transporters, we unexpectedly found that both hUGT and DmUGT could transport UDP-GalNAc as well as UDP-Gal. When amino-acid residues that are conserved among human, murine, fission yeast and Drosophila UGTs, but are distinct from corresponding ones conserved among CMP-Sia transporters (CSTs), were substituted by those found in CST, the mutant transporters were still active in transporting UDP-Gal. One of these mutants in which Asn47 was substituted by Ala showed aberrant intracellular distribution with concomitant destabilization of the protein product. However, this mutation was suppressed by an Ile51 to Thr second-site mutation. Both residues were localized within the first transmembrane helix, suggesting that the structure of the helix contributes to the stabilization and substrate recognition of the UGT molecule.     

Alpha-keto acids are novel siderophores in the genera Proteus, Providencia, and Morganella and are produced by amino acid deaminases.

Growth promotion and iron transport studies revealed that certain alpha-keto acids generated by amino acid deaminases, by enterobacteria of the Proteus-Providencia-Morganella group (of the tribe Proteeae), show significant siderophore activity. Their iron-binding properties were confirmed by the chrome azurol S assay and UV spectra. These compounds form ligand-to-metal charge transfer bands in the range of 400 to 500 nm. Additional absorption bands of the enolized ligands at 500 to 700 nm are responsible for color formation. Siderophore activity was most pronounced with alpha-keto acids possessing an aromatic or heteroaromatic side chain, like phenylpyruvic acid and indolylpyruvic acid, resulting from deamination of phenylalanine and tryptophan, respectively. In addition, alpha-keto acids possessing longer nonpolar side chains, like alpha-ketoisocaproic acid or alpha-ketoisovaleric acid and even alpha-ketoadipic acid, also showed siderophore activity which was absent or negligible with smaller alpha-keto acids or those possessing polar functional groups, like pyruvic acid, alpha-ketobutyric acid, or alpha-ketoglutaric acid. The fact that deaminase-negative enterobacteria, like Escherichia coli and Salmonella spp., could not utilize alpha-keto acids supports the view that specific iron-carboxylate transport systems have evolved in members of the tribe Proteeae and are designed to recognize ferric complexes of both alpha-hydroxy acids and alpha-keto acids, of which the latter can easily be generated by L-amino acid deaminases in an amino acid-rich medium. Exogenous siderophores, like ferric hydroxamates (ferrichromes) and ferric polycarboxylates (rhizoferrin and citrate), were also utilized by members of the tribe Proteeae.     

Trk2 Is Required for Low Affinity K(+) Transport in Saccharomyces Cerevisiae

TRK1, the gene encoding the high affinity K+ transporter in Saccharomyces cerevisiae, is nonessential due to the existence of a functionally independent low affinity transporter. To identify the gene(s) encoding the low affinity K+ transporter, we screened trk1 delta cells for mutants (Kla-) that require higher concentrations of K+ in the medium to support growth. trk1 delta trk2 mutants require up to tenfold higher concentrations of K+ to exhibit normal growth compared to trk1 delta TRK2 cells. K+ and 86Rb+ transport assays demonstrate that the mutant phenotype is due to defective K+ transport (uptake). Each of 38 independent mutants contains a mutation in the same gene, TRK2. Cells deficient for both high and low affinity K+ transport (trk1 delta trk2) exhibit hypersensitivity to low extracellular pH that can be suppressed by high concentrations of K+ but not Na+. TRK1 completely suppresses both the K+ transport defect and low pH hypersensitivity of trk2 cells, suggesting that TRK1 and TRK2 are functionally independent.     

Wide tolerance to amino acids substitutions in the OCTN1 ergothioneine transporter

BackgroundOrganic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease.MethodsHere we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy.ResultsSubstitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased V_max, with modest changes in K_m toward ergothioneine.ConclusionsOur results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity.General significanceThe widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role.     

Identification of Conformationally Sensitive Residues Essential for Inhibition of Vesicular Monoamine Transport by the Noncompetitive Inhibitor Tetrabenazine

Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.     

Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342-557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 +/- 0.5 to 141 +/- 19 microM. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341-454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341-454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 +/- 5 microM). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 +/- 4.5 microm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition.     

Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3

Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were approximately 10-, approximately 48-, and approximately 32-fold enhanced in oocytes expressing rOAT3 and were only approximately 2-, approximately 6-, and approximately 5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP(+), in comparison to PAH (MPP(+) uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.     

Molecular Determinants for Functional Differences between Alanine-Serine-Cysteine Transporter 1 and Other Glutamate Transporter Family Members

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter Glt_Ph. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and Glt_Ph. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and Glt_Ph, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and Glt_Ph. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of Glt_Ph, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.