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

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.

Alanine scanning mutagenesis of a high-affinity nitrate transporter highlights the requirement for glycine and asparagine residues in the two nitrate signature motifs.

Common to all of the nitrate nitrite porter family are two conserved motifs in transmembrane helices 5 and 11 termed NS (nitrate signature) 1 and NS2. Although perfectly conserved substrate-interacting arginine residues have been described in transmembrane helices 2 and 8, the role of NSs has not been investigated. In the present study, a combination of structural modelling of NrtA (nitrate transporter from Aspergillus nidulans) with alanine scanning mutagenesis of residues within and around the NSs has been used to shed light on the probable role of conserved residues in the NSs. Models show that Asn(168) in NS1 and Asn(459) in NS2 are positioned approximately midway within the protein at the central pivot point in close proximity to the substrate-binding residues Arg(368) and Arg(87)respectively, which lie offset from the pivot point towards the cytoplasmic face. The Asn(168)/Arg(368)and Asn(459)/Arg(87) residue pairs are relatively widely separated on opposite sides of the probable substrate translocation pore. The results of the present study demonstrate the critical structural contribution of several glycine residues in each NS at sites of close helix packing. Given the relative locations of Asn(168)/Arg(368)and Asn(459)/Arg(87)pairs, the validity of the models and possible role of the NSs together with the substrate-binding arginine residues are discussed.

Physiological and biochemical characterization of AnNitA, the Aspergillus nidulans high-affinity nitrite transporter.

High-affinity nitrite influx into mycelia of Aspergillus nidulans has been characterized by use of (13)NO(2)(-), giving average K(m) and V(max) 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 (r(2) = 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.

Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtA, NrtB in Aspergillus nidulans.

Disruption of the Aspergillus nidulans high-affinity nitrate transporter genes (nrtA and nrtB) prevents growth on nitrate but not nitrite. We identified a distinct nitrite transporter (K(m)=4.2+/-1 microM, V(max)=168+/-21 nmolmg(-1)DW(-1)h(-1)), designated NitA. Disruption of nrtA, nrtB and nitA blocked growth on nitrite, despite low rates of nitrite depletion we ascribe to passive nitrous acid permeation. Growth of the single mutant nitA16 on nitrite was wild-type, suggesting that NrtA and/or NrtB transports nitrite as well as nitrate. Indeed, NrtA and NrtB transport nitrite at higher rates than NitA; K(m) and V(max) values were 16+/-4 microM and 808+/-67 nmolmg(-1)DW(-1)h(-1) (NrtA) and 11+/-1 microM and 979+/-17 nmolmg(-1)DW(-1)h(-1) (NrtB). We suggest that NrtA is a nitrate/nitrite transporter, NrtB absorbs nitrite in preference to nitrate and NitA is exclusively a nitrite transporter.

Differential regulation of multiple alternatively spliced transcripts of MyoD.

Splice variants of the basic helix-loop-helix myoblast determination factor (myoD) have not been previously found in vertebrates. Here we report the identification and characterization of three alternative transcripts of a myoD paralogue from the tiger pufferfish (Takifugu rubripes). The T. rubripes myoD1 gene (TmyoD1) has 3 exons and 2 introns and it is present on scaffold 104, in a region of conserved synteny with zebrafish. The isoform TMyoD1-alpha is a putative protein of 281 residues that contains the basic, helix-loop-helix and helix III domains and shares 61%, 56%, 51%, 49% and 56% overall identity with zebrafish, Xenopus, mouse, human and chicken MyoD1, respectively. TMyoD1-beta arises from an alternative 3' splice site and differs from TMyoD1-alpha by a 26-residue insertion adjacent to helix III, which is one of the functional domains required for chromatin remodelling. The third alternative transcript, TmyoD1-gamma, retains intron I and has two premature termination codons far from the 3'-most exon-exon junction. TmyoD1-gamma is therefore likely to be degraded by nonsense-mediated decay, an important widespread post-transcriptional mechanism that regulates transcript levels. Analysis of gene expression by qPCR revealed that TmyoD1-alpha was the most abundant transcript in fast and slow myotomal muscle. TmyoD1-alpha expression was 2-fold higher in fast muscle of juvenile fish that were actively producing new myotubes compared to adult stages that had stopped recruiting fast muscle fibres. A similar expression pattern was observed for TmyoD1-alpha in slow muscle but the differences were not significant. Transcript levels of TmyoD1-gamma only varied significantly in fast muscle and were 5-fold higher in adult compared to juvenile stages. Significant differences in expression of TmyoD1 splice variants were also observed during embryonic development. The differential expression of three alternative transcripts of myoD1 in developing and adult myotomal muscle of T. rubripes supports the hypothesis that diversity generated by alternative splicing may be of functional significance in muscle development in this species.

Characterization of two paralogous muscleblind-like genes from the tiger pufferfish (Takifugu rubripes).

Muscleblind-like (Mbnl) proteins are required for terminal muscle differentiation in mammals. In this study we have identified two mbnl paralogues from the tiger pufferfish, tmbnl2a and tmbnl3, which are the first examples of non-mammalian mbnl genes. Tmbnl2a and tmbnl3 were found in regions of conserved synteny and had a high degree of global conservation with their mammalian homologues. Phylogenetic analysis showed that the T. rubripes genome contains one mbnl3 gene and two copies of mbnl1 and mbnl2. Moreover, the mbnl1 and mbnl3 paralogues are derived from duplication of a common ancestral gene. The average rates of synonymous substitutions between T. rubripes, mouse and human mbnl2 and mbnl3 genes were much higher than the corresponding rates of non-synonymous mutations, suggesting that Mbnl2 and Mbnl3 are subjected to strong purifying selection. Quantitation of tmbnl2a and tmbnl3 transcripts by real-time PCR revealed that these two paralogues are differentially expressed in fast and slow myotomal muscle, heart, liver, skin, brain and testes. Tmbnl2a was expressed at similar levels in all tissues examined, as was the mouse orthologue. Tmbnl3 was expressed at higher levels than tmbnl2a, with a ubiquitous tissue distribution. Expression of tmbnl3 remained high in adult pufferfish muscle whereas the mouse orthologue was down-regulated in adults, perhaps reflecting the indeterminate and determinate growth patterns of these taxa, respectively.

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter.

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.

A genomic approach to reveal novel genes associated with myotube formation in the model teleost, Takifugu rubripes.

Little is known about the transcriptional networks that regulate myotube production in vertebrates. In the present study, we have used a genomic approach to discover novel genes associated with myotube formation in fast muscle of the tiger puffer fish, Takifugu rubripes. The number of fast muscle fibers per myotome increased until 1.2 kg body mass, and subsequent growth was by fiber hypertrophy alone. Forward and reverse subtracted cDNA libraries were prepared from a 180-g (myotube +) and a 3.4-kg (myotube -) fish, and 1,452 expressed sequence tags (ESTs) were obtained. After these ESTs were grouped into nonredundant clusters and housekeeping and structural genes were eliminated, 57 genes were selected and quantitative PCR was used to investigate their expression levels in different tissues from independent groups of myotube(-) and myotube(+) fish acclimated to the same environmental conditions and diet. Eleven novel genes were found to be consistently differentially expressed, but only four showed appropriate tissue-specific expression. These four genes were upregulated 5-25 times in fast muscle of myotube(-) relative to myotube(+) growth stages, while their expression remained unchanged in the other tissues studied. The novel genes identified, which are also present in other vertebrate genomes, may play a role in inhibiting myotube formation in vertebrate muscle.

Mutational analysis of the gephyrin-related molybdenum cofactor biosynthetic gene cnxE from the lower eukaryote Aspergillus nidulans.

We report the identification of a number of mutations that result in amino acid replacements (and their phenotypic characterization) in either the MogA-like domain or domains 2 and 3 of the MoeA-like region of the Aspergillus nidulans cnxE gene. These domains are functionally required since mutations that result in amino acid substitutions in any one domain lead to the loss or to a substantial reduction in all three identified molybdoenzyme activities (i.e., nitrate reductase, xanthine dehydrogenase, and nicotinate hydroxylase). Certain cnxE mutants that show partial growth with nitrate as the nitrogen source in contrast do not grow on hypoxanthine or nicotinate. Complementation between mutants carrying lesions in the MogA-like domain or the MoeA-like region, respectively, most likely occurs at the protein level. A homology model of CnxE based on the dimeric structure of E. coli MoeA is presented and the position of inactivating mutations (due to amino acid replacements) in the MoeA-like functional region of the CnxE protein is mapped to this model. Finally, the activity of nicotinate hydroxylase, unlike that of nitrate reductase and xanthine dehydrogenase, is not restored in cnxE mutants grown in the presence of excess molybdate.

Embryonic temperature affects muscle fibre recruitment in adult zebrafish: genome-wide changes in gene and microRNA expression associated with the transition from hyperplastic to hypertrophic growth phenotypes.

We investigated the effects of embryonic temperature (ET) treatments (22, 26 and 31 degrees C) on the life-time recruitment of fast myotomal muscle fibres in zebrafish Danio rerio L. reared at 26/27 degrees C from hatching. Fast muscle fibres were produced until 25 mm total length (TL) at 22 degrees C ET, 28 mm TL at 26 degrees C ET and 23 mm TL at 31 degrees C ET. The final fibre number (FFN) showed an optimum at 26 degrees C ET (3600) and was 19% and 14% higher than for the 22 degrees C ET (3000) and 31 degrees C ET (3100) treatments, respectively. Further growth to the maximum TL of approximately 48 mm only involved fibre hypertrophy. Microarray experiments were used to determine global changes in microRNA (miRNA) and mRNA expression associated with the transition from the hyperplasic myotube-producing phenotype (M(+), 10-12 mm TL) to the hypertrophic growth phenotype (M(-), 28-31 mm TL) in fish reared at 26-27 degrees C over the whole life-cycle. The expression of miRNAs and mRNAs obtained from microarray experiments was validated by northern blotting and real-time qPCR in independent samples of fish with the M(+) and M(-) phenotype. Fourteen down-regulated and 15 up-regulated miRNAs were identified in the M(-) phenotype together with 34 down-regulated and 30 up-regulated mRNAs (>2-fold; P<0.05). The two most abundant categories of down-regulated genes in the M(-) phenotype encoded contractile proteins (23.5%) and sarcomeric structural/cytoskeletal proteins (14.7%). In contrast, the most highly represented up-regulated transcripts in the M(-) phenotype were energy metabolism (26.7%) and immune-related (20.0%) genes. The latter were mostly involved in cell-cell interactions and cytokine pathways and included beta-2-microglobulin precursor (b2m), an orthologue of complement component 4, invariant chain-like protein 1 (iclp), CD9 antigen-like (cd9l), and tyrosine kinase, non-receptor (tnk2). Five myosin heavy chain genes that were down-regulated in the M(-) phenotype formed part of a tandem repeat on chromosome 5 and were shown by in situ hybridisation to be specifically expressed in nascent myofibres. Seven up-regulated miRNAs in the M(-) phenotype showed reciprocal expression with seven mRNA targets identified in miRBase Targets version 5 (http://microrna.sanger.ac.uk/targets/v5/), including asporin (aspn) which was the target for four miRNAs. Eleven down-regulated miRNAs in the M(-) phenotype had predicted targets for seven up-regulated genes, including dre-miR-181c which had five predicted mRNA targets. These results provide evidence that miRNAs play a role in regulating the transition from the M(+) to the M(-) phenotype and identify some of the genes and regulatory interactions involved.

FoxK1 splice variants show developmental stage-specific plasticity of expression with temperature in the tiger pufferfish.

FoxK1 is a member of the highly conserved forkhead/winged helix (Fox) family of transcription factors and it is known to play a key role in mammalian muscle development and myogenic stem cell function. The tiger pufferfish (Takifugu rubripes) orthologue of mammalian FoxK1 (TFoxK1) has seven exons and is located in a region of conserved synteny between pufferfish and mouse. TFoxK1 is expressed as three alternative transcripts: TFoxK1-alpha, TFoxK1-gamma and TFoxK1-delta. TFoxK1-alpha is the orthologue of mouse FoxK1-alpha, coding for a putative protein of 558 residues that contains the forkhead and forkhead-associated domains typical of Fox proteins and shares 53% global identity with its mammalian homologue. TFoxK1-gamma and TFoxK1-delta arise from intron retention events and these transcripts translate into the same 344-amino acid protein with a truncated forkhead domain. Neither are orthologues of mouse FoxK1-beta. In adult fish, the TFoxK1 splice variants were differentially expressed between fast and slow myotomal muscle, as well as other tissues, and the FoxK1-alpha protein was expressed in myogenic progenitor cells of fast myotomal muscle. During embryonic development, TFoxK1 was transiently expressed in the developing somites, heart, brain and eye. The relative expression of TFoxK1-alpha and the other two alternative transcripts varied with the incubation temperature regime for equivalent embryonic stages and the differences were particularly marked at later developmental stages. The developmental expression pattern of TFoxK1 and its localisation to mononuclear myogenic progenitor cells in adult fast muscle indicate that it may play an essential role in myogenesis in T. rubripes.

Evidence for post-translational regulation of NrtA, the Aspergillus nidulans high-affinity nitrate transporter.

Here, influx and efflux of (13)NO(3)(-), and net fluxes of (14)NO(3)(-) and (14)NO(2)(-), were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity NO(3)(-) transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. (13)NO(3)(-) influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. This is the first demonstration of diminished high-affinity NO(3)(-) influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of NO(3)(-) transport at the protein level, reduces the extent of futile cycling of NO(3)(-) that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of NH(4)(+) to mycelia absorbing NO(3)(-).

Myogenin in model pufferfish species: Comparative genomic analysis and thermal plasticity of expression during early development.

Myogenin (Myog) is a muscle-specific basic helix-loop-helix transcription factor that plays an essential role in the specification and differentiation of myoblasts. The myogenin genes from the tiger pufferfish, Takifugu rubripes, and green-spotted pufferfish, Tetraodon nigroviridis, were cloned and a comparative genomic analysis performed. The gene encoding myogenin is composed of three exons and has a relatively similar genomic structure in T. rubripes, T. nigroviridis and human. Introns 1 and 2 were approximately 2-fold and 8-fold longer respectively in human than pufferfish. Myogenin is located in a 100 kb region of conserved synteny between these organisms, corresponding to chromosome 1 in human, chromosome 11 in T. nigroviridis and scaffold 208 in T. rubripes. Pufferfish myogenin contained a serine-rich region at the carboxyl terminus that is highly conserved amongst teleosts. During embryonic development of T. rubripes, myogenin was expressed in a rostral-caudal gradient in the developing somites and subsequently during the pharyngula period in the pectoral fin bud primordia, jaw muscles and extraocular muscle precursors. In T. rubripes, the time required to form a somite pair during the linear phase of somitogenesis ( identical withsomite-interval) was 122 min, 97 min and 50 min in embryos incubated at 15, 18 and 21 degrees C, respectively. Myogenin mRNA transcripts were quantified using qPCR and normalised to the highest level of expression. Peak myogenin expression occurred later with respect to developmental stage (standardised using somite-intervals) and was over 2-fold higher at 21 degrees C than at either 18 or 15 degrees C. Changes in the relative timing and intensity of myogenin expression are a potential mechanism for explaining thermal plasticity of muscle phenotype in larvae via effects on the differentiation programme.

Determination of the essentiality of the eight cysteine residues of the NrtA protein for high-affinity nitrate transport and the generation of a functional cysteine-less transporter.

All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K(m) value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.

The Gibberella fujikuroi niaD gene encoding nitrate reductase: isolation, sequence, homologous transformation and electrophoretic karyotype location.

The Gibberella fujikuroi niaD gene, encoding nitrate reductase, has been isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on niaD selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic transformations, giving frequencies of up to 200 transformants per microgram DNA. Of 15 transformants analysed by Southern blots, six showed homologous integration whilst the remaining nine integrated at heterologous sites, indicating that the vector may be used reliably for both types of integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of defective mutants, and also for gene disruption experiments. Electrophoretic karyotype determination suggested at least 11 chromosomes ranging from 2 to 6 Mb, the total genome size being at least 37 Mb. The niaD gene was assigned to chromosome V by Southern blot analysis. The niaD gene is interrupted by one intron, and remarkably the promoter sequence, but not the 3' untranslated sequence, is highly homologous to that of the corresponding Fusarium oxysporum gene. This situation appears to be unique with respect to the promoter regions of corresponding genes in related species of filamentous fungi.

Mutation and functional analysis of the Aspergillus nidulans ammonium permease MeaA and evidence for interaction with itself and MepA.

The movement of ammonium across biological membranes is mediated in both prokaryotic and eukaryotic systems by ammonium transport proteins which constitute a family of related sequences (called the AMT/MEP family). Interestingly, recent evidence suggests that human and mouse Rhesus proteins which display significant relatedness to AMT/MEP sequences may function as ammonium transporters. To add to the functional understanding of ammonium transport proteins, the sequence changes in 37 loss-of-function mutations within the Aspergillus nidulans ammonium permease gene, meaA, were characterized. Together with the identification of conserved AMT/MEP residues and regions, the mutational analysis predicted regions important for uptake activity. Specifically, a major facilitator superfamily like motif (161-GAVAERGR-168 in MeaA) may be important for the translocation of ammonium across the membrane as may the conserved Pro186 residue. A specific Gly447 to Asp mutation was introduced into MeaA and this mutant protein was found to trans-inhibit the activity of endogenous MeaA and the other A. nidulans ammonium transporter, MepA. These results suggest that MeaA may interact with itself and with MepA, although any hetero-interaction is not required for ammonium transport function. In addition, cross-feeding studies showed that MeaA and to a lesser extent MepA are also required for the retention of intracellular ammonium.

Nitrate reductase activity is required for nitrate uptake into fungal but not plant cells.

The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer (13)NO(3)(-) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed.

Two perfectly conserved arginine residues are required for substrate binding in a high-affinity nitrate transporter.

This study represents the first attempt to investigate the molecular mechanisms by which nitrate, an anion of significant ecological, agricultural, and medical importance, is transported into cells by high-affinity nitrate transporters. Two charged residues, R87 and R368, located within hydrophobic transmembrane domains 2 and 8, respectively, are conserved in all 52 high-affinity nitrate transporters sequenced thus far. Site-directed replacements of either of R87 or R368 residues by lysine were found to be tolerated, but such residue changes increased the K(m) for nitrate influx from micromolar to millimolar values. Seven other amino acid substitutions of R87 or R368 all led to loss of function and lack of growth on nitrate. No evidence was obtained of R87 or R368 forming a salt-bridge with conserved acidic residues. Remarkably, the phenotype of loss-of-function mutant R87T was found to be alleviated by an alteration to lysine of N459, present in the second copy of the nitrate signature (transmembrane domain 11), suggesting a structural or functional interplay between residues R87 and N459 in the three-dimensional NrtA protein structure. Failure of the potential reciprocal second site suppressor N168K (in the first nitrate signature copy of transmembrane domain 5) to revert R368T was observed. Taken with recent structural studies of other major facilitator superfamily proteins, the results suggest that R87 and R368 are involved in substrate binding and probably located in a region of the protein close to N459.

The regulation of nitrate and ammonium transport systems in plants.

Inorganic nitrogen concentrations in soil solutions vary across several orders of magnitude among different soils and as a result of seasonal changes. In order to respond to this heterogeneity, plants have evolved mechanisms to regulate and influx. In addition, efflux analysis using (13)N has revealed that there is a co-ordinated regulation of all component fluxes within the root, including biochemical fluxes. Physiological studies have demonstrated the presence of two high-affinity transporter systems (HATS) for and one HATS for in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for and five members of the AMT1 family encoding putative HATS for. The induction of high-affinity transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of, while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated and may act post-transcriptionally on transporter function. The present challenge is to resolve the functions of all of these genes. In Aspergillus nidulans and Chlamydomonas reinhardtii there are but two high-affinity transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of absorption over the wide range of concentration normally found in nature. Such kinetic differentiation may also have occurred among higher plant transporters. The characterization of transporter function in higher plants is currently being inferred from patterns of gene expression in roots and shoots, as well as through studies of heterologous expression systems and knockout mutants.