A mutation in the Arabidopsis KT2/KUP2 potassium transporter gene affects shoot cell expansion

Potassium ions (K(+)) are the most abundant cations in plants and are necessary for cell growth. Arabidopsis shy3-1 mutant plants have a short hypocotyl, small leaves, and a short flowering stem, and these defects result from decreased cell expansion. The semidominant shy3-1 mutation changes an amino acid in KT2/KUP2, a K(+) transporter related to the Escherichia coli Kup protein. Second mutations in the KT2/KUP2/SHY3 gene, including presumed null mutations, suppress the shy3-1 phenotypes. Plants with these intragenic suppressor mutations appear similar to wild-type plants, suggesting that KT2/KUP2/SHY3 acts redundantly with other genes. Expression of the shy3-1 mutant version of KT2/KUP2/SHY3 in wild-type plants confers shy3-1-like phenotypes, indicating that shy3-1 probably either causes a gain of function or creates an interfering protein. The shy3-1 mutation does not eliminate the ability of the KT2/KUP2 cDNA to rescue the growth of a potassium transport-deficient E. coli mutant. A P(SHY3)::GUS fusion is expressed in growing portions of the plant. These results suggest that KT2/KUP2/SHY3 mediates K(+)-dependent cell expansion in growing tissues.     

Comparative functional features of plant potassium HvHAK1 and HvHAK2 transporters

Plant K(+) transporters of the HAK family belong to four rather divergent phylogenetic clusters, although most of the transporters belong to clusters I or II. A simple phylogenetic analysis of fungal and plant HAK transporters suggests that an original HAK gene duplicated even before fungi and plants diverged, generating transporters that at present fulfill different functions in the plant. The HvHAK1 transporter belongs to cluster I and mediates high-affinity K(+) uptake in barley roots, but no function is known for the cluster II transporter, HvHAK2, which is not functional in yeast. The function of HvHAK2 was investigated by constructing HvHAK1-HAK2 chimeric transporters, which were not functional even when they included only short fragments of HvHAK2. Then, amino acids characteristic of cluster II in the N terminus and in the first transmembrane domain were introduced into HvHAK1. All of these changes increased the Rb(+) K(m), introducing minimal changes in the Na(+) K(m), which suggested that HvHAK2 is a low-affinity, Na(+)-sensitive K(+) transporter. Using a K(+)-defective Escherichia coli mutant, we functionally expressed HvHAK2 and found that the predicted characteristics were correct, as well as discovering that the bacterial expression of HvHAK2 is functional at pH 5.5 but not at 7.5. We discuss whether HvHAK2 may be a tonoplast transporter effective for vacuolar K(+) depletion in K(+) starved plants.     

TRH1 encodes a potassium transporter required for tip growth in Arabidopsis root hairs

Root hair initiation involves the formation of a bulge at the basal end of the trichoblast by localized diffuse growth. Tip growth occurs subsequently at this initiation site and is accompanied by the establishment of a polarized cytoplasmic organization. Arabidopsis plants homozygous for a complete loss-of-function tiny root hair 1 (trh1) mutation were generated by means of the T-DNA-tagging method. Trichoblasts of trh1 plants form initiation sites but fail to undergo tip growth. A predicted primary structure of TRH1 indicates that it belongs to the AtKT/AtKUP/HAK K(+) transporter family. The proposed function of TRH1 as a K(+) transporter was confirmed in (86)Rb uptake experiments, which demonstrated that trh1 plants are partially impaired in K(+) transport. In line with these results, TRH1 was able to complement the trk1 potassium transporter mutant of Saccharomyces, which is defective in high-affinity K(+) uptake. Surprisingly, the trh1 phenotype was not restored when mutant seedlings were grown at high external potassium concentrations. These data demonstrate that TRH1 mediates K(+) transport in Arabidopsis roots and is responsible for specific K(+) translocation, which is essential for root hair elongation.     

Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1

K(+)-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAAT1 shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na(+), K(+), Li(+)) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(II)(1,10-phenanthroline)(3), showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na(+) protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.     

Molecular and functional characterization of a Na(+)-K(+) transporter from the Trk family in the ectomycorrhizal fungus Hebeloma cylindrosporum

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K(+) transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K(+) uptake is mainly dependent on the activity of Trk transporters thought to mediate H(+):K(+) symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K(+) and Na(+), and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na(+) and K(+) might result in Na(+):K(+) co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K(+) and Na(+) nutrition of the fungus (and of the plant) in nutrient-poor soils.     

A potassium transporter of the yeast Schwanniomyces occidentalis homologous to the Kup system of Escherichia coli has a high concentrative capacity.

The yeast Schwanniomyces occidentalis has a high-affinity K+ uptake system with a high concentrative capacity, which is able to deplete the external K+ to < 0.03 microM. We have cloned the gene HAK1 of S.occidentalis which complements defective K+ uptake by trk1 and trk1 trk2 mutants of Saccharomyces cerevisiae. When HAK1 was expressed in a trk1 trk2 S.cerevisiae mutant, transport affinities for K+ and other alkali cations resembled those of S.occidentalis. The predicted amino acid sequence of the HAK1 protein shows significant homology with the hydrophobic region of the Kup transporter of Escherichia coli. In S.occidentalis HAK1 expresses in K(+)-limiting conditions. Our data indicate that in K(+)-starved cells the system encoded by HAK1 is the major K+ transporter of S.occidentalis.     

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.     

Functional and physiological evidence for a rhesus-type ammonia transporter in Nitrosomonas europaea

Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The K(m) value for the MA uptake of N. europaea was 1.8+/-0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [K(i)(NH(4) (+)) 0.3+/-0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH(4) (+) trap.     

Identification and characterization of cDNAs encoding pentatricopeptide repeat proteins in the basal land plant, the moss Physcomitrella patens

A large gene family encoding proteins with a pentatricopeptide repeat (PPR) motif exists in flowering plants but not in algae, fungi, or animals. This suggests that PPR protein genes expanded vastly during the evolution of the land plants. To investigate this possibility, we analysed PPR protein genes in the basal land plant, the moss Physcomitrella patens. An extensive survey of the Physcomitrella expressed sequence tag (EST) databases revealed 36 ESTs encoding PPR proteins. This indicates that a large gene family of PPR proteins originated before the divergence of the vascular plant and moss lineages. We also characterized five full-length cDNAs encoding PPR proteins, designated PPR513-10, PPR566-6, PPR868-14, PPR986-12, and PPR423-6. Intracellular localization analysis demonstrated two PPR proteins in chloroplasts (cp), whereas the cellular localization of the other three PPR proteins is unclear. The genes of the cp-localized PPR513-10 and PPR566-6 were expressed differentially in protonemata grown under different light-dark conditions, suggesting they have distinctive functions in cp. This is the first report and analysis of genes encoding PPR proteins in bryophytes.     

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.     

Identification of a pH regulated Na(+)/H(+) antiporter of Methanococcus jannaschii

The genome of the hyperthermophilic archaeon Methanococcus jannaschii contains three Na(+)/H(+) antiporter related genes Mj0057, Mj1521 and Mj1275. Comparative sequence alignments revealed that Mj0057 and Mj1521 belong to the NhaP family whereas Mj1275 is a member of the NapA family. The genes were cloned and expressed in the double mutant Escherichia coli strain Frag144 (DeltanhaA, DeltanhaB) to analyze their capability of mediating DeltapH driven Na(+) flux in everted vesicles. From the tested clones only Mj0057 displayed Na(+) (Li(+))/H(+) antiporter activity. The transport was pH dependent and occurred at pH 7.0 and below. At pH 6.0 the apparent K(m) values for Na(+) and Li(+) were approximately 10 and 2.5 mM, respectively.     

A chloroplast phosphate transporter,PHT2;1, influences allocation of phosphate within the plant and phosphate-starvation responses

The uptake and distribution of Pi in plants requires multiple Pi transport systems that must function in concert to maintain homeostasis throughout growth and development. The Pi transporter PHT2;1 of Arabidopsis shares similarity with members of the Pi transporter family, which includes Na(+)/Pi symporters of fungal and animal origin and H(+)/Pi symporters of bacterial origin. Sequence comparisons between proteins of this family revealed that plant members possess extended N termini, which share features with chloroplast transit peptides. Localization of a PHT2;1-green fluorescent protein fusion protein indicates that it is present in the chloroplast envelope. A Pi transport function for PHT2;1 was confirmed in yeast using a truncated version of the protein lacking its transit peptide, which allowed targeting to the plasma membrane. To assess the in vivo role of PHT2;1 in phosphorus metabolism, we identified a null mutant, pht2;1-1. Analysis of the mutant reveals that PHT2;1 activity affects Pi allocation within the plant and modulates Pi-starvation responses, including the expression of Pi-starvation response genes and the translocation of Pi within leaves.     

Analysis of the transport activity of barley sucrose transporter HvSUT1

Localization studies indicate that barley (Hordeum vulgare) sucrose transporter HvSUT1 functions in sucrose uptake into seeds during grain filling. To further understand the physiological function of HvSUT1, we have expressed the HvSUT1 cDNA in Xenopus laevis oocytes and analyzed the transport activity by two-electrode voltage clamping. Consistent with a H(+)-coupled transport mechanism, sucrose induced large inward currents in HvSUT1-expressing oocytes with a K (0.5) of 3.8 mM at pH 5.0 and a membrane potential of -157 mV. Of 21 other sugars tested, four glucosides were also transported by HvSUT1. These glucosides were maltose, salicin (2-(hydroxymethyl) phenyl beta-D-glucoside), alpha-phenylglucoside and alpha-paranitrophenylglucoside. Kinetic analysis of transport of these substrates by HvSUT1 was performed and K (0.5) values were measured. The apparent affinity for all substrates was dependent on membrane potential and pH with lower K (0.5) values at lower external pH and more negative membrane potentials. HvSUT1 was more selective for alpha-glucosides over beta-glucosides than the Arabidopsis sucrose transporter AtSUC2. Several substrates transported by AtSUC2 (beta-phenylglucoside, beta-paranitrophenylglucoside, alpha-methylglucoside, turanose, and arbutin (hydroquinone beta-D-glucoside)) showed low or undetectable transport by HvSUT1. Of these, beta-paranitrophenylglucoside inhibited sucrose transport by HvSUT1 indicating that it interacts with the transporter while arbutin and alpha-methyl glucoside did not inhibit. The results demonstrate significant differences in substrate specificity between HvSUT1 and AtSUC2.     

Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.