Cu+-specific CopB transporter: Revising P1B-type ATPase classification.

The copper-transporting P1B-ATPases, which play a key role in cellular copper homeostasis, have been divided traditionally into two subfamilies, the P1B-1-ATPases or CopAs and the P1B-3-ATPases or CopBs. CopAs selectively export Cu+ whereas previous studies and bioinformatic analyses have suggested that CopBs are specific for Cu2+ export. Biochemical and spectroscopic characterization of Sphaerobacter thermophilus CopB (StCopB) show that, while it does bind Cu2+, the binding site is not the prototypical P1B-ATPase transmembrane site and does not involve sulfur coordination as proposed previously. Most important, StCopB exhibits metal-stimulated ATPase activity in response to Cu+, but not Cu2+, indicating that it is actually a Cu+ transporter. X-ray absorption spectroscopic studies indicate that Cu+ is coordinated by four sulfur ligands, likely derived from conserved cysteine and methionine residues. The histidine-rich N-terminal region of StCopB is required for maximal activity, but is inhibitory in the presence of divalent metal ions. Finally, reconsideration of the P1B-ATPase classification scheme suggests that the P1B-1- and P1B-3-ATPase subfamilies both comprise Cu+ transporters. These results are completely consistent with the known presence of only Cu+ within the reducing environment of the cytoplasm, which should eliminate the need for a Cu2+ P1B-ATPase.

Plant HKT Channels: An Updated View on Structure, Function and Gene Regulation.

HKT channels are a plant protein family involved in sodium (Na+) and potassium (K+) uptake and Na+-K+ homeostasis. Some HKTs underlie salt tolerance responses in plants, while others provide a mechanism to cope with short-term K+ shortage by allowing increased Na+ uptake under K+ starvation conditions. HKT channels present a functionally versatile family divided into two classes, mainly based on a sequence polymorphism found in the sequences underlying the selectivity filter of the first pore loop. Physiologically, most class I members function as sodium uniporters, and class II members as Na+/K+ symporters. Nevertheless, even within these two classes, there is a high functional diversity that, to date, cannot be explained at the molecular level. The high complexity is also reflected at the regulatory level. HKT expression is modulated at the level of transcription, translation, and functionality of the protein. Here, we summarize and discuss the structure and conservation of the HKT channel family from algae to angiosperms. We also outline the latest findings on gene expression and the regulation of HKT channels.

Comparative genomics revealed the gene evolution and functional divergence of magnesium transporter families in Saccharum.

BACKGROUND: Sugarcane served as the model plant for discovery of the C4 photosynthetic pathway. Magnesium is the central atom of chlorophyll, and thus is considered as a critical nutrient for plant development and photosynthesis. In plants, the magnesium transporter (MGT) family is composed of a number of membrane proteins, which play crucial roles in maintaining Mg homeostasis. However, to date there is no information available on the genomics of MGTs in sugarcane due to the complexity of the Saccharum genome. RESULTS: Here, we identified 10 MGTs from the Saccharum spontaneum genome. Phylogenetic analysis of MGTs suggested that the MGTs contained at least 5 last common ancestors before the origin of angiosperms. Gene structure analysis suggested that MGTs family of dicotyledon may be accompanied by intron loss and pseudoexon phenomena during evolution. The pairwise synonymous substitution rates corresponding to a divergence time ranged from 142.3 to 236.6 Mya, demonstrating that the MGTs are an ancient gene family in plants. Both the phylogeny and Ks analyses indicated that SsMGT1/SsMGT2 originated from the recent ρWGD, and SsMGT7/SsMGT8 originated from the recent σ WGD. These 4 recently duplicated genes were shown low expression levels and assumed to be functionally redundant. MGT6, MGT9 and MGT10 weredominant genes in the MGT family and werepredicted to be located inthe chloroplast. Of the 3 dominant MGTs, SsMGT6 expression levels were found to be induced in the light period, while SsMGT9 and SsMTG10 displayed high expression levels in the dark period. These results suggested that SsMGT6 may have a function complementary to SsMGT9 and SsMTG10 that follows thecircadian clock for MGT in the leaf tissues of S. spontaneum. MGT3, MGT7 and MGT10 had higher expression levels Insaccharum officinarum than in S. spontaneum, suggesting their functional divergence after the split of S. spontaneum and S. officinarum. CONCLUSIONS: This study of gene evolution and expression of MGTs in S. spontaneum provided basis for the comprehensive genomic study of the entire MGT genes family in Saccharum. The results are valuable for further functional analyses of MGT genes and utilization of the MGTs for Saccharum genetic improvement.

The maize CorA/MRS2/MGT-type Mg transporter, ZmMGT10, responses to magnesium deficiency and confers low magnesium tolerance in transgenic Arabidopsis.

KEY MESSAGE: ZmMGT10 was specifically expressed in maize roots and induced by a deficiency of magnesium. Overexpression of ZmMGT10 restored growth deficiency of the Salmonella typhimurium MM281 strain and enhanced the tolerance in Arabidopsis to stress induced by low magnesium levels by increasing uptake of Mg2+ via roots. CorA/MRS2/MGT-type Mg2+ transporters play a significant role in maintaining magnesium (Mg) homeostasis in plants. Although the maize CorA/MRS2/MGT family comprises of 12 members, currently no member has been functionally characterized. Here, we report the isolation and functional characterization of ZmMGT10 from the maize MRS2/MGT gene family. ZmMGT10 has a typical structure feature which includes two conserved TMs near the C-terminal end and an altered AMN tripeptide motif. The high sequence similarity and close phylogenetic relationship indicates that ZmMGT10 is probably the counterpart of Arabidopsis AtMGT6. The complementation of the Salmonella typhimurium mutated MM281 strain indicates that ZmMGT10 possesses the ability to transport Mg2+. ZmMGT10 was specifically expressed in the plant roots and it can be stimulated by a deficiency of Mg. Transgenic Arabidopsis plants which overexpressed ZmMGT10 grew more vigorously than wild-type plants under low Mg conditions, exhibited by longer root length, higher plant fresh weight and chlorophyll content, suggesting ZmMGT10 was essential for plant growth and development under low Mg conditions. Further investigations found that high accumulation of Mg2+ occurred in transgenic plants attributed to improved Mg2+ uptake and thereby enhanced tolerance to Mg deficiency. Results from this investigation illustrate that ZmMGT10 is a Mg transporter of maize which can enhance the tolerance to Mg deficient conditions by improving Mg2+ uptake in the transgenic plants of Arabidopsis.

Zinc transporter ZIP10 forms a heteromer with ZIP6 which regulates embryonic development and cell migration.

There is growing evidence that zinc and its transporters are involved in cell migration during development and in cancer. In the present study, we show that zinc transporter ZIP10 (SLC39A10) stimulates cell motility and proliferation, both in mammalian cells and in the zebrafish embryo. This is associated with inactivation of GSK (glycogen synthase kinase)-3α and -3ß and down-regulation of E-cadherin (CDH1). Morpholino-mediated knockdown of zip10 causes delayed epiboly and deformities of the head, eye, heart and tail. Furthermore, zip10 deficiency results in overexpression of cdh1, zip6 and stat3, the latter gene product driving transcription of both zip6 and zip10 The non-redundant requirement of Zip6 and Zip10 for epithelial to mesenchymal transition (EMT) is consistent with our finding that they exist as a heteromer. We postulate that a subset of ZIPs carrying prion protein (PrP)-like ectodomains, including ZIP6 and ZIP10, are integral to cellular pathways and plasticity programmes, such as EMT.

Current understanding of ZIP and ZnT zinc transporters in human health and diseases.

Zinc transporters, the Zrt-, Irt-like protein (ZIP) family and the Zn transporter (ZnT) family transporters, are found in all aspects of life. Increasing evidence has clarified the molecular mechanism, in which both transporters play critical roles in cellular and physiological functions via mobilizing zinc across the cellular membrane. In the last decade, mutations in ZIP and ZnT transporter genes have been shown to be implicated in a number of inherited human diseases. Moreover, dysregulation of expression and activity of both transporters has been suggested to be involved in the pathogenesis and progression of chronic diseases including cancer, immunological impairment, and neurodegenerative diseases, although comprehensive understanding is far from complete. The diverse phenotypes of diseases related to ZIP and ZnT transporters reflect the multifarious biological functions of both transporters. The present review summarizes the current understanding of ZIP and ZnT transporter functions from the standpoint of human health and diseases. The study of zinc transporters is currently of great clinical interest.

Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants.

BACKGROUND: Nitrogen uptake, reallocation within the plant, and between subcellular compartments involves ammonium, nitrate and peptide transporters. Ammonium transporters are separated into two distinct families (AMT1 and AMT2), each comprised of five members on average in angiosperms. Nitrate transporters also form two discrete families (NRT1 and NRT2), with angiosperms having four NRT2s, on average. NRT1s share an evolutionary history with peptide transporters (PTRs). The NRT1/PTR family in land plants usually has more than 50 members and contains also members with distinct activities, such as glucosinolate and abscisic acid transport. RESULTS: Phylogenetic reconstructions of each family across 20 land plant species with available genome sequences were supplemented with subcellular localization and transmembrane topology predictions. This revealed that both AMT families diverged prior to the separation of bryophytes and vascular plants forming two distinct clans, designated as supergroups, each. Ten supergroups were identified for the NRT1/PTR family. It is apparent that nitrate and peptide transport within the NRT1/PTR family is polyphyletic, that is, nitrate and/or peptide transport likely evolved multiple times within land plants. The NRT2 family separated into two distinct clans early in vascular plant evolution. Subsequent duplications occurring prior to the eudicot/monocot separation led to the existence of two AMT1, six AMT2, 31 NRT1/PTR, and two NRT2 clans, designated as groups. CONCLUSION: Phylogenetic separation of groups suggests functional divergence within the angiosperms for each family. Distinct groups within the NRT1/PTR family appear to separate peptide and nitrate transport activities as well as other activities contained within the family, for example nitrite transport. Conversely, distinct activities, such as abscisic acid and glucosinolate transport, appear to have recently evolved from nitrate transporters.

Expansion of the APC superfamily of secondary carriers.

The amino acid-polyamine-organoCation (APC) superfamily is the second largest superfamily of secondary carriers currently known. In this study, we establish homology between previously recognized APC superfamily members and proteins of seven new families. These families include the PAAP (Putative Amino Acid Permease), LIVCS (Branched Chain Amino Acid:Cation Symporter), NRAMP (Natural Resistance-Associated Macrophage Protein), CstA (Carbon starvation A protein), KUP (K⁺ Uptake Permease), BenE (Benzoate:H⁺ Virginia Symporter), and AE (Anion Exchanger). The topology of the well-characterized human Anion Exchanger 1 (AE1) conforms to a UraA-like topology of 14 TMSs (12 α-helical TMSs and 2 mixed coil/helical TMSs). All functionally characterized members of the APC superfamily use cation symport for substrate accumulation except for some members of the AE family which frequently use anion:anion exchange. We show how the different topologies fit into the framework of the common LeuT-like fold, defined earlier (Proteins. 2014 Feb;82(2):336-46), and determine that some of the new members contain previously undocumented topological variations. All new entries contain the two 5 or 7 TMS APC superfamily repeat units, sometimes with extra TMSs at the ends, the variations being greatest within the CstA family. New, functionally characterized members transport amino acids, peptides, and inorganic anions or cations. Except for anions, these are typical substrates of established APC superfamily members. Active site TMSs are rich in glycyl residues in variable but conserved constellations. This work expands the APC superfamily and our understanding of its topological variations.

Characterization of OsLCT1, a cadmium transporter from indica rice (Oryza sativa).

Molecular understanding of cadmium (Cd) transport in indica rice (Oryza sativa) is still insufficient, although indica rice generally has a potential to accumulate higher Cd in shoots and grains than japonica rice. We have previously demonstrated that OsLCT1 is a Cd transporter gene responsible for grain Cd accumulation in the japonica model cultivar Nipponbare. In this study, we isolated OsLCT1 cDNA from Kasalath, a model indica (aus subgroup) cultivar and conducted cation transport activity assays in yeast and mRNA expression analysis in plants. The deduced amino acid sequence of Kasalath OsLCT1 is 91.2% identical and 93.8% similar to that of Nipponbare OsLCT1. We established the yeast heterologous system expressing the Kasalath allele of OsLCT1. Elemental profiling of the yeast cells suggested an efflux activity of Kasalath OsLCT1 for Cd, K, Mg, Ca and Mn, but not for Fe, Zn, Cu and Na. This substrate specificity was identical to that of the Nipponbare version. Quantitative real time-polymerase chain reaction (RT-PCR) showed that expression of OsLCT1 in Kasalath was higher in reproductive stage than in vegetative stage. The expression level of OsLCT1 was significantly higher in Kasalath than in Nipponbare. Phylogenetic analysis found several LCT1-like genes only in grass plants. OsLCT1 is the sole copy in the rice genome and is conserved among each rice subgroup. These newly found low-affinity cation transporter (LCT) homologs will provide a basis for further understanding of LCT-mediated Cd transport.

Expression and functional analysis of the CorA-MRS2-ALR-type magnesium transporter family in rice.

Maintenance of an appropriate magnesium ion (Mg(2+)) concentration is essential for plant growth. In Arabidopsis thaliana, the CorA-MRS2-ALR-type proteins, named MRS2/MGT family proteins, are reportedly localized in various membranes and they function in Mg transport. However, knowledge of this family in other plant species is extremely limited. Furthermore, differential diversification among dicot and monocot plants suggested by phylogenetic analysis indicates that the role of the Arabidopsis MRS2/MGT family proteins is not the same in monocot plants. For a further understanding of this family in higher plants, functional analysis and gene expression profiling of rice MRS2/MGT family members were performed. A phylogenetic tree based on the isolated mRNA sequences of nine members of the OsMRS2 family confirmed that the MRS2/MGT family consists of five clades (A-E). A complementation assay in the yeast CM66 strain showed that four of the nine members possessed the Mg(2+) transport ability. Transient green fluorescent protein (GFP) expression in the isolated rice protoplast indicated that OsMRS2-5 and OsMRS2-6, belonging to clades D and A, respectively, localized in the chloroplast. Expression levels of these genes were low in the unexpanded yellow-green leaf, but increased considerably with leaf maturation. In addition, diurnal oscillation of expression was observed, particularly in OsMRS2-6 expression in the expanded leaf blade. We conclude that OsMRS2 family members function as Mg transporters and suggest that the genes belonging to clade A encode the chloroplast-localized Mg(2+) transporter in plants.

Knockouts of Physcomitrella patens CHX1 and CHX2 transporters reveal high complexity of potassium homeostasis.

This study aims to increase our understanding of the functions of CHX transporters in plant cells using the model plant Physcomitrella patens, in which four CHX genes have been identified, PpCHX1-PpCHX4. Two of these genes, PpCHX1 and PpCHX2, are expressed at approximately the same level as the PpACT5 gene, but the other two genes show an extremely low expression. PpCHX1 and PpCHX2 restored growth of Escherichia coli mutants on low K(+)-containing media, suggesting that they mediated K+ uptake that may be energized by symport with H+. In contrast, these genes suppressed the defect associated with the kha1 mutation in Saccharomyces cerevisiae, which suggests that they might mediate K+/H+ antiport. PpCHX1-green fluorescent protein (GFP) fusion protein transiently expressed in P. patens protoplasts co-localized with a Golgi marker. In similar experiments, the PpCHX2-GFP protein appeared to localize to tonoplast and plasma membrane. We constructed the ΔPpchx1 and ΔPpchx2 single mutant lines, and the ΔPpchx2 ΔPphak1 double mutant. Single mutant plants grew normally under all the conditions tested and exhibited normal K+ and Rb+ influxes; the ΔPpchx2 mutation did not increase the defect of ΔPphak1 plants. In long-term experiments, ΔPpchx2 plants showed slightly higher Rb+ retention than wild-type plants, which suggests that PpCHX2 mediates the transfer of Rb+ either from the vacuole to the cytosol or from the cytosol to the external medium in parallel with other transporters. The distinction between these two possibilities is technically difficult. We suggest that K+ transporters of several families are involved in the pH homeostasis of organelles by mediating either K+/H+ antiport or K(+)-H(+) symport.

Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.).

High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 µM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

Magnesium deficiency phenotypes upon multiple knockout of Arabidopsis thaliana MRS2 clade B genes can be ameliorated by concomitantly reduced calcium supply.

Plant MRS2 membrane protein family members have been shown to play important roles in magnesium uptake and homeostasis. Single and double knockouts for two Arabidopsis thaliana genes, AtMRS2-1 and AtMRS2-5, have previously not shown significant phenotypes even under limiting Mg(2+) supply although both are strongly expressed already in early seedlings. Together with AtMRS2-10, these genes form clade B of the AtMRS2 gene family. We now succeeded in obtaining homozygous AtMRS2-1/10 double and AtMRS2-1/5/10 triple knockout lines after selection under increased magnesium supply. Although wilting early, both new mutant lines develop fully and are also fertile under standard magnesium supply, but show severe developmental retardation under limiting Mg(2+) concentrations. To investigate nutrient dependency of germination and seedling development under various conditions, including variable supplies of Mg(2+), Ca(2+), Zn(2+), Mn(2+), Co(2+), Cd(2+) and Cu(2+), in a reproducible and economical way, we employed a small-scale liquid culturing system in 24-well plate set-ups. This allowed the growth and monitoring of individual plantlets of different mutant lines under several nutritional conditions in parallel, and the scoring and statistical evaluation of developmental stages and biomass accumulation. Detrimental effects of higher concentrations of these elements were similar in mutants and the wild type. However, growth retardation phenotypes seen upon hydroponic cultivation under low Mg(2+) could be ameliorated when Ca(2+) concentrations were concomitantly lowered, supporting indications for an important interplay of these two most abundant divalent cations in the nutrient homeostasis of plants.

HAK transporters from Physcomitrella patens and Yarrowia lipolytica mediate sodium uptake.

The widespread presence of Na(+)-specific uptake systems across plants and fungi is a controversial topic. In this study, we identify two HAK genes, one in the moss Physcomitrella patens and the other in the yeast Yarrowia lipolytica, that encode Na(+)-specific transporters. Because HAK genes are numerous in plants and are duplicated in many fungi, our findings suggest that some HAK genes encode Na(+) transporters and that Na(+) might play physiological roles in plants and fungi more extensively than is currently thought.

Nramp: from sequence to structure and mechanism of divalent metal import.

Mn and Fe are important for energy metabolism and oxidative stress resistance and cells maintain adequate stores for survival and prevention of toxicity. Membrane permeases of the natural resistance-associated macrophage protein (Nramp) family importing protons and divalent metals are conserved from bacteria to man. Nramp hydrophobic core relates structurally to a superfamily of cation-driven carriers with inverted symmetry. Molecular phylogeny and sequence features support Nramp pseudo-symmetric three-dimensional (3D) model, and remote ancestry to the LeuT superfamily. Genetic analyses suggest conservation of Nramp sequence marks the transition from a phylogenetic out-group and may relate to divalent metal selectivity. Three phylogroups of bacterial proton-dependent manganese transporters (MntH) demonstrate specific patterns of sequence conservation suggesting functional constraints linked to ecological or taxonomical distributions, which may contribute to bacterial virulence. Nramp 3D model is supported experimentally by transmembrane topology and structure-function studies of Escherichia coli and mouse homologs as well as peptide structure analyses. Eukaryotic Nramps are required for Mn and Fe homeostasis, contributing in multicellular organisms to subcellular and systemic metal traffic and intercellular signaling. Nramps are subjected to elaborate regulation including developmental control of gene expression, protein subcellular targeting, dynamic metallo-dependent control of messenger RNA and protein stability and trafficking. Several human pathologies may result from defects in Nramp-dependent Fe(2+) or Mn(2+) transport, including iron overload, neurodegenerative diseases and innate susceptibility to infectious diseases.

Differential sodium and potassium transport selectivities of the rice OsHKT2;1 and OsHKT2;2 transporters in plant cells.

Na(+) and K(+) homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na(+) transport or Na(+) and K(+) transport when expressed in Xenopus laevis oocytes and yeast. However, the Na(+)/K(+) selectivities of the K(+)-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5' untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K(+) permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na(+) uptake, but little Rb(+) uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na(+)-K(+) cotransport in plant cells such that extracellular K(+) stimulated OsHKT2;2-mediated Na(+) influx and vice versa. Furthermore, at millimolar Na(+) concentrations, OsHKT2;2 mediated Na(+) influx into plant cells without adding extracellular K(+). This study shows that the Na(+)/K(+) selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K(+) and Ca(2+) down-regulated OsHKT2;1-mediated Na(+) influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function.

Cu(+)-ATPases play a key role in bacterial Cu(+) homeostasis by participating in Cu(+) detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P(1B-1) type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu(+) with high affinity in a trigonal planar geometry. The cytoplasmic Cu(+) chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu(+) is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu(+)-ATPases drive cytoplasmic Cu(+) efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu(+)-efflux pumps responsible for Cu(+) tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.

A Trk/HKT-type K+ transporter from Trypanosoma brucei.

The molecular mechanisms of K(+) homeostasis are only poorly understood for protozoan parasites. Trypanosoma brucei subsp. parasites, the causative agents of human sleeping sickness and nagana, are strictly extracellular and need to actively concentrate K(+) from their hosts' body fluids. The T. brucei genome contains two putative K(+) channel genes, yet the trypanosomes are insensitive to K(+) antagonists and K(+) channel-blocking agents, and they do not spontaneously depolarize in response to high extracellular K(+) concentrations. However, the trypanosomes are extremely sensitive to K(+) ionophores such as valinomycin. Surprisingly, T. brucei possesses a member of the Trk/HKT superfamily of monovalent cation permeases which so far had only been known from bacteria, archaea, fungi, and plants. The protein was named TbHKT1 and functions as a Na(+)-independent K(+) transporter when expressed in Escherichia coli, Saccharomyces cerevisiae, or Xenopus laevis oocytes. In trypanosomes, TbHKT1 is expressed in both the mammalian bloodstream stage and the Tsetse fly midgut stage; however, RNA interference (RNAi)-mediated silencing of TbHKT1 expression did not produce a growth phenotype in either stage. The presence of HKT genes in trypanosomatids adds a further piece to the enigmatic phylogeny of the Trk/HKT superfamily of K(+) transporters. Parsimonial analysis suggests that the transporters were present in the first eukaryotes but subsequently lost in several of the major eukaryotic lineages, in at least four independent events.

Developmental expression of sideroflexin family genes in Xenopus embryos.

Emerging evidence indicates that mitochondrial carriers are not only crucial for metabolism, but also important for embryonic development. Sideroflexin is a novel family of mitochondrial tricarboxylate carrier proteins, of which the in vivo function is largely unknown. Here, we report on the expression patterns of five sideroflexin genes in Xenopus embryos. Whole-mount in situ hybridization analysis reveals that while sideroflexin2 is expressed in the pancreas, sideroflexin1 and 3 display a complex expression in the central nervous system, somites, pronephros, liver, and pancreas. In contrast, only a weak expression of sideroflexin4 and 5 was detected in embryonic brain. Taken together, the five sideroflexin genes show both overlapping and nonoverlapping expression during Xenopus embryogenesis. As the primary structures of the five sideroflexin proteins are also quite similar, their functional redundancy should be taken into consideration for gene targeting studies.

Divalent metal transporter 1 is involved in amyloid precursor protein processing and Abeta generation.

The amyloid-beta precursor protein (APP) and its pathogenic byproduct beta-amyloid peptide (Abeta) play central roles in the pathogenesis of Alzheimer's disease (AD). Reduction in levels of the potentially toxic Abeta is one of the most important therapeutic goals in AD. Recent studies have shown that bivalent metals such as iron, copper, and zinc are involved in APP expression, Abeta deposition, and senile plaque formation in the AD brain. However, the underlying mechanisms involved in abnormal homeostasis of bivalent metals in AD brain remain unclear. In the present study, we found that two isoforms of the divalent metal transporter 1 (DMT1), DMT1-IRE, and DMT1-nonIRE, were colocalized with Abeta in the plaques of postmortem AD brain. Using the APP/PS1 transgenic mouse model, we found that the levels of both DMT1-IRE and DMT1-nonIRE were significantly increased in the cortex and hippocampus compared with wild type-control. We further verified the proposed mechanisms by which DMT1 might be involved in APP processing and Abeta secretion by using the SH-SY5Y cell line stably overexpressing human APP Swedish mutation (APPsw) as a cell model. We found that overexpression of APPsw resulted in increased expression levels of both DMT1-IRE and DMT1-nonIRE in SH-SY5Y cells. Interestingly, silencing of endogenous DMT1 by RNA interference, which reduced bivalent ion influx, led to reductions of APP expression and Abeta secretion. These findings suggest both that DMT1 plays a critical role in ion-mediated neuropathogenesis in AD and that pharmacological blockage of DMT1 may provide novel therapeutic strategies against AD.