Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe³⁺-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe³⁺-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe³⁺ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe³⁺ or Fe²⁺ forms or Fe³⁺ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.     

The CorA Mg2+ transporter does not transport Fe2+

corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea. Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity. Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+. Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells. As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+. Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele. Although excess Fe2+ was slightly toxic to S. enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain. We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect.     

The copper transporter (Ctr) family of Cu+ uptake systems

Copper (Cu(+)) transporters of the Ctr family are sequence diverse eukaryotic proteins that function by an unknown mechanism of action. We have conducted bioinformatic analyses of sequenced Ctr proteins. Multiple paralogues are found in single organisms, and these may be either closely or distantly related to each other. Protein phylogeny generally correlates with organismal source and protein size with proteins of each cluster being derived from a specific eukaryotic kingdom and exhibiting characteristic domain arrangements. Some homologues exhibit repeats of the basic 3 TMS unit. Regions of conserved hydrophobicity and amphipathicity suggest functional roles, particularly for putative TMSs 2 and 3 which possess a nearly fully conserved M X(3) M motif in putative TMS2. We propose that this motif comprises the transmembrane Cu(+)-binding site in oligomeric channels that take up Cu(+) by a passive, membrane potential-dependent mechanism.     

The drug/metabolite transporter superfamily.

Previous work defined several families of secondary active transporters, including the prokaryotic small multidrug resistance (SMR) and rhamnose transporter (RhaT) families as well as the eukaryotic organellar triose phosphate transporter (TPT) and nucleotide-sugar transporter (NST) families. We show that these families as well as several other previously unrecognized families of established or putative secondary active transporters comprise a large ubiquitous superfamily found in bacteria, archaea and eukaryotes. We have designated it the drug/metabolite transporter (DMT) superfamily (transporter classification number 2.A.7) and have shown that it consists of 14 phylogenetic families, five of which include no functionally well-characterized members. The largest family in the DMT superfamily, the drug/metabolite exporter (DME) family, consists of over 100 sequenced members, several of which have been implicated in metabolite export. Each DMT family consists of proteins with a distinctive topology: four, five, nine or 10 putative transmembrane alpha helical spanners (TMSs) per polypeptide chain. The five TMS proteins include an N-terminal TMS lacking the four TMS proteins. The full-length proteins of 10 putative TMSs apparently arose by intragenic duplication of an element encoding a primordial five-TMS polypeptide. Sequenced members of the 14 families are tabulated and phylogenetic trees for all the families are presented. Sequence and topological analyses allow structural and functional predictions.     

The ion transporter superfamily

We define a novel superfamily of secondary carriers specific for cationic and anionic compounds, which we have termed the ion transporter (IT) superfamily. Twelve recognized and functionally defined families constitute this superfamily. We provide statistical sequence analyses demonstrating that these families were in fact derived from a common ancestor. Further, we characterize the 12 families in terms of (1) the known substrates transported, (2) the modes of transport and energy coupling mechanisms used, (3) the family sizes (in numbers of sequenced protein members in the current NCBI database), (4) the organismal distributions of the members of each family, (5) the size ranges of the constituent proteins, (6) the predicted topologies of these proteins, and (7) the occurrence of non-homologous auxiliary proteins that may either facilitate or be required for transport. No member of the superfamily is known to function in a capacity other than transport. Proteins in several of the constituent families are shown to have arisen by tandem intragenic duplication events, but topological variation has resulted from a variety of dissimilar genetic fusion, splicing and insertional events. The evolutionary relationships between the members of each family are defined, leading to predictions of functionally relevant orthologous relationships. Some but not all of the families include functionally dissimilar paralogues that arose by early extragenic duplication events.     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

Regulation of iron uptake in Saccharomyces cerevisiae. The ferrireductase and Fe(II) transporter are regulated independently.

Iron is required for the growth of Saccharomyces cerevisiae. High concentrations of iron, however, are toxic, forcing this yeast to tightly regulate its concentration of intracellular free iron. We demonstrate that S. cerevisiae accumulates iron through the combined action of a plasma membrane ferrireductase and an Fe(II) transporter. This transporter is highly selective for Fe(II). Several other transition metals did not inhibit iron uptake when these metals were present at a concentration 100-fold higher than the Km (0.15 microM) for iron transport. Pt(II) inhibited ferrireductase activity but not the ability of cells to transport iron that was chemically reduced to Fe(II). Incubation of cells in a synthetic iron-limited media resulted in the induction of both ferrireductase and Fe(II) transporter activities. In complex media, Fe(II) transport activity was regulated in response to media iron concentration, while the activity of the ferrireductase was not. When stationary phase cells were inoculated into fresh media, ferrireductase activity increased independent of the iron content of the media; in contrast, transporter activity varied inversely with iron levels. These results demonstrate that the ferrireductase and Fe(II) transporter are separately regulated and that iron accumulation may be limited by changes in either activity.     

Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+

Only graminaceous monocots possess the Strategy II iron (Fe)-uptake system in which Fe is absorbed by roots as an Fe3+-phytosiderophore. In spite of being a Strategy II plant, however, rice (Oryza sativa) contains the previously identified Fe2+ transporter OsIRT1. In this study, we isolated the OsIRT2 gene from rice, which is highly homologous to OsIRT1. Real-time PCR analysis revealed that OsIRT1 and OsIRT2 are expressed predominantly in roots, and these transporters are induced by low-Fe conditions. When expressed in yeast (Saccharomyces cerevisiae) cells, OsIRT2 cDNA reversed the growth defects of a yeast Fe-uptake mutant. This was similar to the effect of OsIRT1 cDNA. OsIRT1- and OsIRT2-green fluorescent protein fusion proteins localized to the plasma membrane when transiently expressed in onion (Allium cepa L.) epidermal cells. OsIRT1 promoter-GUS analysis revealed that OsIRT1 is expressed in the epidermis and exodermis of the elongating zone and in the inner layer of the cortex of the mature zone of Fe-deficient roots. OsIRT1 expression was also detected in the ccompanion cells. Analysis using the positron-emitting tracer imaging system showed that rice plants are able to take up both an Fe3+-phytosiderophore and Fe2+. This result indicates that, in addition to absorbing an Fe3+-phytosiderophore, rice possesses a novel Fe-uptake system that directly absorbs the Fe2+, a strategy that is advantageous for growth in submerged conditions.     

The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily

The malaria parasite's chloroquine resistance transporter (CRT) is an integral membrane protein localized to the parasite's acidic digestive vacuole. The function of CRT is not known and the protein was originally described as a transporter simply because it possesses 10 transmembrane domains. In wild-type (chloroquine-sensitive) parasites, chloroquine accumulates to high concentrations within the digestive vacuole and it is through interactions in this compartment that it exerts its antimalarial effect. Mutations in CRT can cause a decreased intravacuolar concentration of chloroquine and thereby confer chloroquine resistance. However, the mechanism by which they do so is not understood. In this paper we present the results of a detailed bioinformatic analysis that reveals that CRT is a member of a previously undefined family of proteins, falling within the drug/metabolite transporter superfamily. Comparisons between CRT and other members of the superfamily provide insight into the possible role of the protein and into the significance of the mutations associated with the chloroquine resistance phenotype. The protein is predicted to function as a dimer and to be oriented with its termini in the parasite cytosol. The key chloroquine-resistance-conferring mutation (K76T) is localized in a region of the protein implicated in substrate selectivity. The mutation is predicted to alter the selectivity of the protein such that it is able to transport the cationic (protonated) form of chloroquine down its steep concentration gradient, out of the acidic vacuole, and therefore away from its site of action.     

A tobacco plasma membrane calmodulin-binding transporter confers Ni2+ tolerance and Pb2+ hypersensitivity in transgenic plants

All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin-binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K+ and to non-selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni2+ and hypersensitivity to Pb2+, which are associated with reduced Ni2+ accumulation and enhanced Pb2+ accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb2+. We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.     

Effect of Ca2+ and Mg2+ on the uptake of Fe3+ by mouse intestinal mucosa

Initial Fe3+ uptake rates by mouse intestinal fragments were determined in vitro. Uptake was dependent primarily on the Fe3+-nitrilotriacetate complex concentration. Addition of Ca2+ and Mg2+ to the incubation medium had only small effects on the Fe3+ uptake rate. Duodenal fragments from hypoxic animals showed enhanced uptake of Fe3+; this increase was more pronounced with a divalent cation-free medium. Ca2+ markedly diminished the Fe3+ uptake by mucosa from hypoxic mice; Mg2+ had no appreciable effect. Distal ileal fragments exhibited lower uptake rates compared to the duodenum, but were more sensitive to the effects of added Ca2+. The ileal fragments did not show an adaptive response of Fe3+ uptake to hypoxia. These results suggest the existence of more than one pathway for mucosal Fe3+ uptake. One pathway, sensitive to Ca2+ and not stimulated by hypoxia, may be present in the duodenum and ileum. A second pathway, inhibited by Ca2+ and exhibiting an adaptive response to hypoxia, occurs only in the duodenum. This latter pathway is more sensitive to the effects of metabolic inhibitors.     

Effect of Fe2+, Mn2+, Zn2+ and Pb2+ on H+/K+ fluxes in excisedPistia stratiotes roots

Pistia stratiotes is used for the epuration of domestic sewage in the Biyem Assi phytopurification station. During the process, Fe²⁺, Mn²⁺, Zn²⁺ and Pb²⁺ are absorbed in substantial amounts by the plant. These metals modify the H⁺/K⁺ exchange system at the root level. H⁺ efflux is inhibited by Fe²⁺ and by Zn²⁺ and enhanced by Mn²⁺ and Pb²⁺. K⁺ influx is inhibited by Fe²⁺, by Zn²⁺ and by Pb²⁺ and enhanced by Mn²⁺. It is shown that the purification capacity ofPistia stratiotes can vary with the composition of the heavy metals in the surrounding medium.     

Cholate-soluble and -insoluble iron binding components of rabbit duodenal brush-border membrane. Relevance to Fe2+ uptake by brush-border membrane vesicles

Fe2+ uptake by brush-border membrane vesicles from rabbit duodenum has been investigated and found to show similar qualitative properties to those previously demonstrated with mouse proximal intestine brush-border membrane vesicles (Simpson, R.J. and Peters, T.J. (1986) Biochim. Biophys. Acta 856, 109-114). In particular, a relatively low affinity (Km(app) approx. 83 microM), NaCl and pH sensitive transport component is present. The disruption of 59Fe2+-laden vesicles with sodium cholate, followed by gel filtration or centrifugal analysis reveals that cholate insoluble material (Mr greater than 10(6)) is the major destination for 59Fe2+ taken up by intact vesicles. Analysis of cholate extracts for Fe2+ binding ability reveals a single high-capacity (49.8 +/- 15.6 nmol/mg vesicle protein (S.E., n = 3)), high-affinity (Kd(app) less than 5 microM) binding component with an Mr equivalent to approx. 10(4) on gel filtration in the presence of cholate. This binding component is extracted into chloroform/methanol (2:1, v/v) is relatively heat and protease resistant and thus appears to be a lipid.     

The Mg2+ transporter MagT1 partially rescues cell growth and Mg2+ uptake in cells lacking the channel-kinase TRPM7

Magnesium (Mg(2+)) transport across membranes plays an essential role in cellular growth and survival. TRPM7 is the unique fusion of a Mg(2+) permeable pore with an active cytosolic kinase domain, and is considered a master regulator of cellular Mg(2+) homeostasis. We previously found that the genetic deletion of TRPM7 in DT40 B cells results in Mg(2+) deficiency and severe growth impairment, which can be rescued by supplementation with excess extracellular Mg(2+). Here, we show that gene expression of the Mg(2+) selective transporter MagT1 is upregulated in TRPM7(-/-) cells. Furthermore, overexpression of MagT1 in TRPM7(-/-) cells augments their capacity to uptake Mg(2+), and improves their growth behavior in the absence of excess Mg(2+).     

Mouse intestinal Fe3+ uptake kinetics in vivo. The significance of brush-border membrane vesicle transport in the mechanism of mucosal Fe3+ uptake

Initial rates of mucosal uptake of Fe3+ from luminal Fe3+-nitrilotriacetate solutions by tied segments of mouse intestine in vivo have been measured. Duodenal uptake showed an approximately hyperbolic dependence of uptake on Fe3+ complex concentration (Km(app) 66 microM, Vmax 6.2 pmol/min per mg intestine) with little dependence on nitrilotriacetate:Fe3+ ratio or on added Ca2+. Duodenal uptake was greatly stimulated by hypoxic treatment of mice. Uptake rates by distal ileum were lower than by duodenum and more sensitive to added Ca2+. These results show that isolated duodenal brush-border membrane Fe3+ transport characteristics (Simpson, R.J. and Peters, T.J. (1984) Biochim. Biophys. Acta 772, 220-226) are inadequate to explain duodenal Fe3+ uptake in vivo. However, ileal uptake can be explained by the properties of isolated ileal brush-border membrane (Simpson, R.J., Raja, K.B. and Peters, T.J. (1985) Biochim. Biophys. Acta 814, 8-12).     

拟南芥K+转运蛋白AtKup1基因的DNA改组

采用同源重组法制备钾离子转运蛋白TRK1和TRK2缺失的酿酒酵母钾营养缺陷型,通过RNA 反转录PCR方法从拟南芥幼根扩增获得片段长度为2139bp 的Atkup1基因,以此片段为模板,采用DNA 改组技术,经Dnase I降解,Primerless PCR , PrimerPCR,建立Atkup1 基因突变库。将突变库和未经DNA 重排处理的Atkup1基因分别构建酵母穿梭载体导入K+转运蛋白基因TRK1和TRK2缺失的酿酒酵母中,分别在低钾(5.0mM KCl)不含色氨酸的培养基上筛选转化子, 突变基因库酵母转化子中获得2株长势明显好于Atkup1 基因转化子的突变基因转化菌株,菌株质粒上的突变Atkup1基因核苷酸测序结果发现突变基因Atkup1发生2个碱基的置换,造成2个氨基酸的改变,转化烟草烟叶化学成分分析证实突变基因的吸钾活性显著提高。     

Zn、Cd及其复合对小麦幼苗吸收Ca、Fe、Mn的影响

研究了溶液培养条件下Cd、Zn及其复合对小麦幼苗吸收Ca、Fe、Mn的影响.结果表明,小麦幼苗对Zn、Cd的吸收随溶液中Cd²⁺、Zn²⁺浓度的升高而增加,Cd、Zn同时存在时与其单独作用时幼苗对它们的吸收不同,Zn影响幼苗对Cd的吸收,Cd对Zn的吸收起抑制作用.Ca、Mn的吸收随溶液中Cd²⁺、Zn²⁺浓度升高而呈下降趋势,在Cd单独处理组和Zn单独处理组中Fe的吸收随Cd²⁺、Zn²⁺浓度升高而增加,但在Zn+Cd处理组中,Fe的吸收则呈下降趋势,其效应方式还与作物具体部位有关.