Role of iron in Nramp1-mediated inhibition of mycobacterial growth.
Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth. (+info)
Characterisation of copper-binding to the second sub-domain of the Menkes protein ATPase (MNKr2).
The Menkes ATPase (MNK) has an essential role in the translocation of copper across cellular membranes. In a complementary manner, the intracellular concentration of copper regulates the activity and cellular location of the ATPase through its six homologous amino-terminal domains. The roles of the six amino-terminal domains in the activation and cellular trafficking processes are unknown. Understanding the role of these domains relies on the development of an understanding of their metal-binding properties and structural properties. The second conserved sub-domain of MNK was over-expressed, purified and its copper-binding properties characterised. Reconstitution studies demonstrate that copper binds to MNKr2 as Cu(I) with a stoichiometry of one copper per domain. This is the first direct evidence of copper-binding to the MNK amino-terminal repeats. Circular dichroism studies suggest that the binding or loss of copper to MNKr2 does not cause substantial changes to the secondary structure of the protein. (+info)
The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.
The natural resistance associated macrophage protein (Nramp) gene family is composed of two members in mammals, Nramp1 and Nramp2. Nramp1 is expressed primarily in macrophages and mutations at this locus cause susceptibility to infectious diseases. Nramp2 has a much broader range of tissue expression and mutations at Nramp2 result in iron deficiency, indicating a role for Nramp2 in iron metabolism. To get further insight into the function and mechanism of action of Nramp proteins, we have generated isoform specific anti-Nramp1 and anti-Nramp2 antisera. Immunoblotting experiments indicate that Nramp2 is present in a number of cell types, including hemopoietic precursors, and is coexpressed with Nramp1 in primary macrophages and macrophage cell lines. Nramp2 is expressed as a 90-100-kD integral membrane protein extensively modified by glycosylation (>40% of molecular mass). Subcellular localization studies by immunofluorescence and confocal microscopy indicate distinct and nonoverlapping localization for Nramp1 and Nramp2. Nramp1 is expressed in the lysosomal compartment, whereas Nramp2 is not detectable in the lysosomes but is expressed primarily in recycling endosomes and also, to a lower extent, at the plasma membrane, colocalizing with transferrin. These findings suggest that Nramp2 plays a key role in the metabolism of transferrin-bound iron by transporting free Fe2+ across the endosomal membrane and into the cytoplasm. (+info)
Genetic selection of mutations in the high affinity K+ transporter HKT1 that define functions of a loop site for reduced Na+ permeability and increased Na+ tolerance.
Potassium is an important macronutrient required for plant growth, whereas sodium (Na+) can be toxic at high concentrations. The wheat K+ uptake transporter HKT1 has been shown to function in yeast and oocytes as a high affinity K+-Na+ cotransporter, and as a low affinity Na+ transporter at high external Na+. A previous study showed that point mutations in HKT1, which confer enhancement of Na+ tolerance to yeast, can be isolated by genetic selection. Here we report on the isolation of mutations in new domains of HKT1 showing further large increases in Na+ tolerance. By selection in a Na+ ATPase deletion mutant of yeast that shows a high Na+ sensitivity, new HKT1 mutants at positions Gln-270 and Asn-365 were isolated. Several independent mutations were isolated at the Asn-365 site. N365S dramatically increased Na+ tolerance in yeast compared with all other HKT1 mutants. Cation uptake experiments in yeast and biophysical characterization in Xenopus oocytes showed that the mechanisms underlying the Na+ tolerance conferred by the N365S mutant were: reduced inhibition of high affinity Rb+ (K+) uptake at high Na+ concentrations, reduced low affinity Na+ uptake, and reduced Na+ to K+ content ratios in yeast. In addition, the N365S mutant could be clearly distinguished from less Na+-tolerant HKT1 mutants by a markedly decreased relative permeability for Na+ at high Na+ concentrations. The new mutations contribute to the identification of new functional domains and an amino acid in a loop domain that is involved in cation specificity of a plant high affinity K+ transporter and will be valuable for molecular analyses of Na+ transport mechanisms and stress in plants. (+info)
Overexpression of a novel Arabidopsis gene related to putative zinc-transporter genes from animals can lead to enhanced zinc resistance and accumulation.
We describe the isolation of an Arabidopsis gene that is closely related to the animal ZnT genes (Zn transporter). The protein encoded by the ZAT (Zn transporter of Arabidopsis thaliana) gene has 398 amino acid residues and is predicted to have six membrane-spanning domains. To obtain evidence for the postulated function of the Arabidopsis gene, transgenic plants with the ZAT coding sequence under control of the cauliflower mosaic virus 35S promoter were analyzed. Plants obtained with ZAT in the sense orientation exhibited enhanced Zn resistance and strongly increased Zn content in the roots under high Zn exposure. Antisense mRNA-producing plants were viable, with a wild-type level of Zn resistance and content, like plants expressing a truncated coding sequence lacking the C-terminal cytoplasmic domain of the protein. The availability of ZAT can lead to a better understanding of the mechanism of Zn homeostasis and resistance in plants. (+info)
Ion channels: structure of a molecular brake.
A combination of crystallographic and mutagenesis studies on the HERG K+ channel, a key determinant of cardiac excitability, has suggested how the protein's extramembraneous amino-terminal domain might act as a 'molecular brake' that slows down channel deactivation. (+info)
Cloning and characterization of the transport modifier RS1 from rabbit which was previously assumed to be specific for Na+-D-glucose cotransport.
Previously we cloned membrane associated polypeptides from pig and man (pRS1, hRS1) which altered rate and glucose dependence of Na+-d-glucose cotransport expressed by SGLT1 from rabbit and man. This paper describes the cloning of a related cDNA sequence from rabbit intestine (rbRS1) which encodes a gene product with about 65% amino acid identity to pRS1 and hRS1. Hybridization of endonuclease-restricted genomic DNA with cDNA fragments of rbRS1 showed that there is only one gene with similarity to rbRS1 in rabbit, and genomic PCR amplifications revealed that the rbRS1 gene is intronless. Comparing the transcription of rbRS1 and rbSGLT1 in various tissues and cell types, different mRNA patterns were obtained for both genes. In Xenopus oocytes the Vmax of expressed Na+-d-glucose cotransport was increased or decreased when rbRS1 was coexpressed with rbSGLT1 or hSGLT1, respectively. After coexpression with hSGLT1 the glucose dependence of the expressed transport was changed. By coexpression of rbRS1 with the human organic cation transporter hOCT2 the expressed cation uptake was not altered; however, the expressed cation uptake was drastically decreased when hRS1 was coexpressed with hOCT2. The data show that RS1 can modulate the function of transporters with non-homologous primary structures. (+info)
Mechanism of increased iron absorption in murine model of hereditary hemochromatosis: increased duodenal expression of the iron transporter DMT1.
Hereditary hemochromatosis (HH) is a common autosomal recessive disorder characterized by tissue iron deposition secondary to excessive dietary iron absorption. We recently reported that HFE, the protein defective in HH, was physically associated with the transferrin receptor (TfR) in duodenal crypt cells and proposed that mutations in HFE attenuate the uptake of transferrin-bound iron from plasma by duodenal crypt cells, leading to up-regulation of transporters for dietary iron. Here, we tested the hypothesis that HFE-/- mice have increased duodenal expression of the divalent metal transporter (DMT1). By 4 weeks of age, the HFE-/- mice demonstrated iron loading when compared with HFE+/+ littermates, with elevated transferrin saturations (68.4% vs. 49.8%) and elevated liver iron concentrations (985 micrograms vs. 381 micrograms). By using Northern blot analyses, we quantitated duodenal expression of both classes of DMT1 transcripts: one containing an iron responsive element (IRE), called DMT1(IRE), and one containing no IRE, called DMT1(non-IRE). The positive control for DMT1 up-regulation was a murine model of dietary iron deficiency that demonstrated greatly increased levels of duodenal DMT1(IRE) mRNA. HFE-/- mice also demonstrated an increase in duodenal DMT1(IRE) mRNA (average 7.7-fold), despite their elevated transferrin saturation and hepatic iron content. Duodenal expression of DMT1(non-IRE) was not increased, nor was hepatic expression of DMT1 increased. These data support the model for HH in which HFE mutations lead to inappropriately low crypt cell iron, with resultant stabilization of DMT1(IRE) mRNA, up-regulation of DMT1, and increased absorption of dietary iron. (+info)