Distinct targeting and recycling properties of two isoforms of the iron transporter DMT1 (NRAMP2, Slc11A2)

The metal transporter DMT1 (Slc11a2) plays a vital role in iron metabolism. Alternative splicing of the 3' exon generates two DMT1 isoforms with different C-terminal protein sequences and a 3' untranslated region harboring (isoform I, +IRE) or not (isoform II, -IRE), an iron-responsive element. Isoform I is expressed at the plasma membrane of certain epithelial cells including the duodenum brush border, where it is essential for the absorption of nutritional iron. Isoform II is expressed in many cells and is essential for the acquisiton of transferrin iron from acidified endosomes. The targeting and trafficking properties of DMT1 isoforms I and II were studied in transfected LLC-PK(1) kidney cells, with respect to isoform-specific differences in function, subcellular localization, endocytosis kinetics, and fate upon internalization. Isoform I showed higher surface expression and was internalized from the plasma membrane with slower kinetics than that of isoform II. As opposed to isoform II, which is efficiently sorted to recycling endosomes upon internalization, isoform I was not efficiently recycled and was targeted to lysosomes. Thus, alternative splicing of DMT1 critically regulates the subcellular localization and site of Fe(2+) transport.     

Carboxyl-terminus determinants of the iron transporter DMT1/SLC11A2 isoform II (-IRE/1B) mediate internalization from the plasma membrane into recycling endosomes

Mutations in DMT1 (Nramp2 and Slc11a2) impair iron metabolism and cause microcytic anemia. DMT1 is expressed at the duodenal brush border where it controls uptake of dietary iron and is present at the plasma membrane and in recycling endosomes of most cells, where it is necessary for acquisition of transferrin-associated iron. The goal of this study was to identify signal(s) in the cytoplasmic segments of DMT1 responsible for its subcellular targeting and internalization from the plasma membrane into recycling endosomes. We introduced mutations in the amino terminus (DeltaNT), carboxyl terminus (DeltaCT), as well as in NPAY28-31, YSCF62-65, and YLLNT555-559 motifs of a DMT1 construct bearing an exofacial epitope tag, which allowed labeling of the transporter at the cell surface for kinetic studies. Mutants were stably expressed in LLC-PK1 kidney cells and were studied for transport activity, subcellular localization, cell-surface and recycling pool distribution, and internalization from the plasma membrane. Kinetic studies showed that carboxyl-terminus mutants (DeltaCT and DeltaYLLNT) had an increased fraction of the "recycling pool" that was expressed at the cell surface because of impaired internalization from the plasma membrane. Further cell-surface-labeling and immunofluorescence studies in intact cells showed that the DeltaYLLNT and DeltaCT mutants were targeted to the lysosomal compartment upon internalization. These results suggest that the major signal for internalization and recycling of DMT1 isoform II (-IRE/1B) resides in its carboxyl terminus and that removal of this signal leads to a default lysosomal targeting.     

Differential localization of syntaxin isoforms in polarized Madin-Darby canine kidney cells.

Syntaxins, integral membrane proteins that are part of the ubiquitous membrane fusion machinery, are thought to act as target membrane receptors during the process of vesicle docking and fusion. Several isoforms of the syntaxin family have been previously identified in mammalian cells, some of which are localized to the plasma membrane. We investigated the subcellular localization of these putative plasma membrane syntaxins in polarized epithelial cells, which are characterized by the presence of distinct apical and basolateral plasma membrane domains. Syntaxins 2, 3, and 4 were found to be endogenously present in Madin-Darby canine kidney cells. The localization of syntaxins 1A, 1B, 2, 3, and 4 in stably transfected Madin-Darby canine kidney cell lines was studied with confocal immunofluorescence microscopy. Each syntaxin isoform was found to have a unique pattern of localization. Syntaxins 1A and 1B were present only in intracellular structures, with little or no apparent plasma membrane staining. In contrast, syntaxin 2 was found on both the apical and basolateral surface, whereas the plasma membrane localization of syntaxins 3 and 4 were restricted to the apical or basolateral domains, respectively. Syntaxins are therefore the first known components of the plasma membrane fusion machinery that are differentially localized in polarized cells, suggesting that they may play a central role in targeting specificity.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron's damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

The transcytosis of divalent metal transporter 1 and apo-transferrin during iron uptake in intestinal epithelium

Caco-2 cells grown in bicameral chambers are a model system to study intestinal iron absorption. Caco-2 cells exhibit constitutive transport of iron from the apical (luminal) chamber to the basal (serosal) chamber that is enhanced by apo-transferrin in the basal chamber, with the apo-transferrin undergoing endocytosis to the apical portion of the cell. With the addition of iron to the apical surface, divalent metal transporter 1 (DMT1) on the brush-border membrane (BBM) undergoes endocytosis. These findings suggest that in Caco-2 cells DMT1 and apo-transferrin may cooperate in iron transport through transcytosis. To prove this hypothesis, we determined by confocal microscopy that, after addition of iron to the apical chamber, DMT1 from the BBM and Texas red apo-transferrin from the basal chamber colocalized in a perinuclear compartment. Colocalization was also observed by isolating endosomes from Caco-2 cells after ingestion of ultra-small paramagnetic particles from either the basal or apical chamber. The isolated endosomes contained both transferrin and DMT1 independent of the chamber from which the paramagnetic particles were endocytosed. These findings suggest that iron transport across intestinal epithelia may be mediated by transcytosis.     

Mitochondria represent another locale for the divalent metal transporter 1 (DMT1)

The divalent metal transporter (DMT1) is well known for its roles in duodenal iron absorption across the apical enterocyte membrane, in iron efflux from the endosome during transferrin-dependent cellular iron acquisition, as well as in uptake of non-transferrin bound iron in many cells. Recently, using multiple approaches, we have obtained evidence that the mitochondrial outer membrane is another subcellular locale of DMT1 expression. While iron is of vital importance for mitochondrial energy metabolism, its delivery is likely to be tightly controlled due to iron''s damaging redox properties. Here we provide additional support for a role of DMT1 in mitochondrial iron acquisition by immunofluorescence colocalization with mitochondrial markers in cells and isolated mitochondria, as well as flow cytometric quantification of DMT1-positive mitochondria from an inducible expression system. Physiological consequences of mitochondrial DMT1 expression are discussed also in consideration of other DMT1 substrates, such as manganese, relevant to mitochondrial antioxidant defense.     

Ferroportin 1 is expressed basolaterally in rat kidney proximal tubule cells and iron excess increases its membrane trafficking

Ferroportin 1 (FPN1) is an iron export protein expressed in liver and duodenum, as well as in reticuloendothelial macrophages. Previously, we have shown that divalent metal transporter 1 (DMT1) is expressed in late endosomes and lysosomes of the kidney proximal tubule (PT), the nephron segment responsible for the majority of solute reabsorption. We suggested that following receptor mediated endocytosis of transferrin filtered by the glomerulus, DMT1 exports iron liberated from transferrin into the cytosol. FPN1 is also expressed in the kidney yet its role remains obscure. As a first step towards determining the role of renal FPN1, we localized FPN1 in the PT. FPN1 was found to be located in association with the basolateral PT membrane and within the cytosolic compartment. FPN1 was not expressed on the apical brush‐border membrane of PT cells. These data support a role for FPN1 in vectorial export of iron out of PT cells. Furthermore, under conditions of iron loading of cultured PT cells, FPN1 was trafficked to the plasma membrane suggesting a coordinated cellular response to export excess iron and limit cellular iron concentrations.     

Rapid regulation of divalent metal transporter (DMT1) protein but not mRNA expression by non-haem iron in human intestinal Caco-2 cells.

A divalent metal transporter, DMT1, located on the apical membrane of intestinal enterocytes is the major pathway for the absorption of dietary non-haem iron. Using human intestinal Caco-2 TC7 cells, we have shown that iron uptake and DMT1 protein in the plasma membrane were significantly decreased by exposure to high iron for 24 h, in a concentration-dependent manner, whereas whole cell DMT1 protein abundance was unaltered. This suggests that part of the response to high iron involved redistribution of DMT1 between the cytosol and cell membrane. These events preceded changes in DMT1 mRNA, which was only decreased following 72 h exposure to high iron.     

Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion

Rap1 and Ras are closely related GTPases that share some effectors but have distinct functions. We studied the subcellular localization of Rap1 and its sites of activation in living cells. Both GFP-tagged Rap1 and endogenous Rap1 were localized to the plasma membrane (PM) and endosomes. The PM association of GFP-Rap1 was dependent on GTP binding, and GFP-Rap1 was rapidly up-regulated on this compartment in response to mitogens, a process blocked by inhibitors of endosome recycling. A novel fluorescent probe for GTP-bound Rap1 revealed that this GTPase was transiently activated only on the PM of both fibroblasts and T cells. Activation on the PM was blocked by inhibitors of endosome recycling. Moreover, inhibition of endosome recycling blocked the ability of Rap1 to promote integrin-mediated adhesion of T cells. Thus, unlike Ras, the membrane localizations of Rap1 are dynamically regulated, and the PM is the principle platform from which Rap1 signaling emanates. These observations may explain some of the biological differences between these GTPases.     

Iron Imports. V. Transport of iron through the intestinal epithelium

Iron absorption across the brush-border membrane requires divalent metal transporter 1 (DMT1), whereas ferroportin (FPN) and hephaestin are required for exit across the basolateral membrane. However, how iron passes across the enterocyte is poorly understood. Both chaperones and transcytosis have been postulated to account for intracellular iron transport. With iron feeding, DMT1 undergoes endocytosis and FPN translocates from the apical cytosol to the basolateral membrane. The fluorescent metallosensor calcein offered to the basolateral surface of enterocytes is found in endosomes in the apical compartment, and its fluorescence is quenched when iron is offered to the apical surface. These experiments are consistent with vesicular iron transport as a possible pathway for intracellular iron transport.     

Apical distribution of HFE–β2-microglobulin is associated with inhibition of apical iron uptake in intestinal epithelia cells

Mutations in the HFE gene result in hereditary hemochromatosis, a disorder of iron metabolism characterized by increased intestinal iron absorption. Based on the observation that ectopic expression of HFE strongly inhibits apical iron uptake (Arredondo et al., 2001, FASEB J 15, 1276–1278), a negative regulation of HFE on the apical membrane transporter DMT1 was proposed as a mechanism by which HFE regulates iron absorption. To test this hypothesis, we investigated: (i) the effect of HFE antisense oligonucleotides on apical iron uptake by polarized Caco-2 cells; (ii) the apical/basolateral membrane distribution of HFE, β-2 microglobulin and DMT1; (iii) the putative molecular association between HFE and DMT1. We found that HFE antisense treatment reduced HFE expression and increased apical iron uptake, whereas transfection with wild-type HFE inhibited iron uptake. Thus, an inverse relationship was established between HFE levels and apical iron uptake activity. Selective apical or basolateral biotinylation indicated preferential localization of DMT1 to the apical membrane and of HFE and β-2 microglobulin (β2m) to the basolateral membrane. Ectopic expression of HFE resulted in increased distribution of HFE–β2m to the apical membrane. The amount of HFE–β2m in the apical membrane inversely correlated with apical iron uptake rates. Immunoprecipitations of HFE or β2m with specific antibodies resulted in the co-precipitation of DMT1. These results sustain a model by which direct interaction between DMT1 and HFE–β2m in the apical membrane of Caco-2 cells result in down-regulation of apical iron uptake activity.     

The iron transporter DMT1

Divalent metal transporter 1 (DMT1) is the first mammalian transmembrane iron transporter to be identified. In 1997, parallel experiments from two groups provided compelling evidence of its function. Fleming and colleagues identified mutations in DMT1 (formerly known as Nramp2 and DCT1) in mice and rats with defects in intestinal iron absorption and red blood cell iron utilization. Gunshin and co-workers (H Gunshin, B MacKenzie, UV Berger, Y Gunshin, MF Romero, WF Boron, S. Nussberger, JL Gollan, MA Hediger, Cloning and characterization of a mammalian proton-coupled metal-ion transporter, Nature 388 (1997) 482-488.) isolated DMT1 through an expression cloning strategy looking for mRNAs that stimulated iron uptake by Xenopus oocytes. Taken together, these data indicate that the twelve transmembrane domain protein DMT1 transfers iron across the apical surface of intestinal cells and out of transferrin cycle endosomes. Human DMT1 may be a good target for pharmacological intervention in patients with iron overload disorders attributable to increased iron absorption.     

Distribution and function of AP-1 clathrin adaptor complexes in polarized epithelial cells

Expression of the epithelial cell-specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD mu subunit, mu1A, being replaced by the closely related mu1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged mu1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.     

Effect of iron status on DMT1 expression in duodenal enterocytes from beta2-microglobulin knockout mice

Divalent metal transporter I (DMT1) is thought to be involved in transport of iron across the apical cell membrane of villus duodenal cells. To determine its role in hereditary hemochromatosis (HH), we used beta2-microglobulin knockout (B2M-/-) mice that accumulate iron as in HH. The B2M-/- and control C57BL/6 (B2M+/+) mice were fed diets with different iron contents. Increasing the iron availability increased plasma iron levels in both B2M+/+ and B2M-/- mice. Reducing the iron availability decreased the plasma iron concentration in B2M+/+ mice but was without effect on plasma iron in B2M-/- mice. DMT1 was not detectable in mice fed normal or iron-loaded diets when using immunohistochemistry. In Western blots, however, the protein was consistently observed regardless of the dietary regimen. DMT1 expression was increased to the same extent in B2M+/+ and B2M-/- mice when fed an iron-poor diet. In both strains of mice fed an iron-poor diet, DMT1 was evenly distributed in the differentiated enterocytes from the base to the tip of the villi but was absent from the crypts of Lieberkühn. These data suggest that the observed effects were due to the state of iron deficiency in mucosal cells rather than genetic defect.     

Apical-basal polarity in Drosophila neuroblasts is independent of vesicular trafficking

The possession of apical-basal polarity is a common feature of epithelia and neural stem cells, so-called neuroblasts (NBs). In Drosophila, an evolutionarily conserved protein complex consisting of atypical protein kinase C and the scaffolding proteins Bazooka/PAR-3 and PAR-6 controls the polarity of both cell types. The components of this complex localize to the apical junctional region of epithelial cells and form an apical crescent in NBs. In epithelia, the PAR proteins interact with the cellular machinery for polarized exocytosis and endocytosis, both of which are essential for the establishment of plasma membrane polarity. In NBs, many cortical proteins show a strongly polarized subcellular localization, but there is little evidence for the existence of distinct apical and basolateral plasma membrane domains, raising the question of whether vesicular trafficking is required for polarization of NBs. We analyzed the polarity of NBs mutant for essential regulators of the main exocytic and endocytic pathways. Surprisingly, we found that none of these mutations affected NB polarity, demonstrating that NB cortical polarity is independent of plasma membrane polarity and that the PAR proteins function in a cell type-specific manner.     

Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane.