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Prepublished online as a Blood First Edition Paper on January 9, 2003; DOI 10.1182/blood-2002-07-2108.
RED CELLS
From the Department of Biochemistry, McGill Cancer
Center and Center for Host Resistance, McGill University,
Montreal, QC.
Mutations at natural resistance-associated macrophage protein 1 (Nramp1) impair phagocyte function and cause susceptibility to infections while mutations at Nramp2 (divalent metal
transporter 1 [DMT1]) affect iron homeostasis and cause severe
microcytic anemia. Structure-function relationships in the Nramp
superfamily were studied by mutagenesis, followed by functional
characterization in yeast and in mammalian cells. These studies
identify 3 negatively charged and highly conserved residues in
transmembrane domains (TM) 1, 4, and 7 as essential for cation
transport by Nramp2/DMT1. The introduction of a charged residue
(Gly185Arg) in TM4 found in the naturally occurring microcytic anemia
mk (mouse) and Belgrade (rat) mutants is shown
to cause a partial or complete loss of function in mammalian and yeast
cells, respectively. A pair of mutation-sensitive and highly conserved
histidines (His267, His272) was identified in TM6. Surprisingly,
inactive His267 and His272 mutants could be rescued by lowering the pH
of the transport assay. This indicates that His267/His272 are not
directly involved in metal binding but, rather, play an important role
in pH regulation of metal transport by Nramp proteins.
(Blood. 2003;101:3699-3707) Natural resistance-associated macrophage protein 2 (Nramp2) (divalent metal transporter 1 [DMT1],
divalent cation transporter 1 [DCT1]) is essential for
nutritional iron (Fe2+) uptake by the duodenum brush
border1-3 and for iron transport across the endosomal
membrane in peripheral tissues.3-5 Nramp2 is an integral
membrane protein composed of 12 predicted transmembrane (TM)
domains.6 The Nramp2 gene produces 2 mRNAs by
alternative splicing of the terminal 3' exon that show different 3'
untranslated regions containing (isoform I, +IRE) or not (isoform II,
The Nramp2 homolog, Nramp1,6,20 is
expressed in the lysosomal compartment of macrophages and
neutrophils21 and is recruited to the membrane of
pathogen-containing phagosomes formed in these cells,22,23
where it may function as a Mn2+ efflux pump.24
A naturally occurring mutation in predicted TM4 of Nramp1 (Gly169Asp)
impairs maturation and membrane targeting of the protein and causes
susceptibility to infection with unrelated intracellular
pathogens.20,25 Nramp1 and Nramp2
define a large superfamily of membrane transporters highly conserved
from bacteria to humans.6,26,27 Demonstration of divalent
cation transport by distant Nramp homologs from bacteria (MntH,
Mramp), yeast (Smf1, Smf2, Smf3), fly (Mlv),
and plant (AtNramp) has highlighted functional conservation
in this family.6,28-30 In addition, expression of mouse
Nramp2 protein in a double smf1/smf2 mutant can restore the
ability of this mutant to grow at alkaline pH and on medium containing
metal chelators.31,32 Likewise, expression of human NRAMP1 in the fly mutant malvolio corrects the
taste discrimination defect of this mutant,30 in a manner
similar to that produced by increasing dietary Fe2+ or
Mn2+.33
In the present study, we have used multiple sequence alignments to
identify highly conserved residues in the Nramp superfamily. We have
studied the functional role of conserved charged TM domain residues in
substrate selectivity and pH regulation of Nramp2.
Materials
Plasmids and constructs
Yeast transformation and smf1/smf 2 complementation assays The Saccharomyces cerevisiae smf1/smf 2 double mutant is a mutant in which the SMF1 and SMF2 genes have been insertionally inactivated (MATa ura3-52 leu2-3 112 gal2
SMF1::LEU2,
SMF2::LEU2).32 This mutant cannot grow on alkaline medium or on medium containing metal chelators.31,32 Yeast cells were transformed with
pVT/Nramp2 constructs,35 and
ura+ transformants were grown as mass populations, and crude membrane fractions36 were prepared for
Nramp2 protein expression by immunoblotting using the mouse
anti-cMyc monoclonal antibody 9E10 (Babco, Berkeley, CA).9
To test for possible complementation of the growth defects of the
smf1/smf2 mutant by Nramp2 variants, duplicate
aliquots (1 mL) of ura+ transformants were resuspended in
either YPD medium or in alkaline YPD medium (pH 7.9, OD595 = 0.02) in 96-well plates (100 µL per well).
Growth was measured after 24 hours of incubation at 30°C using an
enzyme-linked immunosorbent assay (ELISA) microplate reader
(Bio-Rad model 450). Sensitivity to alkaline pH was measured as
relative growth of each transformant (expressed as a percentage) in
alkaline YPD 7.9 compared with growth of the same transformant in
normal YPD medium (pH 5.5 to 6.0). For complementation studies on
alkaline agar, cells grown to saturation in normal YPD were diluted to OD595 = 0.2, 0.02, 0.002, 0.0002, and 0.000 02 in YPD
7.9, and 20 µL of each dilution was spotted on alkaline YPD agar
plates and grown for 36 to 48 hours at 30°C before photography.
Cell culture and transfection LR-73 Chinese hamster ovary (CHO) cells were routinely grown in -minimum essential medium (-MEM) supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 µg/mL streptomycin
(Invitrogen, Burlington, ON, Canada). All pCB6 (neo) plasmid
constructs were transfected into cells as calcium phosphate
coprecipitates, according to a procedure we have previously
described.37 Clones of stable transfectants were selected
in medium containing Geneticin (G418, 770 µg/mL; Invitrogen) and were
picked after 8 to 13 days of selection.
Crude membrane preparation from CHO transfectants Cell pellets were resuspended in 250 µL TNE buffer (100 mM NaCl; 10 mM Tris [tris(hydroxymethyl)aminomethane]-Cl, pH 7.0; 10 mM EDTA [ethylenediaminetetraacetic acid]) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 µM pepstatin, 0.3 µM aprotinin, 1 µM leupeptin). Cells were homogenized by 20 passages through a 25-gauge, 5/8-inch needle, followed by centrifugation (4°C, 2000g, 10 minutes) to eliminate nuclei and unbroken cells. Membranes were then pelleted from the supernatant by ultracentrifugation (75 000 rpm, TLA-100 rotor [Beckman, Mississauga, ON, Canada], 4°C) and were resuspended in TNE containing 30% glycerol and protease inhibitors. Recombinant Nramp2 protein variants were detected using the mouse monoclonal anti-c-Myc antibody 9E10 (1:1000; Babco) as previously described.13Calcein loading of the cells and divalent metal transport assay CHO Nramp2 transfectants were loaded with the metal-sensitive fluorescent dye calcein-AM, as we have previously described.13 Briefly, CHO transfectants (1 × 106 cells) were incubated with 0.250 µM calcein-AM for 10 minutes at 37°C in 1 mL loading medium (-minimum essential medium, 1 mg/mL bovine serum albumin
[BSA], 20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic
acid], pH 7.4). The cells were washed twice in and
resuspended in 500 µL transport buffer (150 mM NaCl, 20 mM MES
[2-[N-morpholino]ethanesulfonic acid monohydrate], pH 5.0 to 6.5). The cell suspension was transferred to a stirred thermostated
(37°C) semimicrocuvette, and fluorescence was recorded using an
LS-50B fluorescence spectrometer (PerkinElmer, Woodbridge, ON, Canada;
excitation, 488 nm; emission, 517 nm; excitation and emission bandpass,
5 nm; response time, 6 seconds; data interval, 0.5 seconds). Divalent
metals (20 µM final concentration of Fe2+ or
Co2+) were added to the cell suspension after allowing
fluorescence to stabilize for 60 seconds. For assays using iron, a
combination of membrane-permeable (SIH) and membrane-impermeable
(HES-DFO) iron chelators were used at various time points (Figure 5A)
to distinguish intracellular from cell-associated quenchable
fluorescence: After fluorescence was allowed to stabilize for 60 seconds, 20 µM Fe2+ was added to the cell suspension
(Figure 5A, arrow 1). After 240 seconds, the membrane-impermeable iron
chelator HES-DFO was added (Figure 5A, arrow 2) to release
metal-induced quenching of extracellular cell-associated complexed
calcein. At 300 seconds, a membrane-permeable iron chelator SIH was
added (Figure 5A, arrow 3) to release metal-induced quenching of
intracellular calcein fluorescence. Initial rates were calculated from
quenching curves, and the size of the intracellular labile iron pool
was extracted from data obtained with the 2 metal chelators.
Cell surface protein biotinylation CHO Nramp2 transfectants were washed thoroughly with cold phosphate-buffered saline (PBS) (supplemented with 1 mM MgCl2, 0.1 mM CaCl2) and then with cold borate buffer (10 mM boric acid, 154 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl2, pH 9.0). Cells were labeled for 15 minutes on ice with 0.5 mg/mL Sulfo-NHS-SS-Biotin (sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate) (Pierce, Rockford, IL) in cold borate buffer. After washing 3 times with cold quenching buffer (PBS, 200 mM glycine), cells were scraped, collected, and resuspended in 1 mL lysis buffer (1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 10 mM Tris-Cl [pH 7.4], 30% glycerol) plus protease inhibitors. Lysates were incubated on ice for 20 minutes and pelleted (10 minutes, 10 000g, 4°C). Supernatants were collected, and protein levels were quantified by Bradford assay. A total of 500 µg total protein lysate was incubated overnight at 4°C with 50 µL immobilized streptavidin beads (Pierce) in a final volume of 500 µL (with lysis buffer and protease inhibitors). Streptavidin beads were washed 3 times with lysis buffer and then once with PBS. Labeled cell surface proteins were eluted with 50 µL 1 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and separated by SDS-PAGE.
Mutagenesis strategy Alignment of 28 eukaryotic and prokaryotic Nramp sequences (supplement on Blood website; see the Supplemental Document link at the top of the online article) identify a common and conserved hydrophobic core of 10 TM domains (about 30% identity between bacteria and humans)6 that contain 4 absolutely invariant (Asp86, Glu154, Glu299, Arg416) and 5 highly conserved (Arg119, Arg146, Asp161, Asp192, Glu225) charged residues (6 negative, 3 positive) in TM domains (Figure 1, dark blue residues). Another unique feature of the Nramp family is the presence of 2 invariant histidine residues (His267, His272) in the predicted TM domain 6 (Figure 1, red residues). In mice, naturally occurring mutations at adjacent residues in TM4 of Nramp1 and Nramp2 cause susceptibility to infection and microcytic anemia, respectively, and the loss-of-function phenotype of the microcytic anemia Nramp2 variant (Gly185Arg) was studied. Mutant cDNAs were expressed in yeast S cerevisiae and tested for their ability to complement 2 phenotypes of an smf1/smf2 mutant: (a) impaired growth on metal chelators31 and (b) impaired growth at alkaline pH.32 Mutants showing partial or complete loss of function were subsequently expressed in CHO cells and tested for Fe2+ and Co2+ transport at the plasma membrane.13
Conserved charged residues in membrane domains Immunoblotting indicated that all mutants could be stably expressed as 60- to 65-kDa immunoreactive proteins in yeast membrane fractions (Figure 2A, left panel). These results suggest that none of the mutations had a major effect on protein expression or stability in yeast. The ability of each mutant to complement the null smf1/smf2 yeast mutant was tested in parallel by plating serial dilutions of each transformant on YPD agar, pH 7.9 (Figure 2B, left panel), and using a growth inhibition assay in liquid YPD (pH 7.9). Routinely, yeast wild-type (WT) Nramp2 transformants showed a 9-fold stimulation for growth on alkaline medium over negative controls (pVT, smf/;
Figure 3, left panel). Two
Nramp2 mutants previously shown to either abrogate
(Gln394Val) or to have no effect (Gln395Glu) on Nramp2 function in
yeast32 were used as additional controls (Figure 3,
left panel).
In both assays, mutants Arg119Ala, Arg146Ala, Asp161Ala, and Glu225Ala showed full complementation of the smf1/smf2 growth defect, while mutants Asp86Ala, Glu154Ala, Asp192Ala, Glu299Ala, and Arg416Ala failed to do so. In addition, testing these 11 mutants for their ability to complement susceptibility of smf1/smf2 mutant to metal chelator (growth in EGTA [ethylenebis (oxyethylenenitrilo)-tetraacetic acid]-containing liquid medium) showed relative activities similar to that seen for growth at alkaline pH (data not shown). These results identify 5 of the 9 charged residues in TM domains as essential for Nramp2 function and smf1/smf2 complementation in yeast. The Asp86Ala, Glu154Ala, Asp161Ala, Asp192Ala, Glu299Ala, and Arg416Ala
mutants were analyzed for metal transport after transfection in CHO
cells (Figure 4A, left panel).
Transfectants were screened by immunoblotting for expression of the
corresponding Nramp2 variants at the cell membrane. Membranes from
cells transfected with the pCB6 empty vector were used as negative
controls, while 2 previously characterized transfectants13
expressing either low (N2GG) or high (N2.3) levels of Nramp2 protein
were used as positive controls. CHO cell clones stably expressing
mutants Glu154Ala and Arg416Ala could not be obtained in 4 independent
transfections (141 clones screened) for Glu154Ala and 3 independent
transfections (75 clones screened) for Arg416Ala, suggesting possible
effect of these mutations on protein folding/processing and/or toxicity
for the cells. Stable CHO transfectants expressing Asp86Ala, Asp192Ala,
and Glu299Ala could be readily isolated. In these clones the level of
expression varied but was in the range of levels seen in positive
controls expressing WT (N2.3, N2GG) or Asp161Ala proteins (Figure 4A,
left panel). In all cases, variability in the level of expression of individual mutants (including those that could not be expressed) can be
attributed to a combination of the site of integration of the vector in
the host genome and the effect of the introduced mutation on cell
growth and/or survival. In addition, biotin labeling experiments in
intact cells identified cell surface reactivity of all expressed
mutants (Asp86Ala, Asp161Ala, Asp192Ala, Glu299Ala), with levels
approximately proportionate to total expression levels detected in
membrane fractions by immunoblotting (Figure 4B, left panel). These
results indicate that mutations at Asp86, Asp161, Asp192, and Glu299 do
not have a major effect on Nramp2 protein maturation or membrane
targeting.
Transport properties of the mutants were investigated in intact cells
using a calcein-quenching assay.13 Metal transport by
Nramp2 using the calcein-quenching assay and transport of isotopic 55Fe have been shown to be comparable in the identical
control Nramp2 CHO transfectants13 as well as some Nramp2
mutant variants (data not shown). For the calcein-quenching assay, CHO
transfectants were loaded with the metal-sensitive fluorescent dye
calcein, and the effect of externally added divalent cations
Fe2+ or Co2+ on the rate of quenching of
fluorescence was monitored (at optimal pH 6.0), and the slope of the
initial quenching curve was calculated (initial rate13).
Results shown in Figure 5A show a typical set of fluorescence quenching traces for positive and negative controls. Transport activities of mutants are shown in Figure 5B and
are expressed as a relative transport activity (%). Mutants were
classified as having either low (less than 33% of WT), intermediate (between 34% and 67%), or WT activity (above 67%). Expression of WT
Nramp2 in clones N2.3 and N2GG caused a 10-fold and 5-fold stimulation
of calcein quenching and hence Co2+ and Fe2+
transport, respectively, compared with controls. Likewise, cells expressing mutant Asp161Ala showed transport rates similar to WT
(Figure 5B). On the other hand, mutant Asp192Ala in TM4 showed intermediate transport activity when compared with WT for both Fe2+ and Co2+. Finally, mutants
Asp86Ala and Glu299Ala were completely inactive for the 2 substrates
tested. These results identify residues Asp86 and Glu299 in
corresponding TM1 and TM7 as playing key roles in metal transport
by Nramp2.
Gly185Arg mutation of mk mice A naturally occurring mutation at Gly185 in TM4 (Gly185Arg) is associated with severe iron deficiency and microcytic anemia of the mk mouse2 and of the Belgrade rat.3 When expressed in the yeast smf1/smf2 mutant, the Gly185Arg variant of Nramp2 can be expressed in the membrane fraction of these cells at levels similar to those seen for either the WT protein and for the other mutants (Figure 2A). Complementation studies indicated that Gly185Arg could not restore growth in alkaline medium (Figure 3) or in medium containing metal chelators (data not shown). These results suggest that in yeast cells, Gly185Arg is either transport incompetent or is active but mistargeted to an inappropriate transport site. CHO transfectants expressing robust levels of Gly185Arg mutant could be readily isolated (Figure 4). Interestingly, transport experiments in these cells (Figure 5B) indicated that the Gly185Arg mutant retains significant and intermediate transport activity, with 50% (Co2+) and 30% activity (Fe2+) of the WT protein. These results in CHO cells clearly suggest that the Gly185Arg mutation only attenuates but does not eliminate transport function of Nramp2 protein in mammalian cells.Conserved histidine pair in TM6 Histidine pairs can form metal binding sites in soluble38 or in membrane proteins.39,40 The highly conserved histidine pair found in TM6 (His267, His272) of the Nramp superfamily was initially studied in Nramp2 by mutagenesis to the small neutral residue alanine in mutants His267Ala and His272Ala and in the double mutant His267Ala/His272Ala. Metal binding by His residues involves donating imidazole nitrogen lone-pair electrons to the unfilled orbitals of the metal, and cysteines can substitute for His in this process.38,41-43 Thus, mutants His267Cys, His272Cys, and His267Cys/His272Cys were created. The imidazole proton of His has a pKa of 6.6 and can mediate pH-dependent effects in proteins. Thus, a last set of mutants was created in which His was replaced by the positively charged Arg (pKa about 12), which may functionally mimic the protonated His, albeit with a much larger bulk. Mutants were transformed in yeast cells, and immunoblotting analyses show that all 9 single and double mutants could be stably expressed in the membrane fraction of the smf1/smf2 mutant (Figure 2A, right panel). Complementation studies for growth at alkaline pH (Figure 3) and in the presence of metal chelators (data not shown) indicate that His267 is highly mutation sensitive, with replacements to Ala, Cys, and Arg causing severe or complete loss of function. His272 was less mutation sensitive than His267, with His272Ala and His272Cys retaining near WT activity, and only substitution to the bulkier Arg (His272Arg) abrogated complementation. Finally, the 3 double mutants (His267Ala/His272Ala, His267Cys/His272Cys, His267Arg/His272Arg) were completely inactive in yeast.Immunoblotting analysis (Figure 4A, right panel) revealed that all
mutants could be stably expressed after transfection in CHO cells, with
the exception of the double His267/His272Cys mutant (3 independent
transfections, 119 clones screened). The other 8 Nramp2
mutants were expressed at varying levels but generally fell between
those seen in the positive controls expressing low (N2GG) and high
(N2.3) amounts of Nramp2. Biotin labeling experiments in intact cells
identified cell surface reactivity of all expressed mutants, with
levels approximately proportionate to total expression levels detected
in membrane fractions by immunoblotting (Figure 4B, right panel).
However, reduced trafficking of protein to the plasma membrane may have
occurred in His267Cys and His272Ala. Metal transport activity of the
mutants was tested in the calcein-quenching assay for Fe2+
and Co2+ at pH 6.0. Mutants bearing either single mutations
to Arg at either position (His267Arg, His272Arg) or double mutations
(His267A/His272Ala, His267Arg/His272Arg) were completely inactive for
transport (Figure 6). Likewise, mutant
His272Ala was also inactive in these conditions, and only low transport
activity for Co2+ was detected for His267Cys (Figure 6).
Reduced trafficking to the plasma membrane (Figure 4B) may have
contributed to reduced activity of His267Cys and His272Ala. Only
His267Ala and His272Cys retained modest but significant transport
activity toward both metal cations analyzed in the assay, with
His267Ala possibly having a higher selectivity for cobalt.
These results indicate that His267 and His272 play an important role in
Nramp2 metal transport, both in yeast and mammalian cells.
Possible change in ion selectivity for the His267Ala mutant was investigated further through radioisotopic 55Fe transport measurements. Competition experiments with cold iron and cobalt failed to reveal a significant preference for cobalt in His267Ala compared with the WT Nramp2 (data not shown). Therefore, the change in substrate selectivity in His267Ala suggested by the calcein-quenching assay, while statistically significant, could not be validated by more direct measurements with radioisotopic 55Fe. Possible pH effects on transport activity of the mutants were
investigated (Figure 7). Fe2+
(Figure 7A, top row) and Co2+ (data not shown) transport by
Nramp2 in clones N2.3 and N2GG approached maximum at pH of about 6.0, and decreasing the pH to 5.0 had little effect on transport. Likewise,
background uptake of metal in negative controls was not affected by
lowering pH. However, the low level of transport activity detected at
pH 6.0 in mutants His267Ala, His267Cys, and His272Cys (Figure 6) could be significantly enhanced by progressively lowering pH (Figure 7). This
increase occurred gradually over background, pH-insensitive levels
detected in control pCB6 cells. Surprisingly, mutants 272Ala and
267Ala/272Ala, which showed complete loss of function at pH 6.0 (Figure
6), could be rescued by lowering the pH. Interestingly, however,
lowering the pH of the extracellular medium had no effect on
Co2+ (not shown) or Fe2+ transport (Figure 7B)
of any of the histidine-to-arginine mutants (His267Arg, His272Arg,
His267/272Arg). The pH effect seen in the Cys/Ala mutants was specific
for the conserved His pair of TM6 and was not observed in any of the
other inactivating mutations studied (eg, Asp86Ala, Glu299Ala, or
Gly185Arg) (data not shown). Therefore, mutations at the 2 conserved
histidines in TMD6 clearly affect pH sensitivity of Nramp2 transport
and suggest that these residues may be involved in pH regulation
of transport.
We aimed to study the functional role of highly conserved charged residues in the TM domains of Nramp proteins on substrate transport and pH regulation. Such residues were mutated in the backbone of Nramp2, and corresponding mutants were tested in yeast and mammalian cells. With few exceptions, there was good general agreement between smf1/smf2 complementation results in yeast and transport data in mammalian cells. Interestingly, although all mutants could be expressed in the membrane fractions of yeast cells (Figure 2A), several could not be expressed in CHO cell membranes. These results suggest that such TM mutations (1) affected normal protein folding, maturation, and processing, possibly leading to protein instability and/or degradation, or (2) that overexpression of the corresponding mutant protein was somehow toxic for the cells. These results highlight possible differences in protein sorting, maturation, and targeting mechanisms in both cell systems but also stress the importance of maturation and membrane targeting for proper Nramp2 function. Gly185 is invariant in all Nramp orthologs from bacteria to humans. Strikingly, its mutation to Arg arose independently in 2 rodent models of iron deficiency, the microcytic anemia mouse mk 2 and the anemic Belgrade (b) rat.3 The Gly185Arg mutation showed a complex phenotype. In yeast, Gly185Arg could be expressed in yeast membranes but could not complement the smf1/smf2 mutant, suggesting a loss of transport activity, in agreement with results from Su et al16 and from Worthington et al,44 who reported a 95% and 85% reduction in 55Fe2+ transport activity of Gly185Arg in HEK293 cells and COS-7 cells, respectively. Gly185Arg could also be expressed in the membrane fraction of CHO cells (Figure 4), where it retained significant transport activity for both Co2+ (50% of WT) and Fe2+ (35% of WT). Similar transport activity of Gly185Arg has also been observed in stable transfectants in the LLC-PK1 porcine kidney cells (data not shown). This significant transport activity suggests that loss of transport function may not be the sole defect responsible for the mk phenotype in vivo. Rather, the mutation may affect membrane targeting in a cell-specific fashion, perhaps including targeting to a transport-incompetent compartment in yeast cells. Recent studies in vivo support such a tissue or cell-specific effect. Indeed, immunoblotting studies show robust expression of the Gly185Arg isoform in duodenum membrane fractions of mk/mk mice, but immunohistochemistry studies revealed absence of protein targeting to the brush border, the site of active transport.17 Likewise, mk/mk mice show a strong reduction of Nramp2 (Gly185Arg) protein expression in the kidney,12 while mk/mk reticulocytes are completely devoid of Nramp2 expression.10 Parallel analysis of the disease susceptibility Gly169Asp mutation in TM4 of Nramp1 (reconstructed in Nramp2; data not shown) associated with susceptibility to infections also indicated absence of smf1/smf2 complementation in yeast. Also, we could not isolate CHO clones stably expressing this mutant, a situation similar to that seen in vivo in macrophages from Nramp1Gly169Asp mouse strains, where no mature protein is detected.45 Together, these results suggest an important role for this residue and TM segment for transport activity but also suggest that its integrity is required for proper maturation, folding, and/or targeting of the Nramp proteins. Helical wheel projections in the conserved hydrophobic core of Nramp proteins reveal strong amphipathic character for several TM domains, including TM 3, 5, and 9.6 Sequence conservation expressed as a variability moment6 indicates periodicity with strong conservation of the polar face, with the apolar "lipid-accessible" face of the helix being heavily substituted. Several highly charged residues map to the polar side of TM helices. Such an arrangement is characteristic of families of ion transporters and ion channels.6 Mutations at the 9 conserved charges in TM domains had either no effect (Arg119, Arg146, Asp161, Glu225) or caused partial (Asp192) or complete loss of function (Asp86, Glu154, Glu299, Arg416). Asp86, Glu154, Glu299, and Arg416 are the most highly conserved, being invariant in multiple sequence alignments performed (supplemental document). It is striking that 3 of them have negatively charged side chains, raising the possibility that they may mediate interaction with the positively charged divalent cation substrates of Nramp transporters. Alternatively, such residues may be involved in hydrogen bonding, salt bridge formation (dipole), or other interactions in the formation of a water-filled pore or transport path. The absolute conservation of the histidine pair His267/His272 in TM6 in eukaryotic and prokaryotic Nramp sequences suggests an important role. In addition, studies by us (data not shown) and others44 show that Nramp2-mediated transport in transfected cells is sensitive to the action of the histidine-specific reagent diethyl pyrocarbonate (DEPC). Here, we show that both residues are mutation sensitive (in particular, His267), with independent substitutions at either or both residues causing loss of function. Strikingly, several poorly active (His267Ala, His267Cys, His272Cys) or completely inactive His mutants (His272Ala, His272Cys, His267Ala/His272Ala) at pH 6.0 could be rescued by lowering the pH of the transport assay (Figure 7). The observed pH effect was incremental, with maximal transport attained at or below pH 5.0. However, completely inactive histidine-to-arginine mutants (His267Arg, His272Arg, His267/272Arg) could not be rescued by lowering the pH of the transport assay. This may be a result of increased steric hindrance associated with replacement of histidine to bulky arginine. Thus, mutations at either His residues in TM6 shifted the pH required to achieve maximal transport to a more acidic value. Several explanations can be put forward to account for the unique effect of pH on transport properties of the Nramp2 His mutants. First, His267 and His272 may be involved in direct binding of the metal substrate in a pH-dependent fashion. Metal binding by His pairs has been documented in soluble proteins such as transcription factors38 and has also been used in membrane proteins where they have been engineered to study proximity relationships between individual TM domains by electroparamagnetic resonance.46 Although His267 and His272 may indeed form part of a binding site for metals in the membrane portion of Nramp2, it appears unlikely. Indeed, an Nramp2 mutant lacking both histidines (His267Ala/His272Ala) is still transport-active at pH 5.0 (Figure 7B). A second possibility is that His267, His272, or both participate in H+ movement across the membrane in a H+ cotransport mechanism possibly by a proton relay system.1 Such a relay system has been described for the lactose permease of Escherichia coli and involves TM residues Arg302/His322/Glu325.47 Such a proton relay system may exist in Nramp2 and may involve conserved residues such as His267/His272 as well as other negatively and positively charged residues in TM domains. Partial or complete inactivation of this system would be predicted to have a major effect on the pH dependence of transport. A third explanation for the observed pH effect on transport properties of single or double His mutants is that His267/His272 may be implicated in pH regulation of the transporter through gain or loss of the imidazole proton (pKa 5.5 to 6.5). In this favored model, protonation of His267/His272 would be required to maintain the protein in a functional, transport-competent conformation. This effect could be either general and involve additional residues, resulting in a pH-dependent global conformational change from an inactive state (neutral pH) to an active state (acidic pH). In this model, loss of the key His residues would shift the pH for maximal transport to a more acidic value, requiring protonation of other groups or side chains to create the same overall conformational change. Alternatively, either or both His267/His272 could play a more specific role in creating a pH-dependent transport path in the transporter. For example, an interaction with adjacent and highly conserved negatively charged residues in other TM domains could be necessary to open an ion transport path. Loss of His267/His272 would require formation of compensatory interactions of conserved negatively charged residues with other protonated side chains and/or water molecules in the transport path. Although highly speculative, such a mechanism appears to account for the pH dependence of anion transport by the band 3 transporter.48,49
The authors are indebted to Drs S. Grinstein (University of Toronto) and H. R. Kaback (University of California, Los Angeles) for helpful discussions and suggestions during this work and to Dr P. Ponka (McGill University, Montreal) for the generous preparation and gift of iron chelators.
Supported by a research grant from the National Institute of Allergy and Infectious Diseases (RO1 AI35237-08) (P.G.), a studentship from the Canadian Insitutes of Health Research (S.L.), and a Distinguished Scientist salary award from the Canadian Institutes of Health Research (P.G.).
Submitted July 15, 2002; accepted December 19, 2002. Prepublished online as Blood First Edition Paper, January 9, 2003; DOI 10.1182/blood-2002-07-2108.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Philippe Gros, Professor, Department of Biochemistry, McGill University, 3655 Sir William Osler Promenade, Montreal, QC, Canada, H3G-1Y6; e-mail: gros{at}med.mcgill.ca.
1. Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388:482-488[CrossRef][Medline] [Order article via Infotrieve]. 2. Fleming MD, Trenor CC III, Su MA, et al. Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene. Nat Genet. 1997;16:383-386[CrossRef][Medline] [Order article via Infotrieve].
3.
Fleming MD, Romano MA, Su MA, Garrick LM, Garrick MD, Andrews NC.
Nramp2 is mutated in the anemic Belgrade (b) rat: evidence of a role for Nramp2 in endosomal iron transport.
Proc Natl Acad Sci U S A.
1998;95:1148-1153
4.
Gruenheid S, Canonne-Hergaux F, Gauthier S, Hackam DJ, Grinstein S, Gros P.
The iron transport protein NRAMP2 is an integral membrane glycoprotein that colocalizes with transferrin in recycling endosomes.
J Exp Med.
1999;189:831-841
5.
Tabuchi M, Yoshimori T, Yamaguchi K, Yoshida T, Kishi F.
Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells.
J Biol Chem.
2000;275:22220-22228
6.
Cellier M, Prive G, Belouchi A, et al.
Nramp defines a family of membrane proteins.
Proc Natl Acad Sci U S A.
1995;92:10089-10093 7. Lee PL, Gelbart T, West C, Halloran C, Beutler E. The human Nramp2 gene: characterization of the gene structure, alternative splicing, promoter region and polymorphisms. Blood Cells Mol Dis. 1998;24:199-215[CrossRef][Medline] [Order article via Infotrieve]. 8. Fleming MD, Andrews NC. Mammalian iron transport: an unexpected link between metal homeostasis and host defense. J Lab Clin Med. 1998;132:464-468[CrossRef][Medline] [Order article via Infotrieve].
9.
Canonne-Hergaux F, Gruenheid S, Ponka P, Gros P.
Cellular and subcellular localization of the Nramp2 iron transporter in the intestinal brush border and regulation by dietary iron.
Blood.
1999;93:4406-4417
10.
Canonne-Hergaux F, Zhang AS, Ponka P, Gros P.
Characterization of the iron transporter DMT1 (NRAMP2/DCT1) in red blood cells of normal and anemic mk/mk mice.
Blood.
2001;98:3823-3830 11. Tchernitchko D, Bourgeois M, Martin ME, Beaumont C. Expression of the two mRNA isoforms of the iron transporter Nrmap2/DMTI in mice and function of the iron responsive element. Biochem J. 2002;363:449-455[CrossRef][Medline] [Order article via Infotrieve]. 12. Canonne-Hergaux F, Gros P. Expression of the iron transporter DMT1 in kidney from normal and anemic mk mice. Kidney Int. 2002;62:147-156[CrossRef][Medline] [Order article via Infotrieve].
13.
Picard V, Govoni G, Jabado N, Gros P.
Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool.
J Biol Chem.
2000;275:35738-35745
14.
Tandy S, Williams M, Leggett A, et al.
Nramp2 expression is associated with pH-dependent iron uptake across the apical membrane of human intestinal Caco-2 cells.
J Biol Chem.
2000;275:1023-1029 15. Edwards JA, Hoke JE. Defect of intestinal mucosal iron uptake in mice with hereditary microcytic anemia. Proc Soc Exp Biol Med. 1972;141:81-84[CrossRef][Medline] [Order article via Infotrieve].
16.
Su MA, Trenor CC, Fleming JC, Fleming MD, Andrews NC.
The G185R mutation disrupts function of the iron transporter Nramp2.
Blood.
1998;92:2157-2163
17.
Canonne-Hergaux F, Fleming MD, Levy JE, et al.
The Nramp2/DMT1 iron transporter is induced in the duodenum of microcytic anemia mk mice but is not properly targeted to the intestinal brush border.
Blood.
2000;96:3964-3970 18. Andrews NC. Iron metabolism: iron deficiency and iron overload. Annu Rev Genomics Hum Genet. 2000;1:75-98[CrossRef][Medline] [Order article via Infotrieve]. 19. Wessling-Resnick M. Iron transport. Annu Rev Nutr. 2000;20:129-151[CrossRef][Medline] [Order article via Infotrieve]. 20. Vidal SM, Malo D, Vogan K, Skamene E, Gros P. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. Cell. 1993;73:469-485[CrossRef][Medline] [Order article via Infotrieve]. 21. Govoni G, Gauthier S, Billia F, Iscove NN, Gros P. Cell-specific and inducible Nramp1 gene expression in mouse macrophages in vitro and in vivo. J Leukoc Biol. 1997;62:277-286[Abstract]. 22. Cellier M, Shustik C, Dalton W, et al. Expression of the human NRAMP1 gene in professional primary phagocytes: studies in blood cells and in HL-60 promyelocytic leukemia. J Leukoc Biol. 1997;61:96-105[Abstract].
23.
Gruenheid S, Pinner E, Desjardins M, Gros P.
Natural resistance to infection with intracellular pathogens: the Nramp1 protein is recruited to the membrane of the phagosome.
J Exp Med.
1997;185:717-730
24.
Jabado N, Jankowski A, Dougaparsad S, Picard V, Grinstein S, Gros P.
Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.
J Exp Med.
2000;192:1237-1248
25.
Vidal S, Tremblay ML, Govoni G, et al.
The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene.
J Exp Med.
1995;182:655-666 26. Forbes JR, Gros P. Divalent-metal transport by NRAMP proteins at the interface of host-pathogen interactions. Trends Microbiol. 2001;9:397-403[CrossRef][Medline] [Order article via Infotrieve]. 27. Gruenheid S, Gros P. Genetic susceptibility to intracellular infections: Nramp1, macrophage function and divalent cations transport. Curr Opin Microbiol. 2000;3:43-48[CrossRef][Medline] [Order article via Infotrieve].
28.
Agranoff D, Monahan IM, Mangan JA, Butcher PD, Krishna S.
Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.
J Exp Med.
1999;190:717-724 29. Belouchi A, Cellier M, Kwan T, Saini HS, Leroux G, Gros P. The macrophage-specific membrane protein Nramp controlling natural resistance to infections in mice has homologues expressed in the root system of plants. Plant Mol Biol. 1995;29:1181-1196[CrossRef][Medline] [Order article via Infotrieve]. 30. Rodrigues V, Cheah PY, Ray K, Chia W. malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour. EMBO J. 1995;14:3007-3020[Medline] [Order article via Infotrieve].
31.
Supek F, Supekova L, Nelson H, Nelson N.
A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria.
Proc Natl Acad Sci U S A.
1996;93:5105-5110
32.
Pinner E, Gruenheid S, Raymond M, Gros P.
Functional complementation of the yeast divalent cation transporter family SMF by NRAMP2, a member of the mammalian natural resistance-associated macrophage protein family.
J Biol Chem.
1997;272:28933-28938
33.
Orgad S, Nelson H, Segal D, Nelson N.
Metal ions suppress the abnormal taste behavior of the Drosophila mutant malvolio.
J Exp Biol.
1998;201:115-120 34. Urbatsch IL, Julien M, Carrier I, Rousseau ME, Cayrol R, Gros P. Mutational analysis of conserved carboxylate residues in the nucleotide binding sites of P-glycoprotein. Biochemistry. 2000;39:14138-14149[CrossRef][Medline] [Order article via Infotrieve]. 35. Sherman F, Fink GR, Hicks JB. Methods of Yeast Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982. 36. Kwan T, Gros P. Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains. Biochemistry. 1998;37:3337-3350[CrossRef][Medline] [Order article via Infotrieve]. 37. Kast C, Canfield V, Levenson R, Gros P. Membrane topology of P-glycoprotein as determined by epitope insertion: transmembrane organization of the N-terminal domain of mdr3. Biochemistry. 1995;34:4402-4411[CrossRef][Medline] [Order article via Infotrieve]. 38. Nakagama H, Heinrich G, Pelletier J, Housman DE. Sequence and structural requirements for high-affinity DNA binding by the WT1 gene product. Mol Cell Biol. 1995;15:1489-1498[Abstract]. 39. Eng BH, Guerinot ML, Eide D, Saier MH Jr. Sequence analyses and phylogenetic characterization of the ZIP family of metal ion transport proteins. J Membr Biol. 1998;166:1-7[CrossRef][Medline] [Order article via Infotrieve].
40.
Voss J, Hubbell WL, Kaback HR.
Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.
Proc Natl Acad Sci U S A.
1995;92:12300-12303
41.
Omichinski JG, Trainor C, Evans T, Gronenborn AM, Clore GM, Felsenfeld G.
A small single-"finger" peptide from the erythroid transcription factor GATA-1 binds specifically to DNA as a zinc or iron complex.
Proc Natl Acad Sci U S A.
1993;90:1676-1680 42. Ghazaleh FA, Omburo GA, Colman RW. Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase. Biochem J. 1996;317:495-501[Medline] [Order article via Infotrieve].
43.
Worthington MT, Amann BT, Nathans D, Berg JM.
Metal binding properties and secondary structure of the zinc-binding domain of Nup475.
Proc Natl Acad Sci U S A.
1996;93:13754-13759
44.
Worthington MT, Browne L, Battle EH, Luo RQ.
Functional properties of transfected human DMT1 iron transporter.
Am J Physiol Gastrointest Liver Physiol.
2000;279:G1265-G1273 45. Vidal SM, Pinner E, Lepage P, Gauthier S, Gros P. Natural resistance to intracellular infections: Nramp1 encodes a membrane phosphoglycoprotein absent in macrophages from susceptible (Nramp1 D169) mouse strains. J Immunol. 1996;157:3559-3568[Abstract].
46.
Voss J, Salwinski L, Kaback HR, Hubbell WL.
A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme.
Proc Natl Acad Sci U S A.
1995;92:12295-12299
47.
Kaback HR.
Lac permease of Escherichia coli: on the path of the proton.
Philos Trans R Soc Lond B Biol Sci.
1990;326:425-436 48. Muller-Berger S, Karbach D, Kang D, et al. Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport. Biochemistry. 1995;34:9325-9332[CrossRef][Medline] [Order article via Infotrieve]. 49. Muller-Berger S, Karbach D, Konig J, et al. Inhibition of mouse erythroid band 3-mediated chloride transport by site-directed mutagenesis of histidine residues and its reversal by second site mutation of Lys 558, the locus of covalent H2DIDS binding. Biochemistry. 1995;34:9315-9324[CrossRef][Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
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  E. S. Unal, R. Zhao, and I. D. Goldman Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1 Am J Physiol Cell Physiol, July 1, 2009; 297(1): C66 - C74. [Abstract] [Full Text] [PDF]
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 E. S. Unal, R. Zhao, M.-H. Chang, A. Fiser, M. F. Romero, and I. D. Goldman The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1 J. Biol. Chem., June 26, 2009; 284(26): 17846 - 17857. [Abstract] [Full Text] [PDF]
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 L. T. Jensen, M. C. Carroll, M. D. Hall, C. J. Harvey, S. E. Beese, and V. C. Culotta Down-Regulation of a Manganese Transporter in the Face of Metal Toxicity Mol. Biol. Cell, June 15, 2009; 20(12): 2810 - 2819. [Abstract] [Full Text] [PDF]
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P. Courville, E. Urbankova, C. Rensing, R. Chaloupka, M. Quick, and M. F. M. Cellier Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6 J. Biol. Chem., April 11, 2008; 283(15): 9651 - 9658. [Abstract] [Full Text] [PDF]
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M. E. Techau, J. Valdez-Taubas, J.-F. Popoff, R. Francis, M. Seaman, and J. M. Blackwell Evolution of Differences in Transport Function in Slc11a Family Members J. Biol. Chem., December 7, 2007; 282(49): 35646 - 35656. [Abstract] [Full Text] [PDF]
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 V. A Fitsanakis, G. Piccola, A. P. Marreilha dos Santos, J. L Aschner, and M. Aschner Putative proteins involved in manganese transport across the blood-brain barr 1ier Human and Experimental Toxicology, April 1, 2007; 26(4): 295 - 302. [Abstract] [PDF]
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S. Lam-Yuk-Tseung, V. Picard, and P. Gros Identification of a Tyrosine-based Motif (YGSI) in the Amino Terminus of Nramp1 (Slc11a1) That Is Important for Lysosomal Targeting J. Biol. Chem., October 20, 2006; 281(42): 31677 - 31688. [Abstract] [Full Text] [PDF]
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 F. Canonne-Hergaux, A. Donovan, C. Delaby, H.-j. Wang, and P. Gros Comparative studies of duodenal and macrophage ferroportin proteins Am J Physiol Gastrointest Liver Physiol, January 1, 2006; 290(1): G156 - G163. [Abstract] [Full Text] [PDF]
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 M. Priwitzerova, G. Nie, A. D. Sheftel, D. Pospisilova, V. Divoky, and P. Ponka Functional consequences of the human DMT1 (SLC11A2) mutation on protein expression and iron uptake Blood, December 1, 2005; 106(12): 3985 - 3987. [Abstract] [Full Text] [PDF]
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A. K. Mandal, Y. Yang, T. M. Kertesz, and J. M. Arguello Identification of the Transmembrane Metal Binding Site in Cu+-transporting PIB-type ATPases J. Biol. Chem., December 24, 2004; 279(52): 54802 - 54807. [Abstract] [Full Text] [PDF]
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Y. Nevo and N. Nelson The Mutation F227I Increases the Coupling of Metal Ion Transport in DCT1 J. Biol. Chem., December 17, 2004; 279(51): 53056 - 53061. [Abstract] [Full Text] [PDF]
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 K. M. Papp and M. E. Maguire The CorA Mg2+ Transporter Does Not Transport Fe2+ J. Bacteriol., November 15, 2004; 186(22): 7653 - 7658. [Abstract] [Full Text] [PDF]
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N. Touret, N. Martin-Orozco, P. Paroutis, W. Furuya, S. Lam-Yuk-Tseung, J. Forbes, P. Gros, and S. Grinstein Molecular and cellular mechanisms underlying iron transport deficiency in microcytic anemia Blood, September 1, 2004; 104(5): 1526 - 1533. [Abstract] [Full Text] [PDF]
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 A. Cohen, Y. Nevo, and N. Nelson The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity PNAS, September 16, 2003; 100(19): 10694 - 10699. [Abstract] [Full Text] [PDF]
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J. R. Forbes and P. Gros Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and Nramp2 (Slc11a2) expressed at the plasma membrane Blood, September 1, 2003; 102(5): 1884 - 1892. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
