PHAGOCYTES
Iron, manganese, and cobalt transport by Nramp1 (Slc11a1) and
Nramp2 (Slc11a2) expressed at the plasma membrane
John R. Forbes and Philippe Gros
Mutations in the Nramp1 gene (Slc11a1)
cause susceptibility to infection by intracellular pathogens. The Nramp1 protein
is expressed at the phagosomal membrane of macrophages and neutrophils
and is a paralog of the Nramp2 (Slc11a2)
iron transporter. The Nramp1 transport
mechanism at the phagosomal membrane has remained controversial. An
Nramp1 protein modified by insertion of a
hemagglutinin epitope into the predicted
TM7/8 loop was expressed at the plasma
membrane of Chinese hamster ovary cells
as demonstrated by immunofluorescence
and surface biotinylation. Experiments in
Nramp1HA transfectants using the metalsensitive fluorophors calcein and Fura2
showed that Nramp1HA can mediate Fe2ⴙ,
Mn2ⴙ, and Co2ⴙ uptake. Similar results
were obtained in transport studies using radioisotopic 55Fe2ⴙ and 54Mn2ⴙ.
Nramp1HA transport was dependent on
time, temperature, and acidic pH, occurring down the proton gradient. These
experiments suggest that Nramp1HA may
be a more efficient transporter of Mn2ⴙ
compared to Fe2ⴙ and a more efficient
Mn2ⴙ transporter than Nramp2HA. The
membrane topology and transport properties of Nramp1HA and Nramp2HA
were indistinguishable, suggesting that
Nramp1 divalent-metal transport at the
phagosomal membrane is mechanistically similar to that of Nramp2 at the
membrane of acidified endosomes. These
results clarify the mechanism by which
Nramp1 contributes to phagocyte defenses against infections. (Blood. 2003;
102:1884-1892)
© 2003 by The American Society of Hematology
Introduction
Mutations in the mouse Nramp1 gene (natural resistance associated
macrophage protein 1, also known as Slc11a1; OMIM [Online
Mendelian Inheritance in Man] #600266) cause susceptibility to
infection by several intracellular pathogens including Mycobacterium, Leishmania, and Salmonella.1 Likewise, polymorphic variants of NRAMP1 are associated with human susceptibility to
tuberculosis and leprosy in endemic areas of disease.2,3 Nramp1
mRNA is abundant in mouse macrophages and human neutrophils,
where it encodes a 90- to 100-kDa integral membrane phosphoglycoprotein4 present in the Lamp1-positive late endosomes and
lysosomes5 and in gelatinase-positive tertiary granules,6 respectively. Following phagocytosis, Nramp1 is rapidly recruited to and
remains associated with the membrane of phagosomes containing
either inert particles5 or live bacteria/parasites.7-9 Recruitment of
Nramp1 to the membrane of Mycobacteria containing phagosomes
is associated with bacteriostasis, bacterial damage, and appears to
antagonize the ability of Mycobacterium to block phagolysosomal
fusion and acidification.10,11 Similarly, Nramp1 appears to impair
the ability of Salmonella to shelter in a vacuole that does not fuse to
early endosomes and that remains negative for the mannose-6phosphate receptor.9
Nramp1 has a close mammalian paralog Nramp2 (OMIM
#600523; also known as Dmt1, Dct1, Slc11a2)12 that has been
functionally characterized. Transport studies using Xenopus laevis
oocytes13 and mammalian cell lines14,15 have demonstrated that
Nramp2 is a broad specificity divalent-metal transporter that
functions in a pH-dependent fashion, stimulated by acidic pH,
which is suggestive of a proton/metal-symport mechanism.13 In
mice, Nramp2 is expressed at the duodenum brush border, where it
is responsible for transferrin-independent uptake of dietary iron
from the intestinal lumen.16 Nramp2 also colocalizes with transferrin in the recycling endosomes of many cell types, including
reticulocytes,17,18 where it transports iron from the acidified lumen
of the endosomes into the cytoplasm.19-21 A mutation in Nramp2
impairs both of these aspects of iron homeostasis and causes
microcytic anemia in mk mice and Belgrade rats.17,20,22
These studies of Nramp2 have suggested a role for Nramp1 in
metal transport at the phagosomal membrane. However, controversy regarding the mechanism of Nramp1 metal transport with
respect to protein topology and the direction of metal transport in
relation to the proton gradient has persisted.23 Previously, we used a
metal-sensitive fluorophor (Fura-FF6) chemically coupled to zymosan particles to monitor divalent-metal flux by means of real-time
microfluorescence imaging across the membrane of single phagosomes formed in live Nramp1⫹/⫹ and Nramp1⫺/⫺ primary macrophages.24 Nramp1-positive phagosomes exhibited reduced intraphagosomal accumulation of externally added Mn2⫹ ions. Likewise,
Nramp1-positive phagosomes showed increased release of Mn2⫹
ions from preloaded Fura-FF6–zymosan particles. Mn2⫹ transport
by Nramp1 was abrogated by bafilomycin (vacuolar H⫹/ATPase
From the Department of Biochemistry, Center for the Study of Host Resistance,
Cancer Center, McGill University, Montreal, QC, Canada.
Reprints: P. Gros, Department of Biochemistry, McGill University, 3655 Sir
William Osler Promenade, Montreal, QC, Canada, H3G1Y6; e-mail:
[email protected].
Submitted February 7, 2003; accepted May 6, 2003. Prepublished online as
Blood First Edition Paper, May 15, 2003; DOI 10.1182/blood-2003-02-0425.
Supported by research grant RO1 AI35237-08 from the National Institute of
Allergy and Infectious Diseases (P.G.). J.R.F. is supported by a fellowship from
the Canadian Institutes of Health Research, and P.G. is supported by a
Distinguished Scientist salary award from the Canadian Institutes of
Health Research.
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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.
© 2003 by The American Society of Hematology
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
inhibitor), suggesting that Nramp1 functions to efflux Mn2⫹ ions in
a pH-dependent fashion from acidified phagosomes down the
proton gradient. This mechanism is similar to Fe2⫹ transport by
Nramp2 across the membrane of acidified endosomes, implying
that Nramp1/2 share a common transport mechanism and suggests
that Nramp1 exerts its antimicrobial activity through depletion of
divalent metals from the phagosomal space. This hypothesis is in
agreement with results from other independent studies.25-28
In contrast, increased Nramp1-dependent accumulation/binding
of isotopic Fe2⫹ into isolated phagosomes containing either Latex
beads or M avium has been reported.29-31 Increased Fe2⫹ accumulation was blocked by anti-Nramp1 antibodies, suggesting that
Nramp1 may transport cytoplasmic Fe2⫹ into phagosomes. In an
independent study, injection of Nramp1 mRNA into X laevis
oocytes induced small Zn2⫹-dependent inward currents suggestive
of metal uptake.32 Additional studies of pH-dependent transport of
isotopic Zn2⫹ led these authors to conclude that Nramp1 may
transport cytoplasmic metals into the phagosome by a proton/
divalent-metal antiport mechanism. These authors proposed that
increased phagosomal Fe2⫹ would stimulate oxygen radical production in situ via the Fenton reaction resulting in increased bactericidal activity.29-32 However, phagosomal metal influx mediated by
Nramp1 requires that Nramp1 would have to be mechanistically
distinct from Nramp2, with respect to direction of transport, use of
the transmembrane pH gradient, and/or membrane topology of
the proteins.
In order to address these differing conclusions regarding the
transport function of Nramp1, we sought to directly compare
Nramp1 and Nramp2 proteins. Should Nramp1/2 have the same
membrane organization and work by the same mechanism, ectopic
expression of Nramp1 at the plasma membrane of whole cells
would, like Nramp2, be expected to result in the pH-dependent
uptake of metals from the extracellular milieu. To test this proposal,
independent Chinese hamster ovary (CHO) transfectants expressing HA-tagged Nramp1 protein at the plasma membrane were
created. Comparative analysis of Nramp1 and Nramp2 activity was
performed with respect to transport function, pH dependence, and
metal ion selectivity. These studies show that the 2 proteins are
mechanistically indistinguishable but may have different substrate
selectivity.
Materials and methods
Cell culture
A mammalian expression plasmid (Nramp1HA-pCB6) containing a neomycin resistance gene together with the complete Nramp1 coding sequence
modified by the in-frame insertion of a hemagglutinin (HA) epitope
(YPYDVPDYAS) at amino-acid position 330 was constructed as was
previously described for the corresponding HA-tagged Nramp2 construct
(Nramp2HA-pCB6).15 CHO LR73 cells33 were cultured as previously
described15 and transfected with Nramp1HA-pCB6 using a calciumphosphate coprecipitation method.34 Stably transfected clones were selected (geneticin, 1 mg/mL; Invitrogen, Burlington, ON, Canada) for 10
days, followed by isolation and expansion of individual clones. Total
protein extracts were prepared from CHO transfectants, and Nramp1HA
protein expression was analyzed by immunoblotting with anti-HA monoclonal antibodies. The CHO transfectant N2-310a stably expressing Nramp2HA
was previously reported,15 as were the CHO transfectants stably expressing
Nramp1/2 proteins modified with a C-terminal c-Myc epitope (Nramp1Myc,
DIVALENT-METAL TRANSPORT BY Nramp1
1885
Nramp2Myc).5,18 All chemicals were purchased from Sigma Chemical
(Oakville, ON, Canada) unless otherwise noted.
Protein preparations and immunoblotting
Total cell protein extracts were prepared by solubilizing cell pellets in Tris
[tris(hydroxymethyl)aminomethane]-buffered saline (TBS; 100 mM TrisHCl pH 7.5, 150 mM NaCl) containing 1% Triton X-100, 1 mM PMSF
(phenylmethanesulfonyl fluoride), 2 ␮g/mL leupeptin, 2 ␮g/mL aprotinin, 1
␮g/mL pepstatin, 2 mM EDTA (ethylenediaminetetraacetic acid), and 20%
glycerol (20 minutes, on ice), and the insoluble material was removed by
centrifugation (16 000 g, 10 minutes, 4°C). Crude membrane fractions were
prepared as previously described.35 Protein concentrations were determined
using the Bradford assay (BioRad, Missisauga, ON, Canada). Discontinuous sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) was done with a 4% polyacrylamide stacking gel and a 10%
separating gel.36 Proteins were mixed with sample buffer36 and denatured
(20°C, 20-30 minutes) prior to loading onto gels. Prestained molecular mass
markers (BioRad) were included in all SDS-PAGE experiments.
For immunoblotting, proteins separated by SDS-PAGE were transferred
electrophoretically to polyvinylidine difluoride membranes (4°C, 450 V/hr;
Schleicher and Schuell, Keene, NH) in buffer containing 20% methanol and
0.01% SDS.37 Membranes were blocked (30 minutes-1 hour, 20°C) in TBS
containing 0.25% Tween-20 (TBST) and 5% nonfat skim milk. Membranes
were then incubated in blocking buffer for either 3 hours at 20°C or 16
hours at 4°C with one of the following primary antibodies: mouse
monoclonal anti–HA 16B12 or anti–c-Myc 9E10 antibodies (both used at
1/1000; Covance, Princeton, NJ), or affinity purified polyclonal rabbit
anti-Nramp2NT or anti-Nramp1NT antibodies (used at 1/1000), which are
directed against the amino-terminus of Nramp2 and Nramp1, respectively.4,18 Membranes were washed in TBST (3 ⫻ 5 minutes, 20°C) prior to
incubation with horseradish peroxidase–conjugated goat anti–rabbit IgG or
anti–mouse IgG (1/10 000; Perkin-Elmer, Woodbridge, ON, Canada)
secondary antibodies (1 hour, 20°C) in blocking buffer. Membranes were
washed in TBST (4 ⫻ 5 minutes, 20°C), and specific immune complexes
were revealed by enhanced chemiluminescence (ECL; Perkin-Elmer) and
autoradiography (Kodak, Rochester, NY).
Immunofluorescence
Immunofluorescence on nonpermeabilized whole cells was done as previously described.15 Live cells grown on coverslips were blocked (15
minutes, 4°C) and then incubated (1 hour, 4°C) with mouse anti–HA
monoclonal antibodies (diluted 1/50) or with affinity-purified rabbit polyclonal anti–Nramp1NT antiserum (diluted 1/50). Cells were then washed
extensively, fixed with 4% paraformaldehyde (20 minutes, on ice), and
incubated (1 hour, 20°C) with either goat anti–mouse IgG Cy3 (1/200;
Jackson Laboratories, West Grove, PA) or goat anti–rabbit IgG rhodamine
(1/100; Jackson Laboratories) secondary antibodies. Following extensive
washing, the coverslips were mounted onto glass slides using Permafluor
antifade reagent (Shandon, Pittsburgh, PA). The cells were photographed by
epifluorescence microscopy using a Nikon (Tokyo, Japan) Eclipse E800
microscope mounted with a 60 ⫻ oil-immersion objective lens and a Nikon
DXM1200 digital camera.
Surface biotinylation
Attached cells were rinsed twice with ice-cold phosphate-buffered saline
(PBS) and once with ice-cold borate buffer (10.0 mM boric acid, 154 mM
NaCl, 7.2 mM KCl, 1.8 mM CaCl2, pH 9.0), and then incubated (15
minutes, on ice) in the same buffer containing Sulfo-NHS-LC-biotin (0.5
mg/mL; Pierce, Milwaukee, WI). Unreacted biotin was removed by 3
washes with TBS containing 0.2 M glycine and one wash with TBS. Total
cell protein extracts were prepared from the biotinylated cells. Biotinylated
proteins were captured by incubation (16 hours, 4°C) of 100 ␮g of total cell
extracts with 50 ␮L of a 50% (wt/vol) slurry of streptavidin-agarose beads
(Pierce) in 500 ␮L (total) of TBS buffer containing 1% Triton X-100 and
protease inhibitors. Beads were washed 5 times with TBS/1% Triton X-100,
and bound proteins were eluted in 50 ␮L of sample buffer36 containing 5%
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FORBES and GROS
␤-mercaptoethanol (30 minutes, 20°C). Captured proteins were analyzed by
SDS-PAGE and immunoblotted with either anti–HA or anti–cMyc monoclonal antibodies, or with anti–Nramp1NT or anti–Nramp2NT affinitypurified antisera.
Divalent-metal transport by fluorescence quenching
Cells were detached using PBS/citrate (5-10 minutes, 37°C) and resuspended in loading medium (␣-MEM [minimum essential media], 0.5
mg/mL BSA [bovine serum albumin], 25 mM HEPES [N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid] pH 7.4). Cells (1 ⫻ 106 per assay)
were centrifuged and resuspended in prewarmed (37°C) loading medium
containing either 0.25 ␮M calcein-AM (acetoxy-methylester) or 2 ␮M
Fura2-AM (1 mM stock solutions in 100% DMSO [dimethyl sulfoxide];
Molecular Probes, Eugene, OR) followed by incubation at 37°C for 10
minutes or 1 hour, respectively. After this loading period, cells were washed
and resuspended in loading medium. Transport assays were performed with
a Perkin-Elmer LS-50B fluorometer equipped with a stirring and waterjacketed cuvette holder. To measure metal-dependent calcein/Fura-2 quenching, the fluorometer settings were excitation ␭ ⫽ 488/360 nm and emission
␭ ⫽ 517/510 nm with 5/7.5 micron bandpass slit widths. Immediately prior
to each transport assay, an aliquot of cells was washed once in PBS (37°C),
resuspended in 500 ␮L transport buffer (37°C; 150 mM NaCl, MES
[2-(N-morpholino)ethanesulfonic acid] pH 5-6 or HEPES pH 7-8), transferred into a prewarmed cuvette, and thereafter cell-associated fluorescence
was recorded continuously (0.5-second intervals). After a stabilization
period (60 seconds), divalent-metal was added and cell-associated fluorescence was recorded for an additional 120 seconds. Divalent-metals MnCl2
and CoCl2 were prepared as 2 mM stock solutions in water. Iron was
prepared fresh as a 2 mM FeNH4SO4 stock solution in transport buffer with
a 25:1 molar ratio of sodium ascorbate (50 mM) to maintain the metal in its
reduced form. Fluorescence quenching data were normalized for individual
samples to the fluorescence value taken at 70 seconds to facilitate visual
presentation of the data.
Radioisotopic divalent-metal transport assay
Cells were harvested as described for the fluorescent assay and resuspended
(107 cells per assay) in 1.5 mL of transport buffer (25 mM Tris, 25 mM
MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl2, pH 5.5).
Transport was initiated by addition of 1 mL of radioisotope buffer, followed
by incubation at 20°C. Mn2⫹ radioisotope buffer was transport buffer
containing 0.45 ␮M 54Mn (54MnCl2, 13.4 Ci/mmol [495.8 GBq/mmol];
Perkin-Elmer) and 22.05 ␮M MnCl2 (22.5 ␮M total Mn, 49:1 molar ratio of
cold MnCl2:54Mn), giving a final concentration of 9 ␮M Mn2⫹ in each
transport reaction. Fe2⫹ radioisotope buffer contained 1.125 ␮M 55Fe
(55FeCl3, 3.012 Ci/mmol [111.4 GBq/mmol]; Perkin-Elmer) and 21.375
␮M FeNH4SO4 (22.5 ␮M total Fe, 19:1 molar ratio of cold FeNH4SO4/
55Fe) together with 1.125 mM sodium ascorbate (50:1 molar ratio ascorbate–
Fe) giving a final concentration of 9 ␮M Fe2⫹. At predetermined time
intervals (0, 5, 15, 30 minutes), 500 ␮L cell aliquots were transferred to
microcentrifuge tubes containing a 200-␮L oil cushion (4:1 silicon oilmineral oil). Cells were pelleted by centrifugation (12 000 g, 10 seconds)
through the oil cushion, the aqueous phase was removed, and the walls of
the tube were washed with transport buffer. The oil cushion was removed,
and the cell pellets were digested with 0.1N NaOH. Lysates were
neutralized by addition of an equal volume of 0.1N HCl, and cell-associated
radioactivity was determined by liquid scintillation counting. The protein
content of each lysate was determined using the Bradford assay (BioRad).
Background radioisotope binding was determined by parallel control
transport experiments performed on ice. For metal ion selectivity studies,
cells (3 ⫻ 106) were resuspended in 375 ␮L transport buffer, and transport
was initiated by addition of an equal volume of radioisotope buffer. Stock
Mn2⫹ radioisotope buffer contained 0.4 ␮M 54Mn and 19.6 ␮M MnCl2 (20
␮M total Mn), and stock Fe2⫹ radioisotope buffer contained 1 ␮M 55Fe and
19 ␮M FeNH4SO4 (20 ␮M total Fe) in 1 mM sodium ascorbate. Cells were
incubated for 10 minutes with serial 2-fold dilutions (0.3125-10 ␮M final)
of these stocks, and the amount of cell-associated radioactivity was
determined as described above.
Results
PM expression of Nramp1HA detected by immunofluorescence
The aim of the present study was to express Nramp1 at the plasma
membrane (PM) of CHO cells, where its transport properties could
be studied and compared to Nramp2. Previous studies done in
primary macrophage, together with RAW264.7 and/or CHO cells
transfected with Nramp1 and Nramp2 constructs modified by the
addition of a C-terminal c-Myc tag (Nramp1Myc, Nramp2Myc),
indicated different subcellular distributions of the 2 proteins.5,18
Nramp2Myc was found to be expressed at the PM and in recycling
endosomes, whereas Nramp1Myc was not found at the PM but was
detected in lysosomes/late endosomes. Likewise, an Nramp2
protein modified by the insertion of an HA epitope (Nramp2HA)
into the predicted loop delineated by putative transmembrane (TM)
domains 7 and 8 was shown to be functional and expressed at the
PM of CHO cells with the HA epitope extracellularly located and
accessible to antibodies added to the external medium.15 In an
attempt to identify cell lines expressing Nramp1 at the PM,
Nramp1 cDNA was similarly modified by insertion of HA epitope
in the TM7/8 loop, followed by transfection into CHO cells.
Several transfectants stably expressing Nramp1HA protein were
identified (Figures 1-3; N1-1816, N1-94, N1-116, and N1-123).
The possibility of PM localized Nramp1HA protein was examined
by immunofluorescent detection of the HA epitope in nonpermeabilized cells, and representative images are shown in Figure 1. In
nonpermeabilized N1-1816 cells, Nramp1HA staining was detected at the cell periphery by extracellular anti–HA monoclonal
antibodies (Figure 1B), revealing a staining pattern similar to that
observed in control N2-310a cells expressing Nramp2HA (Figure
1C). PM staining of N1-1816 cells was Nramp1HA-specific, as it
was absent in untransfected CHO cell controls (Figure 1A) and
in N1-1816 cells stained with the secondary antibody alone
Figure 1. Detection of Nramp1HA and Nramp2HA proteins by immunofluorescence in nonpermeabilized cells. Mouse Nramp1 and Nramp2 cDNAs were
modified by in-frame insertion of a hemagglutinin (HA) epitope (YPYDVPDYAS) into
the predicted loop delineated by putative TM7 and TM8 followed by transfection into
CHO cells. Surface expression was monitored by immunofluorescent detection of the
HA epitope in nonpermeabilized cells with the mouse monoclonal anti–HA antibody
16B12. Control CHO cells (A), Nramp2HA-expressing CHO cell line N2-310a (C),
and Nramp1HA-expressing CHO cell line N1-1816 (B,D,E,F) were grown on glass
coverslips and incubated (1 hour, 4°C) with mouse anti–HA monoclonal primary
antibodies (A-C; 1/50 dilution), affinity-purified rabbit polyclonal anti–Nramp1NT
antibodies (E; diluted 1/50), or without primary antibody (D). Cells were then fixed,
and incubated with goat anti–mouse-IgG-Cy3 (A-D; 1/200) or goat anti–rabbit
IgG-rhodamine (E-F; 1/100) secondary antibodies. In panel F, N1-1816 cells were
fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100 prior
to addition of primary antibodies to detect both intracellular and PM-localized
Nramp1HA protein. Original magnification, ⫻ 600 for all panels.
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
Figure 2. Cell-surface biotinylation of CHO cells expressing Nramp1/2HA or
Nramp1/2Myc proteins. Cell-surface biotinylation was used to assess plasma
membrane expression of Nramp1 (N1) and Nramp2 (N2) proteins modified either by
insertion of an HA epitope into the loop delineated by predicted TM7/8 (panel A) or by
a c-Myc epitope at the C-terminus (panel B). Live cells were labeled with membrane
impermeant Sulfo-NHS-LC-biotin (see “Materials and methods”). Total cell protein
extracts were prepared, and biotinylated proteins were isolated by affinity capture
with streptavidin-agarose beads (from 100 ␮g of cell extract). Captured biotinylated
proteins (B, the entire eluate), and postcapture supernatant (S; 10% of remaining
supernatant volume) from CHO controls and from Nramp1/2HA (panel A) or
Nramp1/2Myc (panel B) transfectants were analyzed by SDS-PAGE and immunoblotted
with the corresponding anti–HA (panel A) or anti–cMyc monoclonal antibodies (panel B).
Panel C shows a direct comparison of biotinylated proteins from CHO control, Nramp1HA,
and Nramp1Myc expressing CHO transfectants immunoblotted with affinity-purified rabbit
anti–Nramp1NT antibodies that recognize both Nramp1HA and Nramp1Myc proteins. The
size of the molecular mass markers is shown to the left of the immunoblots.
(Figure 1D). This demonstrates that the predicted TM7/8 loop of
Nramp1HA is extracellular in N1-1816 cells. Additionally, Nramp1HA
was detected by an anti–Nramp1NT rabbit polyclonal antiserum
directed against the amino terminus of the protein in Triton X-100
permeabilized N1-1816 cells (Figure 1F), but not in intact N1-1816
cells (Figure 1E), demonstrating that the N-terminus of Nramp1HA
in N1-1816 cells was intracellular. Together, these results indicate
that Nramp1HA was expressed at the PM, and that it has a
membrane topology similar to that of Nramp2HA.
DIVALENT-METAL TRANSPORT BY Nramp1
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In previous studies of CHO cells expressing Nramp1/2Myc
proteins, Nramp2Myc was detected by immunofluorescence at the
PM, while Nramp1Myc was not.5,18 Thus, the surface biotinylation
of Nramp1/2Myc (Figure 2B) proteins expressed in CHO cells was
compared to that of Nramp1/2HA (Figure 2A). Both Nramp1Myc
and Nramp2Myc (Figure 2B) were biotinylated and detected in the
streptavidin-captured fraction (“B”) by immunoblotting with an
anti-Myc antibody. However, the relative proportion of Nramp1Myc
in this fraction compared to that of Nramp1HA was considerably
lower (Figure 2A-B, compare “B” to “S”), whereas surfacebiotinylated Nramp2HA and Nramp2Myc proteins appeared to be
detected in streptavidin-captured fractions in similar amounts
under the same conditions (Figure 2A-B, compare “B” to “S”).
Direct comparison of Nramp1HA and Nramp1Myc proteins in
streptavidin-captured fractions by immunoblotting with anti–
Nramp1NT polyclonal antiserum (Figure 2C, compare “B” to “S”)
further illustrates that Nramp1Myc was detectable in surfacebiotinylated fractions but in a significantly lower proportion
compared to Nramp1HA. This suggests that insertion of the HA tag
in the predicted TM7/8 loop of Nramp1 was responsible for the
increased PM localization of Nramp1HA protein.
Immunoblot analysis also showed that in contrast to
Nramp1Myc, which is expressed as a glycoprotein of ⬃ 90 to 100
kDa in CHO cells, Nramp1HA is present as 2 prominent bands of
⬃ 90 to 100 kDa and of ⬃ 50 kDa. The ⬃ 50 kDa was particularly
apparent when the proteins were immunoblotted against anti–
Nramp1NT polyclonal antibodies. Pulse-chase metabolic labeling
of Nramp1HA protein with 35S-methionine followed by immunoprecipitation suggested that the lower ⬃ 50 kDa band consisted of a
mixture of newly synthesized polypeptide as well as degradation
products and/or partially glycosylated Nramp1HA (data not shown).
Three additional stably transfected CHO clones expressing
significant amounts of Nramp1HA (N1-94, N1-116, N1-123) were
identified (Figure 3). Crude membrane preparations from these
clones were analyzed by immunoblotting with anti-Nramp1NT,
anti-Nramp2NT, or anti-HA antibodies (Figure 3A). The results
PM expression of Nramp1HA detected by surface biotinylation
Cell-surface biotinylation was used to verify PM expression of
Nramp1/2HA proteins. Live CHO, N1-1816 (Nramp1HA), and
N2-310a (Nramp2HA) cells were reacted with a membraneimpermeant biotinylating reagent (Sulfo-NHS-LC-biotin), and biotinylated proteins were captured using streptavidin-conjugated
agarose beads. Proteins present in the streptavidin-captured fraction (the entire eluate was loaded; “B” in Figure 2A) were analyzed
together with proteins remaining in the supernatant fraction after
streptavidin-capture (10% [vol] of the postcapture supernatant was
loaded; “S” in Figure 2A) by immunoblotting with anti-HA
antibody. Both Nramp1HA and Nramp2HA proteins were biotinylated and subsequently detected in the streptavidin-captured fraction
(“B”) of the corresponding CHO transfectants (Figure 2A). Nramp1/
2HA protein detection was specific, as it was absent in both biotinylated
untransfected CHO controls and in unbiotinylated Nramp1/2HA cell
extracts incubated with streptavidin beads (Figure 2A). Neither the
abundant cytosolic protein tubulin nor the endo/lysosomal mannose-6phosphate receptor proteins were detected in biotinylated fractions of the
cell lines examined (data not shown). These data demonstrate that
biotinylated Nramp1HA and Nramp2HA proteins in the streptavidincaptured fraction were due to surface labeling of PM-localized Nramp1/
2HA proteins, as opposed to labeling of intracellular protein pools by the
biotinylating reagent.
Figure 3. Immunoblot detection of cell-surface biotinylated Nramp1HA in
independent CHO transfectants. Membrane protein fractions were prepared from
independent CHO transfectants stably expressing Nramp1HA (N1-94, N1-116,
N1-123, N1-1816) or Nramp2HA (N2-310a) and from CHO controls. These proteins
(10 ␮g/lane) were analyzed by immunoblotting with affinity-purified rabbit polyclonal
anti–Nramp1NT or anti–Nramp2NT, or with monoclonal anti–HA antibodies (panel A). In
panel B, the cell lines were labeled by surface biotinylation (as described in “Materials and
methods”), and biotinylated proteins captured with streptavidin-agarose beads were
analyzed by immunoblotting with affinity-purified rabbit polyclonal anti–Nramp1NT or
anti–Nramp2NT antibodies, or monoclonal anti–HA antibodies. The size of the molecular
mass markers is shown to the left of the immunoblots.
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BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
confirmed the presence of a ⬃ 50 kDa and ⬃ 90 to 100 kDa
immunoreactive species in all Nramp1HA transfectants. These
clones were analyzed for presence of Nramp1HA protein at the PM
by surface biotinylation, followed by analysis of the streptavidincaptured proteins by immunoblotting (Figure 3B). N2-310a
(Nramp2HA) and N1-1816 (Nramp1HA) transfectants were used
as positive controls and CHO cells as negative controls. These
experiments confirmed results of transfectant N1-1816 (Figure 2)
by showing surface labeling/PM localization of Nramp1HA in
transfectants N1-94, N1-116, and N1-123. Interestingly, the ⬃ 90
to 100 kDa species was predominantly biotinylated, compared to
the 50 kDa species, indicating that the former comprises most of
the Nramp1HA protein present at the cell surface.
Nramp1HA divalent-metal transport properties monitored by
fluorescence quenching assays
The possibility of Nramp1HA metal transport activity at the PM was
investigated using transfectants N1-94, N1-1816, N1-116, N1-123 that
express different amounts of Nramp1HA. Nramp2HA expressing
transfectant N2-310a and CHO cells were used as positive and negative
controls, respectively (Figure 4). Metal transport was investigated in
whole cells using a fluorescence quenching assay.15 Cells were loaded
with the membrane-permeant acetoxymethylester (AM) form of the
dyes calcein or Fura-2, which are subsequently de-esterified in the
cytosol releasing the fluorescent, membrane-impermeant, metalsensitive probe. The effect of extracellularly added Fe2⫹ (Figure 4A) or
Co2⫹ (Figure 4C) on intracellular calcein fluorescence was measured.
Similarly, the effect of extracellularly added Mn2⫹ on intracellular
Fura-2 fluorescence (Figure 4B) was monitored for 200 seconds.
Experiments were carried out at pH 6.0, a pH known to be optimal for
Nramp2 activity in this assay.15,38 Typical fluorescence quenching traces
are shown in Figure 4A-C, and the rate of quenching was calculated
from the initial linear slope of individual traces generated in 3 to 6
independent experiments (Figure 4D-F). For each of the metal/
fluorophor combinations tested, Nramp1HA-expressing cells demonstrated substantial, rapid, time-dependent, and statistically significant
(Figure 4) fluorescence quenching compared to CHO controls. Background fluorescent quenching in CHO-negative control cells was
identical in 5 independent isolates (data not shown). Examination of the
rate of quenching measured in independent Nramp1HA transfectants for
each divalent-metal/fluorophor combination (Figure 4D-F) suggested
that divalent-metal uptake was proportional to the amount of Nramp1HA
protein expressed in each cell line and to the amount of Nramp1HA
protein localized to the PM as determined by immunoblot analysis of the
corresponding total cell membrane or surface biotinylated fraction
(Figure 2). Clone N1-94, which expresses the largest amount of
Nramp1HA, exhibited initial rates of Fe2⫹, Mn2⫹, and Co2⫹ uptake that
were respectively 2.1 ⫾ 0.1-, 2.7 ⫾ 0.2-, and 2.1 ⫾ 0.1-fold greater than
background measured in CHO cells. These results demonstrate that
Nramp1HAprotein is capable of Fe2⫹, Mn2⫹, and Co2⫹ uptake at the PM.
A hallmark of metal transport by eukaryotic and prokaryotic
members of the Nramp protein superfamily is that transport is pH
dependent, suggesting a proton-symport mechanism.23 Therefore,
the pH dependence of divalent-metal transport by Nramp1HA at
the PM was investigated. For this, CHO and N2-310a (Nramp2HA)
controls together with N1-94 cells (Nramp1HA) were loaded with
Fura-2 and Nramp1/2HA-dependent Mn2⫹ uptake was monitored
by fluorescence quenching at different extracellular pH (Figure 5).
Typical fluorescence quenching traces for each cell line are shown
for pH 5.0 (Figure 5A), 6.0 (Figure 5B), and 7.0 (Figure 5C). The
rate of quenching at each pH was calculated from the initial linear
slope of individual traces generated in 3 to 5 independent experi-
Figure 4. Metal transport by Nramp1HA and Nramp2HA in CHO transfectants
measured by quenching of calcein or Fura2 fluorescence. Metal transport was
measured in independent transfected CHO cell lines stably expressing Nramp1HA
(N1-94, N1-116, N1-123, N1-1816) or Nramp2HA (N2-310a) and in untransfected
CHO controls (color coded and identified in the inset). For Fe2⫹ (A) and Co2⫹ (C)
transport assays, cells were loaded with calcein-AM (0.25 ␮M), and the effect of
extracellular Fe2⫹ (added as sodium ascorbate: Fe2⫹) or Co2⫹ on intracellular calcein
fluorescence was continuously monitored at 37°C, pH 6.0, for 200 seconds
(0.5-second intervals) using a Perkin-Elmer LS-50B fluorometer (excitation ␭ ⫽ 488
nm; emission ␭ ⫽ 517 nm; 5 ␮M bandpass slit width). For Mn2⫹ transport assays (B),
cells were loaded with Fura2-AM (2 ␮M) and the effect of extracellular Mn2⫹ on
intracellular Fura2 fluorescence quenching was monitored (excitation ␭ ⫽ 360 nm,
emission ␭ ⫽ 510 nm, 7.5 ␮M bandpass slit width). Representative quenching curves
are shown in panels A-C as relative fluorescence (Rel. Fluor.) normalized to the
70-second time point (immediately following addition of metal) for all groups. The
average rate (with standard errors) of fluorophor quenching was calculated for Fe2⫹
(D), Mn2⫹ (E), and Co2⫹ (F) from the initial slope of 3-6 individual quenching curves. The
mean quenching rates of all Nramp1/2HAtransfectants were statistically different (P ⬍ .005)
than those of CHO controls with the following (still significant) exceptions: N1-116 (Fe2⫹;
P ⫽ .010), N1-123 (Co2⫹; P ⫽ .05) as determined using the Student t test.
ments (Figure 5D). These rate data are also expressed relative to the
CHO cell quench rate at each pH (Figure 5E). Nramp1HAdependent quenching of Fura-2 fluorescence by Mn2⫹ was strongly
pH dependent: Transport compared to CHO controls was substantial at acidic pH 5.0 and 6.0, but was minimal at neutral pH 7.0
(Figure 5E). This behavior was identical to that seen for Nramp2HA
tested under the same experimental conditions (Figure 5). Similar
results were obtained when Co2⫹ and Fe2⫹ were used as transport
substrates (data not shown). Therefore, results from fluorescence
quenching assays demonstrate that Nramp1HA is transport competent at the PM and indicate that metal transport by Nramp1HA is
pH dependent and mechanistically similar to Nramp2HA.
Nramp1HA transport properties monitored by radioisotopic
metal transport
Divalent-metal transport activity for Nramp1HA also was investigated using radioisotopic 54Mn or 55Fe. For these studies, control
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
DIVALENT-METAL TRANSPORT BY Nramp1
1889
Figure 5. pH-dependence of metal transport by Nramp1HA and Nramp2HA expressed in CHO transfectants. CHO control cells along with Nramp1HA (N1-94) and
Nramp2HA (N2-310a) expressing CHO transfectants were loaded with Fura2-AM, and the quenching of intracellular Fura2 fluorescence by extracellular Mn2⫹ ions was
determined as described in “Materials and methods” and in the legend to Figure 4. Transport was conducted in buffer systems adjusted to pH 5, 6, or 7. Data were collected
(0.5-second intervals) for 200 seconds. Typical fluorescence quenching traces for each cell line are shown for pH 5.0 (A), 6.0 (B), and 7.0 (C). The average rate (with standard
errors) of Nramp1/2-dependent Fura2 quenching by Mn2⫹ was calculated at each pH from the initial slope of 3-5 independent quenching curves (D). These rate data are also
expressed relative to background metal uptake in CHO-negative controls (E). The means of normalized quenching rates (E) of Nramp1/2HA transfectants were statistically
different than those of CHO controls at pH 5, pH 6 (P ⬍ .0005), and pH 7.0 (P ⫽ .0015 and P ⫽ .015, respectively) as determined using the Student t test. The normalized
mean of Nramp1HA transport (E) at pH 5 was not statistically different than that of its transport at pH 6 (P ⫽ .1). However, the normalized means of Nramp1HA transport (E) at
both pH 5 (P ⫽ .0006) and pH 6 (P ⫽ .015) were significantly different than that of its transport at pH 7.0. The mean of normalized Nramp2HA transport (E) at pH 5 was
statistically different than those of its transport at pH 6.0 and 7.0 (P ⬍ .0001). Likewise, the mean of Nramp2HA transport (E) at pH 6.0 was statistically different than that of its
transport at pH 7.0 (P ⫽ .012).
CHO cells, N1-94 (Nramp1HA) and N2-310a (Nramp2HA) transfectants were used. In the first set of experiments, cells were
incubated with tracer amounts of radioisotopic 55Fe or 54Mn in a
total metal concentration of 9 ␮M at pH 5.5. At predetermined time
intervals over a 30-minute period, cells were pelleted by centrifugation through an oil cushion to remove unincorporated metal, and
the cell-associated radioactivity was determined. Results from 3 to
5 independent experiments are shown in Figure 6A (55Fe2⫹) and 6B
(54Mn2⫹). Expression of Nramp1HA or Nramp2HA strongly stimulated accumulation of 55Fe2⫹ and 54Mn2⫹ into transfected CHO
cells. For both proteins, metal uptake was time dependent and
increased steadily over the 30-minute incubation period. Metal
transport was temperature dependent, being abrogated at 4°C
(Figure 6A-B). Subtraction of the nonspecific metal binding to cells
measured at 4°C gives the net amount of metal uptake, which is
shown in Figure 6C (55Fe2⫹) and Figure 6D (54Mn2⫹). Over 30
minutes, Nramp1HA caused a 4- and 10-fold stimulation of 55Fe2⫹
and 54Mn2⫹ uptake, respectively, compared to 3- and 4-fold
stimulation for Nramp2HA-expressing cells. Thus, Nramp1HA
appeared to transport Mn2⫹ to a greater degree than Nramp2HA,
while both proteins showed similar uptake of Fe2⫹.
The substrate selectivity of the Nramp1/2HA transporters for
Fe2⫹ and Mn2⫹ was characterized in dose-response experiments
(Figure 7). In these experiments, the total concentration of Fe2⫹ and
Mn2⫹ in the transport buffer was varied between 0.31 ␮M and 10
␮M. Cell-associated radioactivity was determined (after 10 minutes) at each divalent-metal concentration. Parallel transport assays
were carried out at 20°C and at 4°C. The 4°C binding data were
subtracted from each corresponding 20°C data point. In the case of
Fe2⫹ (Figure 7A), results were very similar for Nramp1HA and
Figure 6. Time and temperature dependence of Mn2ⴙ and Fe2ⴙ transport by
Nramp1HA and Nramp2HA measured by a radioisotopic uptake assay. Incorporation of 55Fe2⫹ (A) and 54Mn2⫹ (B) into CHO control cells, and Nramp1HA expressing
(clone N1-94) or Nramp2 HA expressing (clone N2-310a) CHO cell transfectants was
measured as described in “Materials and methods.” Briefly, cells were resuspended
in transport buffer (107 cells/1.5 mL), and transport was initiated by addition of 1 mL of
radioisotope buffer containing tracer amounts of either 54Mn2⫹ (total Mn2⫹ concentration of 9 ␮M) or 55Fe2⫹ (total Fe2⫹ concentration of 9 ␮M), followed by a 30-minute
incubation at 20°C. At predetermined time points (0, 5, 15, 30 minutes), metal
accumulation was calculated and expressed as picomolar equivalents (pmoleq)
divalent-metal/␮g total cellular protein as shown in panels A (Fe2⫹) and B (Mn2⫹),
which represent the average (with standard error) from 3 or 4 independent
experiments. Parallel experiments were conducted at 4°C to establish the temperatureindependent component of cell-associated radioactivity (binding). These values were
subtracted from the 20°C accumulation data to deduce net uptake values, which are
shown in panels C (Fe2⫹) and D (Mn2⫹).
Figure 7. Divalent-metal selectivity of Nramp1HA and Nramp2HA. The divalentmetal selectivity of Nramp1HA and Nramp2HA was compared in dose-response
experiments. Transfected CHO cell lines N1-94 (Nramp1HA) and N2-310a
(Nramp2HA), along with untransfected CHO cells, were resuspended in transport
buffer (3 ⫻ 106 cells/0.375 mL), and transport was initiated by addition of an equal
volume of radioisotope buffer containing tracer amounts of 55Fe2⫹ (A) or 54Mn2⫹ (B),
followed by incubation at 20°C. The final concentration of Fe2⫹ and Mn2⫹ in the
transport reaction was varied between 0.31 ␮M and 10 ␮M total divalent metal (by
2-fold serial dilution), while the specific radioactivity of each was held constant. For
Fe2⫹ transport buffer, a 50:1 molar excess of ascorbate to Fe2⫹ was used and kept
constant at each Fe2⫹ concentration. Transport was allowed to proceed for 10
minutes at 20°C, and cell-associated radioactivity was determined as described in
“Materials and methods” and in the legend to Figure 6. Parallel transport assays were
conducted at 4°C, and these values were subtracted from those obtained at 20°C to
determine the net amount of metal uptake, which is expressed as pmoleq of
divalent-metal/␮g of total cellular protein.
1890
FORBES and GROS
Nramp2HA with transport reaching a plateau at approximately 1
␮M, closely approximating that previously determined for Nramp2
in fluorescence quenching transport studies.15 In the case of Mn2⫹
(Figure 7B), transport appeared to plateau at approximately 2.5 ␮M
for both Nramp1HA and Nramp2HA. However, Nramp1HA appeared to be a more efficient transporter for Mn2⫹ than Nramp2HA,
with a minimum of 2-fold increases in total cellular accumulation
of the metal over all concentrations tested, in agreement with
results shown in Figure 6B. Together, these results confirm and
extend those obtained in fluorescence quenching studies (Figures
4-5) and establish that Nramp1HA (1) is active at the plasma
membrane, (2) can transport Fe2⫹, Mn2⫹, and Co2⫹, (3) is acid-pH
dependent, (4) is mechanistically indistinguishable from
Nramp2HA, and (5) appears to be a more efficient transporter of
Mn2⫹ than is Nramp2HA.
Discussion
Both the mechanism of transport and the substrate specificity of
Nramp1 at the phagosomal membrane have proven difficult to
study. In order to overcome this difficulty, we have inserted an HA
epitope into the TM7/8 loop of Nramp1 (Nramp1HA) and expressed the recombinant protein at the plasma membrane of CHO
cells. PM expression in CHO transfectants was demonstrated by
direct extracellular accessibility of the HA tag to antibodies in
whole cells using immunofluorescence (Figure 1), as well as by
cell-surface biotinylation experiments (Figures 2-3). These studies
provide topologic information for Nramp1 and establish that the
TM7/8 loop, which bears a number of predicted N-linked glycosylation sites,39 is extracellular when Nramp1HA is expressed at the
PM. By inference, the TM7/8 loop of Nramp1 would be found in
the lumen of phagosomal vesicles. We show also that the Nterminus of Nramp1HA, which is only accessible to anti–
Nramp1NT antibodies in permeabilized but not in whole cells, is
cytoplasmic. Thus, the membrane organization of Nramp1HA at
the PM is identical to that established previously for Nramp2HA,15
in agreement with the high degree of shared sequence similarity
(78%) and identity (64%).12
The PM localization of Nramp1HA in transfected CHO cells is
unique and different from that of the endogenous protein found in
either primary macrophages or neutrophils, where Nramp1 was not
at the PM but was found in lysosomes or tertiary granules,
respectively.5-7 Likewise, recombinant Nramp1Myc expression in
CHO cells or in RAW264.7 macrophages was restricted to the late
endosomes and lysosomes and was not detected at the PM.5
Unsurprisingly, expression of Nramp1Myc in CHO cells did not
stimulate uptake of extracellular divalent-metal.15 This suggests
that insertion of the HA epitope into the TM7/8 loop may directly
alter targeting, maturation, or processing of Nramp1HA, resulting
in its accumulation at the PM. Several endosomal/lysosomal
proteins transiently pass through the PM, prior to final localization
via endocytotic retrieval.40,41 The relatively high level of Nramp1HA
PM accumulation compared to Nramp1Myc detected in transfected
CHO cells may reflect partial or complete uncoupling of this
retrieval process due to the presence of HA tag. Also, the TM7/8
loop is predicted to have several N-glycosylation sites.39 The
structure or processing of these sites may be altered by the inserted
HA tag, thereby contributing to increased PM Nramp1HA accumulation. Indeed, apical PM targeting of Nramp2 in polarized MDCK
(Madin Darby canine kidney) cells has been shown to depend on
N-glycosylation.42 Likewise, the effect of N-glycosylation on
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
lysosomal protein targeting recently has been demonstrated for the
protein endolyn.43 Disruption of N-glycosylation in MDCK cells
was shown to cause redistribution of endolyn from an apical PM
lysosomal sorting pathway to the basolateral PM.
Our results from fluorescence quenching transport assays
(Figures 4-5) and transport studies with isotopic 55Fe2⫹ and 54Mn2⫹
(Figures 6-7) show that Nramp1HA expressed at the PM is
transport competent and functions as a multispecific divalent-metal
transporter. Divalent-metal transport by Nramp1HA was time and
temperature dependent and required an acidic pH. These Nramp1HA
transport characteristics are identical to those demonstrated for
Nramp2HA in our study, and they closely parallel results from
independent transport studies of Nramp2 expressed either in
Xenopus laevis oocytes13 or transfected mammalian cells,14,15 as
well as those reported for other eukaryotic and bacterial Nramp
homologs.23 PM expression of Nramp1HA has permitted the
analysis of its substrate selectivity compared to that of Nramp2HA.
Our experiments have shown that like other Nramp family
transporters,13,44-46 Nramp1HA can transport both Fe2⫹ and Mn2⫹
with apparent affinity in the low micromolar range. These values
are in the physiologically relevant concentration range and are
consistent with the known concentration of chelatable Fe2⫹ in
mammalian cells (0.2-1 ␮M),47 as well as cellular and tissue
concentrations of Mn2⫹ (⬍ 4 ␮M).48 In addition, Nramp1HA
appears to have a preference for Mn2⫹ ions when compared to
Fe2⫹, suggesting that the former may be a preferred substrate at the
phagosomal membrane. More detailed transport studies in membrane vesicles will be required to fully characterize the ion
selectivity of Nramp1 and Nramp2.
Despite this potential difference in substrate preference, our
results indicate that Nramp1 and Nramp2 are functionally equivalent multispecific divalent-metal transporters. The major physiologic difference between the 2 proteins appears to be at the level
of the cell type–specific expression and subcellular site of transport. Nramp2 is expressed in recycling endosomes, where it
transports transferrin-delivered iron from the acidified endosomal
lumen into the cytoplasm down a proton gradient.17-21 This
endosomal Fe2⫹ efflux is impaired in reticulocytes from microcytic
anemia mk mice, which bear a loss-of-function mutation at
Nramp2.17,22 It is very likely that Nramp1 functions in an analogous
fashion to remove divalent metals from the phagosome. Indeed,
microfluorescence imaging studies of Nramp1-positive phagosomes containing zymosan particles labeled with the metalsensitive fluorophor Fura-FF6 have demonstrated pH-dependent
metal efflux by Nramp1 at the phagosomal membrane within live
primary macrophage.24 For both Nramp1 and Nramp2, luminal
acidification is achieved by recruitment of the vacuolar H⫹/
ATPase, which provides the proton gradient necessary for Nramp
protein function.11,24,49 Altogether, these results strongly support a
model in which Nramp1 transports Fe2⫹, Mn2⫹, and likely other
divalent metals from the lumen of acidified phagosomes and into
the cytoplasm.
The mechanism by which Nramp1-mediated depletion of
divalent metals from the phagosomal space affects the survival and
replication of unrelated intracellular parasites is not fully understood. Several lines of evidence indicate that an adequate supply of
divalent metals such as Fe2⫹, Mn2⫹, and Mg2⫹ is critical for
successful intracellular parasitism.50,51 First, bacteria such as
Salmonella express, under different conditions, a surprising number of diverse high- or low-affinity adenosine triphosphate (ATP)–
dependent or proton-coupled iron (Fe2⫹, Fe3⫹) and/or manganese
transporters such as fepBCDG, sitAD, FeoABC, CorAD,52-56 and
BLOOD, 1 SEPTEMBER 2003 䡠 VOLUME 102, NUMBER 5
the Nramp homolog MntH.45,57,58 Second, mutation of several of
these transporter genes abrogate virulence and impair intracellular
replication in vivo.53,59-61 Third, intracellular replication of Salmonella within permissive Nramp1-negative RAW264.7 macrophage
was partly abrogated by incubation with the metal chelator
dipiridyl.59 Interestingly, recent studies using Salmonella-bearing
gene-specific reporter constructs have established that phagosomal
divalent-metal depletion by Nramp1 creates a stressful environment, which is sensed by bacteria within macrophage. The
intracellular bacteria responded to the presence of Nramp1 by the
transcriptional induction of a number of “virulence” genes that map
within Salmonella pathogenicity island 2 (SPI2) including ssrA and
sseJ.62 Similarly, infection of human macrophage by M tuberculosis results in induction of several mycobacterial genes required for
siderophore-mediated iron uptake.63 More specifically, preliminary
comparative transcriptional profiling studies of M bovis–infected
DIVALENT-METAL TRANSPORT BY Nramp1
1891
RAW264.7 macrophages (Nramp1-negative) and RAW264.7Nramp1 transfectants suggest that Nramp1 has a direct effect on the
level of transcriptional induction of the mycobacterial MbtB gene
involved in iron acquisition (J.R.F. and P.G., unpublished data,
2002). Together, these results suggest that Nramp1 can act as an
important antagonist of bacterial metal acquisition systems in the
microenvironment of the phagosome.
Acknowledgments
We acknowledge Dr Francois Canonne-Hergaux and Dr Samantha
Gruenheid for the preparation of anti-Nramp1NT and Nramp2NT
polyclonal antibodies, and Dr Virginie Picard for the kind gift of
cell lines and cDNA. We thank Steven Lam-Yuk-Tseung for advice
with the fluorescent quenching assays.
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