From the Department of Nutrition, Harvard School of Public Health,
Boston, Massachusetts 02115
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INTRODUCTION |
Although the major route for the cellular delivery of iron is via
receptor-mediated endocytosis of diferric transferrin
(Tf),1 several lines of
evidence indicate that mammalian cells also acquire iron through
Tf-independent pathways. Recently, two iron transporters have been
identified by functional expression cloning (1, 2). DCT1/Nramp2 is
thought to be involved in intestinal Fe2+ transport (2, 3)
and appears to mediate uptake of many other divalent cations (2). In
contrast, SFT shows high specificity toward iron for Tf-independent
uptake (1) and is able to stimulate translocation of both
Fe3+ and Fe2+ across membrane bilayers (4). A
functionally important REXXE motif has been found in SFT (1)
that resembles domains in the yeast iron transporter Ftr1 (5) and
ferritin light chains (6) that are implicated to interact with iron.
However, the topological arrangement of SFT within the membrane has yet
to be determined; hence, whether this putative iron-binding domain is
involved in extra- or intracellular functions is unknown.
Kyte-Doolittle analysis suggests that SFT is an intrinsic membrane
protein with six bilayer-spanning domains (1). Using BHK cells stably
expressing this transport protein with epitope tags, the topological
disposition of the N and C termini of SFT, as well as the orientation
of two large extramembranous loop domains, were defined in this
investigation. The membrane topology of SFT predicts that the putative
iron-binding REXXE domain resides within the cytoplasm.
However, while SFT presents specific iron-binding sites on the BHK cell
surface, mutants with Glu
Ala conversions in the REXXE
domain lack extracellular iron-binding activity. The unexpected
observation that depletion of cellular iron blocks SFT-mediated
transport suggests that Glu83/Glu86 may bind
intracellular iron to regulate SFT function and further indicates that membrane transport of iron mediated by SFT is itself an
iron-dependent process.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection--
BHK cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum, 50 units/ml penicillin G, and 50 µg/ml streptomycin.
For transport assays, cells were grown to near confluence in 6-well
(35-mm) plates. For Scatchard analysis and membrane topology studies,
cells were cultured in 24-well (16-mm) plates. BHK cells were
transfected using LipofectAMINETM as the DNA carrier (Life
Technologies, Inc.); generation of stable cells with pGL2 vector (Life
Technologies, Inc.), pGL2-SFT (1), pGL2-SFT(Glu
Ala), or pEFtag-SFT
was the same as described previously (1).
Indirect Immunofluorescence Microscopy--
Transiently
transfected BHK cells grown on coverslips were fixed and incubated with
PBS containing 1 mM MgCl2, 0.1 mM
CaCl2, 0.1% Triton X-100, 1 mg/ml BSA, and either antibody
against GFP (Molecular Probes, Eugene, OR) at 1:400 dilution or anti-HA
antibody (Babco, Richmond, CA) at 1:600 dilution. After rinsing, cells were incubated with a 1:500 dilution of either anti-rabbit
IgG-fluorescein isothiocyanate or anti-mouse IgG-fluorescein
isothiocyanate (both from Jackson ImmunoResearch Laboratories, West
Grove, PA). After copious washing, coverslips were mounted using
Fluoromount-G (Southern Biotechnology Associates Inc., Birmingham, AL)
containing 2.5 mg/ml N-propyl gallate. Immunofluorescence
microscopy was performed using an Axioskop epifluorescence microscope
(Carl Zeiss, Thornwood, NY) at a nominal magnification of × 100.
Construction of HA-tagged SFT and Mutant GFP-SFTGlu
Ala--
PCR was performed using pBSK-SFT (1) as template with
following two primers: 5'-CTGGACGGATCCATGAAAGAAT and
5'-GCGCTCTAGACAAGGGAGAC. The PCR product was digested with
BamHI and XbaI and subcloned into pEFtag, a
generous gift of Dr. A. Rao (7), to generate HA-tagged SFT containing
two repeated epitopes of YPYDVPDYA at its N terminus. Point mutagenesis
to introduce alanines in place of Glu83 and
Glu86 of SFT was as follows. Two complementary primers
(5'-GTGCAGAGCATCCATCATGCGTTAAAAAAT and
5'-ATTTTTTAACGCATGGATTGCTCTGCAC) to introduce
the alanine substitutions were employed in a PCR step with pGL2-SFT (1) as template along with universal SP6 primer and
5'-AATGGCGGCCGCCTTAATTATCAG, thus generating two products of 300 and
800 base pairs. The isolated fragments were then used as templates in a
second PCR reaction with only the last two primers amplifying SFT ORF
(1). After digestion with EcoRI and NotI, the 1.1 kilobase pair product was subcloned into pGL2 vector.
Iron Uptake Measurements--
55FeCl3
was purchased from NEN Life Science Products (>3 mCi/mg) and
[55Fe]nitrilotriacetic acid (NTA) was prepared
essentially as described by Teichmann and Stremmel (8). Uptake assays
were carried out by incubating cells with specified concentrations of
55FeNTA in serum-free medium for the indicated time
periods. Cells were lifted using PBS containing 1 mM EDTA,
and cell-associated radioactivity was measured by scintillation
counting and normalized to protein content (10).
Modified Radioimmunoassay (RIA)--
BHK(GFP-SFT) and
BHK(HA-SFT) grown in 24-well plates were washed with PBS and incubated
overnight at 4 °C with antibodies or preimmune sera at desired
dilutions in PBS containing 0.5% BSA. Specifically, BHK(GFP-SFT) cells
were incubated with anti-GFP antibodies (1:500) (Molecular Probes), and
BHK(HA-SFT) cells were incubated with anti-HA antibodies (1:500)
(Babco), anti-L4 (1:250), anti-L5 (1:250), or preimmune sera (1:250).
Generation of antisera against the two extramembranous loops of SFT, L4
and L5, was described previously (4). After extensive washes with PBS,
antibody binding was detected using 0.5 nM
125I-labeled Protein A (NEN Life Sciences Products,
specific activity >80 mCi/mg of protein); incubation was on ice for 60 min. After three washes with PBS, cells were lifted off the plates in
PBS containing 1 mM EDTA, and cell-associated radioactivity
was determined in duplicate 200-µl samples by
-counting. For some
experiments, cells were permeabilized with 0.5% Triton X-100 to detect
intracellular immunoreactivity.
Iron-binding Assay--
Assay conditions to detect iron binding
were as described previously (9) with the following modifications. BHK
cells were incubated with 55FeNTA in the presence or
absence of 1000-fold excess unlabeled FeNTA for 1 h at 4 °C,
washed once with PBS, then lifted with PBS containing 1 mM
EDTA. Cell-associated radioactivity measured in the presence of
unlabeled FeNTA was subtracted from equivalent samples incubated with
55FeNTA alone to determine specific 55Fe
binding. Binding affinity (Kd) and the number of
iron-binding sites (Bmax) were determined by
Scatchard analysis (11).
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RESULTS |
Expression of HA- and GFP-tagged SFT in BHK Cells--
We have
previously reported that a C-terminal chimera of SFT with GFP is
localized to the plasma membrane and recycling endosomes (1). To
identify potential alterations in the intracellular trafficking and
targeting of N-terminally tagged SFT, immunofluorescence experiments
were performed. Fig. 1a shows
that HA-SFT predominantly localizes to the juxtanuclear recycling
endosomal compartment with rather diffuse distribution at cell surface.
A similar pattern was observed for BHK cells expressing GFP-SFT
(panel b), indicating that modification of the N
or C termini of SFT does not interfere with its biosynthesis, membrane
transport, and intracellular localization. While the epitope
availability in RIA experiments described below suggests that SFT
molecules are evenly divided between cell surface and intracellular
compartments, its distribution within plasma membrane does not appear
to provide a sufficiently intense signal for prominent staining.
Similarly, Tf receptor staining is rather conspicuous in punctate
vesicular compartments wherein protein is concentrated but not on the
cell surface where it is known to reside (1).

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Fig. 1.
Expression of HA-SFT, GFP-SFT, and
GFP-SFTGlu Ala in BHK cells. BHK cells were grown
on coverslips in 6-well plates (35 mm). Transfection, indirect
immunofluorescence, and fluorescence microscopy were performed as
detailed under "Experimental Procedures." Shown are the staining
for HA-SFT (panel a), GFP-SFT (panel
b), and GFP-SFTGlu Ala (panel
c). Microscopy was performed at a nominal magnification
of × 100.
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Comparison of HA-SFT and GFP-SFT Iron Uptake Activities--
To
characterize the function of human SFT as an exogenously expressed
protein and to compare the transport activities of N- and C-terminal
chimeras of SFT, BHK cells stably expressing the GFP-SFT fusion
protein, BHK(GFP-SFT), or SFT with two N-terminal HA tags, BHK(HA-SFT),
were established. Fig. 2 shows the time course of non-Tf-bound Fe uptake for BHK(HA-SFT), BHK(GFP-SFT), and
non-transfected control BHK cells. Time- and
temperature-dependent uptake of 55Fe is
observed; at 4 °C, less than 5% of radioactivity is found associated with cells compared with 55Fe internalized at
37 °C. GFP-SFT and HA-SFT stimulate 55Fe uptake 1.7-fold
and 2.1-fold, respectively. The observation that BHK(HA-SFT) cells
display ~40% higher iron uptake activity is due to greater
expression of HA-SFT (see below). To compare the kinetic determinants
of non-Tf-bound Fe uptake by BHK(HA-SFT), BHK(GFP-SFT), and control BHK
cells, initial rates of uptake were measured as a function of iron
concentration. Double-reciprocal plots of these data are shown in Fig.
3. Control BHK cells have an apparent
Km for non-Tf-bound iron transport of 14.8 ± 3.4 µM (n = 3); this value is ~2- to
3-fold greater than that determined for HeLa cells (4, 13). However,
BHK cells expressing GFP-SFT or HA-SFT display lower
Km values of 5.2 ± 0.2 µM and
4.8 ± 0.3 µM, respectively (n = 3),
consistent with the measured value of SFT-mediated uptake for HeLa
cells (4). While control cells exhibit a Vmax
for non-Tf-bound iron uptake of 7.5 ± 0.8 pmol/min/mg of protein,
expression of SFT increases this kinetic parameter to 9.5 ± 0.8 and 13.6 ± 2.0 pmol/min/mg protein for BHK(GFP-SFT) and
BHK(HA-SFT) cells, respectively. Again, these observations are
consistent with the idea that more iron transporters are expressed in
the latter cell line.

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Fig. 2.
Time course of iron uptake. BHK(HA-SFT)
(circles), BHK(GFP-SFT) (triangles), and control
BHK cells (squares) were incubated at 37 °C
(filled symbols) or 4 °C (open
symbols) in the presence of 1 µM
55FeNTA. At the indicated times, 55Fe uptake
was quenched by rapidly chilling the cells on ice, followed by three
washes with ice-cold PBS. Nonspecific surface-bound 55Fe
was displaced by a brief (20-min) incubation on ice with 1 mM FeNTA in 25 mM HEPES, 150 mM
NaCl, pH 7.4. Cells were then lifted off plates with 600 µl of PBS
containing 1 mM EDTA. Cell-associated radioactivity was
determined in duplicate 200-µl aliquots. The mean value (±S.E.) of
55Fe taken up by BHK cells (fmol/µg of protein) is shown
as a function of time. Results from an individual experiment are
provided and reflect similar data obtained on three separate
occasions.
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Fig. 3.
Kinetic analysis of iron uptake mediated by
SFT. BHK(HA-SFT) (circles), BHK(GFP-SFT)
(triangles), and control BHK cells (squares) were
incubated with different concentrations of 55FeNTA for 10 min. Iron uptake measurements were carried out as detailed for Fig. 1.
The difference between values obtained at 37 and 4 °C was taken as
specific 55Fe transport. Shown is the double-reciprocal
plot of data from a single experiment (±S.E.) representative of four
independent experiments. Initial rate of uptake,
V0 (pmol of 55Fe/min/mg of protein),
is shown as a function of [Fe] (µM).
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Determination of the Membrane Topology of SFT--
Kyte-Doolittle
analysis predicts that SFT is an integral membrane protein with six
transmembranous segments (1). A hypothetical model for SFT topology is
presented in Fig. 4A. To
confirm this topological arrangement, antibodies were raised against
SFT-GST fusion peptides containing residues
Glu149-Met224 and
His246-Leu296, representing the integral
membrane protein's extramembranous loops L4 and L5, respectively.
Previous work has shown that anti-L4 antibody recognizes SFT and
SFT-GFP by Western blot analysis (4); similar results were obtained for
anti-L5 antibody (not shown). As shown in Fig. 4B, preimmune
antisera showed little reactivity against BHK(HA-SFT) cells. Anti-HA
and anti-L4 recognize and bind to these cells, but only upon
permeabilization (solid bars). In contrast,
anti-L5 binds to the surface of nonpermeabilized cells (open
bars), and more immunoreactivity is observed upon addition of detergents (solid bars) due to solubilization
of endosomal compartments and detection of intracellular SFT. Thus, the
L5 domain appears to be extracellularly situated, while L4 and the N
terminus of SFT are occluded from antibody in the absence of detergent,
indicating their cytoplasmic orientation. The enhancement in L5
antibody binding upon the addition of detergent further supports the
endosomal localization of SFT (Fig. 1) and suggests that the protein is
roughly distributed in equal amounts between the cell surface and the
intracellular compartments.

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Fig. 4.
Determination of SFT membrane topology.
Panel A shows a hypothetical model for SFT
membrane structure. Panel B presents the results
of modified RIA experiments. Briefly, BHK(GFP-SFT) and BHK(HA-SFT)
cells were grown in 24-well plates. After washing with PBS three times,
individual wells containing BHK(GFP-SFT) were incubated with anti-GFP;
wells containing BHK(HA-SFT) were incubated overnight with anti-HA,
anti-L4, anti-L5, or preimmune serum in PBS containing 0.5% BSA at
4 °C. Cells were then washed eight times with cold PBS, once with
PBS containing 0.5% BSA, and then incubated with 0.5 nM
125I-labeled protein A for 60 min on ice to detect
immunoreactivity. After three washes with PBS, cell-associated
radioactivity was determined by -counting. To determine antibody
binding to intracellular sites, cells were first permeabilized by
incubation with 0.5% Triton X-100 (solid
bars).
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To examine the orientation of the C-terminal domain, experiments were
performed with BHK(GFP-SFT) cells, and anti-GFP immunoreactivity was
assessed. 125I-Labeled Protein A binding is detected only
upon permeabilization, placing the C terminus within the cell
cytoplasm. Notably, the total amount of SFT is lower in these cells.
BHK(GFP-SFT) cells have 32% lower cell surface immunoreactivity than
BHK(HA-SFT) as detected by anti-L5 (data not shown), supporting the
conclusion that more transporters are expressed at plasma membrane in
the BHK(HA-SFT) cells (see Figs. 2 and 3). Anti-peptide antisera had negligible immunoreactivity in control BHK cells or cells stably expressing cytosolic GFP alone (see Fig.
5B), indicating that endogenous rodent SFT, if expressed, cannot be recognized by these antibodies. Thus, these data confirm the predicted topology of SFT
(Fig. 4A). Although direct evidence to support the
intracellular placement of the REXXE domain is lacking, the
overall hydrophobicity of the M1 to M3 region (~64%), the number of
charged residues within this motif (4 of 5 amino acids), and the
observation that the protein is inserted into membranes
cotranslationally (1) support the conclusion that this putative
iron-binding domain is situated on the cytoplasmic face of the
membrane.

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Fig. 5.
Glu Ala mutant fails to
stimulate iron uptake. In panel A, modified
RIA experiments were performed for BHK(GFP-SFT), BHK(GFP-SFTGlu
Ala), and BHK(GL2) cells as detailed under "Experimental
Procedures" and in Fig. 4. In panel B,
BHK(GFP-SFT) (circles), BHK(GFP-SFTGlu Ala)
(triangles), and BHK(GL2) cells (squares) were
incubated with 1 µM 55Fe at 37 °C
(filled symbols) and 4 °C (open
symbols). 55Fe uptake assays were performed as
detailed in Fig. 1. Results from an independent experiment are provided
here, and similar results were obtained on three separate
occasions.
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Function of the REXXE Motif--
Crystal structure analysis has
implicated a role for REXXE motifs in iron binding by
ferritin (6). Moreover, the REXXE motif of the yeast Ftr1
iron transporter has been shown to play an important functional role
(5). We have previously demonstrated that mutation of the two glutamic
acid residues within the SFT domain eliminates its ability to mediate
iron uptake in Xenopus oocytes (1). To study how this motif
may affect SFT-mediated transport in mammalian cells, stable BHK cells
were established expressing Glu
Ala point mutations in the GFP-SFT
chimera. As shown in Fig. 5A, antibody-binding experiments
demonstrate that nearly equal numbers of SFT molecules are expressed on
the surface of BHK(GFP-SFT) and BHK(GFP-SFTGlu
Ala)
cells as detected by anti-L5 binding. As a control, anti-L5 binding
BHK(GL2) cells was measured and found to be negligible (<25 cpm/µg
of protein). These results indicate that the biosynthesis, membrane
trafficking, and cell surface expression of the mutant is unaffected.
This idea is confirmed by the immunofluorescence data presented in Fig.
1 demonstrating that the cellular distribution of GFP-SFTGlu
Ala is identical to wild-type (compare panel
c to panels a and b). Fig.
5B compares the time course of non-Tf-iron uptake by
BHK(GFP-SFT) and BHK(GFP-SFTGlu
Ala) and demonstrates
that although GFP-SFT (circles) stimulates Fe uptake, Glu
Ala mutations within the REXXE domain impair this
transport activity. The transport activity of BHK(GFP-SFTGlu
Ala) cells is nearly the same as that measured for the control.
The failure of the mutant to stimulate iron transport is consistent with results observed when its expression was studied in
Xenopus oocytes (1) and further identifies that
Glu83 and/or Glu86 are critical residues for
SFT transport function rather than its biosynthesis and
trafficking.
Characterization of Iron-binding Activity of SFT--
To examine
the ability of SFT to bind iron and to determine whether the two
glutamic acid residues are involved in this activity, the association
of 55Fe with the membrane surface of BHK(GL2),
BHK(GFP-SFT), and BHK(GFP-SFTGlu
Ala) cells was
measured. As shown in Fig. 6, saturable
55Fe binding is observed for control BHK(GL2) cells.
Similar activity is detected for BHK(GFP-SFTGlu
Ala)
cells, but BHK(GFP-SFT) cells show enhanced surface 55Fe
binding. Scatchard analysis (Fig. 7)
reveals a single class of binding sites (1.1 × 107/cell) with a Kd of 5.9 ± 1.0 µM determined for BHK(GFP-SFT) cells (n = 3). This value is quite comparable to the apparent Km of transport associated with SFT function which
is ~5 µM (Fig. 3). In contrast, BHK(GFP-SFTGlu
Ala) and BHK(GL2) cells have limited binding sites (~7 × 106/cell), with a Kd of 12.3 ± 2.1 µM and 15.7 ± 3.1 µM, respectively
(n = 3). The latter results suggest that
Glu83 and Glu86 are critical not only for iron
transport, but also for the apparent iron-binding activity of SFT.
Given that the topological data described above contradict the idea
that the REXXE domain would be extracellularly disposed and
thus capable of binding 55Fe, a simple explanation for the
failure of the mutant to bind iron is that protein folding might be
perturbed. To examine whether Glu
Ala mutations impair SFT function
by disrupting its membrane topology, experiments were performed to
determine the orientations of L4, L5, and the C-terminal domain. Fig.
8 shows that anti-GFP and anti-L4 only
bind to the BHK cells stably expressing GFP-SFTGlu
Ala
upon solubilization, while anti-L5 binding is detected for both intact
and permeabilized cells, consistent with wild type membrane topology.

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Fig. 6.
Glu Ala mutations block SFT
iron-binding activity. BHK cells were assayed for cell surface
iron-binding activity as described under "Experimental Procedures."
In brief, BHK(GFP-SFT) (closed circles),
BHK(GFP-SFT Glu Ala) (open
circles), and BHK(GL2) (squares) cells cultured
in 24-well (16-mm) plates were incubated with the indicated
concentrations of 55FeNTA for 1 h at 4 °C in the
absence or presence of 1000-fold molar excess of FeNTA. Nonspecific
binding determined in the presence of excess FeNTA was subtracted to
yield specific iron-binding activity (pmol of 55Fe bound).
Cells were solubilized with 0.5% Triton X-100, and the protein
concentration was determined. Shown are results from a single
experiment representative of data obtained on three separate
occasions.
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Fig. 7.
Scatchard analysis of iron binding by
SFT. Scatchard analysis of the binding data shown in Fig. 6 is
presented. Bound/free is shown as a function of bound 55Fe.
Data are from a single experiment representative of results obtained on
three separate occasions.
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Fig. 8.
Topology of SFT-GFPGlu Ala. Modified RIA experiments were performed essentially as
described for Fig. 4. BHK(GFP-SFTGlu Ala) cells were
incubated with anti-GFP, anti-HA, anti-L4, anti-L5, or preimmune serum
in PBS and cell-associated antibody was measured by
125I-labeled protein A binding. Solid
bars are data for cells permeabilized with 0.5% Triton
X-100.
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Depletion of Intracellular Iron Inhibits SFT Function--
To
further explore the role of the intracellular REXXE motif,
the iron content of BHK cells was depleted by overnight incubation with
50 µM desferrioxamine. 55Fe uptake assays
were then performed to compare the activities of control and
iron-depleted BHK(GFP-SFT), BHK(GFP-SFTGlu
Ala), and
BHK(GL2) cells. As shown in Fig.
9A, 55Fe uptake
was unaffected in control BHK(GL2) and BHK(GFP-SFTGlu
Ala) cells upon iron depletion, consistent with observations
made for fibroblasts (14). Although expression of wild type SFT
stimulates iron import nearly 2-fold over control, treatment with
desferrioxamine (solid bars) blocks SFT-mediated
transport activity, suggesting that the transport function of SFT is
iron-dependent. Incubation of BHK(GFP-SFT) cells with 65 µM FeNTA for 2 h after desferrioxamine treatment
completely restored SFT activity (not shown). Moreover, Fig.
9B shows that iron depletion does not result in lower cell surface 125I-labeled protein A binding as detected by
anti-L5, although surface 55Fe binding is reduced ~50%.
Thus, effects of iron depletion are reversible and are not accounted
for by an iron-dependent decrease of cell surface SFT.

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Fig. 9.
Depletion of cellular iron inhibits SFT
function. In panel A, iron uptake assays
were carried out for BHK(GFP-SFT), BHK(GFP-SFTGlu Ala),
and BHK(GL2) cells as detailed for Fig. 2, except that cells were
incubated with 1 µM 55FeNTA for 20 min. To
deplete cellular iron, cells were incubated overnight with 50 µM desferrioxamine (solid bars). In
panel B, surface 55Fe binding and
anti-L5 immunorecognition for intact BHK(GFP-SFT) cells was determined
exactly as described for Figs. 4 and 6. Cell-surface iron and
antibody-binding activities of BHK(GFP-SFT) cells treated with
desferrioxamine (solid bars) were calculated as
the percent measured for control (non-depleted) BHK(GFP-SFT) cells
(open bars). Results from a single experiment are
shown and represent data obtained on three separate occasions.
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DISCUSSION |
When exogenously expressed in BHK cells, SFT stimulates
non-Tf-bound iron uptake with properties essentially the same as
described for HeLa cells (1). The previously reported
Km (~5 µM) is nearly identical to
values determined for BHK(GFP-SFT) and BHK(HA-SFT) cells by our initial
rate analysis. However, unlike HeLa cells which have an endogenous pool
of SFT (1), BHK cells appear to have an uptake system with different
properties (Km ~15 µM). Complex
kinetics of iron uptake suggesting the presence of two transport
mechanisms in BHK(GFP-SFT) or BHK(HA-SFT) cells are not observed in our
study. Although one interpretation is that Lineweaver-Burk analysis may
not be powerful enough to reveal the existence of multiple uptake
mechanisms under our experimental conditions, it should be noted that
the possibility that SFT modulates the activity of endogenous BHK cell
transporters cannot be excluded. However, the findings that the
biosynthesis, membrane trafficking, and transport characteristics of
this protein are identical whether expressed in rodent or human cells
lend support to the idea that SFT function does not require other
protein cofactors. These observations are in contrast to the
Saccharomyces cerevisiae transporter Ftr1, which requires
Fet3 for proper function (12); moreover, the Schizosaccharomyces
pombe homolog (Fip1) of Ftr1 cannot complement ftr1
cells unless the S. pombe
homolog of Fet3 (Fio1) is co-expressed (12). Thus, we might tentatively
conclude that SFT acts as a direct transport facilitator for iron,
although rigorous reconstitution experiments will be necessary to
confirm its activity.
Previous primary structure analysis predicted SFT to be a hydrophobic
protein with six transmembranous domains (1). Taking advantage of the N
and C terminally tagged SFT constructs and anti-peptide antibodies, we
have confirmed the topology suggested by Kyte-Doolittle analysis (Fig.
4A). This model situates the putative iron-binding
REXXE motif on an intracellular domain. However, Glu
Ala
mutations in this domain block SFT-mediated iron binding and
translocation at the cell surface. Immunofluorescence microscopy
indicates that these mutations do not interfere with biosynthesis and
targeting of SFT (Fig. 1), and loss of function for mutant SFT is not
due to lower amounts of expressed protein at the cell surface (Fig.
5A). In addition, Glu
Ala mutations do not alter the
topological arrangements of L4, L5, and the C terminus (Fig. 8), such
that incorrect membrane folding is unlikely to account for failure of
SFT mutant to bind and take up iron. An unexpected and interesting
observation in our study is that SFT-mediated transport activity is
dependent on intracellular iron status. Based on this finding and
features of the Glu
Ala mutant, we speculate that the L2 domain of
SFT acts as a "sensor" to monitor cellular iron status.
The dependence of SFT function on iron must occur at a
post-translational level, because the activity of exogenously expressed protein appears to be exclusively affected. Moreover, the fact that
this effect is fully reversed by brief incubation of cells with iron
suggests that SFT may directly bind the cation. Thus, the
REXXE motif domain may act to bind cytosolic "free" iron
to maintain SFT in a conformation competent for binding and transport of extracellular iron. Since loss of the ability to sense intracellular levels of iron could result in excessive iron accumulation due to
unchecked SFT activity, the failure of the Glu
Ala mutant to bind
iron may provide a feedback signal that blocks iron uptake. Thus, we
propose that the glutamic acids of the L2 region of SFT not only bind
iron but serve as a sensory domain to regulate SFT activity. The
envisioned sensor mechanism predicts that when the intracellular free
iron level rises, SFT-mediated transport is diminished due to
interaction at the REXXE site. It is known, for example,
that increased cellular iron content decreases Tf receptor number to
limit iron import under these conditions. However, unlike the effects
observed for SFT function, iron chelation up-regulates Tf receptor
expression. Although SFT can stimulate Tf-mediated iron uptake (1), we
have yet to determine whether intracellular iron status influences SFT
function in this pathway. Because iron depletion appears to enhance Tf
receptor number, our results may suggest that SFT function in
Tf-mediated uptake is differentially regulated from that of Nramp2,
which has also been implicated recently to play a role in endosomal
transport (16) and which appears to be up-regulated in response to
diminished iron levels (2).
Post-translational effects of iron on non-Tf-mediated uptake have been
reported previously (14, 15). In the studies of fibroblasts, HeLa, CHO,
Hep-G2, and L-cells, increased levels of extracellular iron were found
to increase iron assimilation (14, 15). Kaplan and co-workers
hypothesized that this enhanced uptake was due to the recruitment of
cryptic intracellular iron transporters to the cell surface, an idea
supported by the enhanced Vmax observed under
these conditions. In our study, cell surface SFT numbers do not change
upon iron depletion, but further experiments are required to study the
influence of high levels of cytosolic iron on the function and
distribution of SFT. Our investigation, however, raises critical
questions regarding the regulation of SFT function by cellular iron
status and provides the basis for future analysis of regulatory
domains. A key to understanding SFT structure-function relationships
will be the ultimate identification of the extracellular iron-binding
and transport site that appears to be influenced by the intracellular
REXXE domain.