From the Department of Nutrition, Harvard School of Public Health,
Boston, Massachusetts 02115
We recently identified a novel factor involved in
cellular iron assimilation called SFT or Stimulator of
Fe Transport (Gutierrez, J. A., Yu, J.,
Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol.
149, 895-905). When stably expressed in HeLa cells, SFT was found to
stimulate the uptake of both transferrin- and nontransferrin-bound Fe
(iron). Assimilation of nontransferrin-bound Fe by HeLa cells stably
expressing SFT was time- and temperature-dependent; both the rate and
extent of uptake was enhanced relative to the activity of control
nontransfected cells. Although the apparent Km for
Fe uptake was unaffected by expression of SFT (5.6 versus
5.1 µM measured for control), the
Vmax of transport was increased from 7.0 to
14.7 pmol/min/mg protein. Transport mediated by SFT was inhibitable by
diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and
Fe2+-specific chelators. Because cellular copper status is
known to influence Fe assimilation, we investigated the effects of Cu
(copper) depletion on SFT function. After 4 days of culture in
Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was
reduced by more than 60%. Both control cells and cells stably
expressing SFT displayed reduced Fe uptake as well; levels of
transferrin-mediated import fell by ~80%, whereas levels of
nontransferrin-bound Fe uptake were ~50% that of Cu-replete cells.
The failure of SFT expression to stimulate Fe uptake above basal levels
in Cu-depleted cells suggests a critical role for Cu in SFT function. A
current model for both transferrin- and nontransferrin-bound Fe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the
divalent cation across the membrane bilayer. SFT expression did not
enhance levels of HeLa cell surface reductase activity; however, Cu
depletion was found to reduce endogenous activity by 60%, suggesting
impaired ferrireductase function may account for the influence of Cu
depletion on SFT-mediated Fe uptake. To test this hypothesis, the
ability of SFT to directly mediate Fe2+ import was
examined. Although expression of SFT enhanced Fe2+ uptake
by HeLa cells, Cu depletion did not significantly reduce this activity.
Thus, we conclude that a ferrireductase activity is required for SFT
function in Fe3+ transport and that Cu depletion reduces
cellular iron assimilation by affecting this activity.
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INTRODUCTION |
Mammalian cells can acquire iron either via the binding of
Fe2-transferrin
(Tf)1 to high affinity
surface receptors and its subsequent internalization into endosomal
compartments or through Tf-independent pathways utilizing plasma
membrane-based transport systems (1, 2). Several lines of evidence
suggest that Tf-dependent and -independent import involves
at least two functional activities as follows: a ferrireductase that
converts Fe3+ to Fe2+ and a carrier mechanism
that subsequently translocates Fe2+ across membrane
bilayers (3-8). Genetic studies in yeast have begun to identify a
number of factors involved in membrane transport of Fe, and at least
two uptake systems have been characterized: a high affinity transport
mechanism involving FTR1 and FET3 (9, 10) and a low affinity activity
provided by FET4 (11, 12). In addition, two ferrireductases, FRE1 and
FRE2, are known to play a role in yeast iron transport (13, 14).
Whereas FET4 can mediate the import of Fe2+ in the absence
of ferrireductase activity (11), FTR1 is believed to be a transporter
for iron that has been first reduced by FRE1 (or FRE2) and then
oxidized by the multi-copper oxidase FET3 (2, 10). However, although it
is presumed that FET3-oxidized Fe3+ is then translocated
across the yeast membrane, the exact valency of transported iron and
the precise function of FTR1 remain poorly defined. Nonetheless, an
interesting connection between Cu and Fe metabolism has been revealed
by the observation that FTR1 biosynthesis is impaired in the absence of
Cu-requiring FET3 (10). The relationship between Cu and Fe transport in
yeast (2) is highly reminiscent of the role of copper for iron
metabolism in mammals (15). Dietary Cu deficiency leads to microcytic
hypochromic anemia (16-18); this apparent iron deficiency appears to
be promoted by the loss of oxidase activity provided by circulating
ceruloplasmin, a Cu-requiring enzyme (19).
Although progress in characterizing the proteins involved in yeast Fe
transport has been made, relatively little is known about mammalian
uptake systems. Recently, two factors were identified to be involved in
this process. DCT1 (or nRAMP2) was defined to function in rat
intestinal iron uptake by the functional expression cloning of
Fe2+ transport activity in Xenopus oocytes (20).
Moreover, the mk mouse, which suffers from microcytic anemia
due to inefficient iron absorption (21), was found to have a defect in
the nRAMP2 gene; this missense mutation is predicted to impair nRAMP2
transport function (22). DCT1/nRAMP2 appears not only to mediate uptake of Fe2+, but it is capable of facilitating the
translocation of other divalent cations as well (20). In contrast, a
second transport protein that we have identified, called SFT or
Stimulator of Fe Transport, enables
Xenopus oocytes to take up iron presented as Fe3+, and this activity is inhibited by Cd2+
but not other divalent cations (23). Our preliminary investigation of
SFT function in mammalian cells revealed its capacity to also stimulate
the assimilation of Fe from the Tf-mediated pathway. Here, we report a
more detailed functional characterization of the stimulation of SFT of
non-Tf-bound Fe uptake in mammalian cells. These studies define the
function of SFT in promoting the uptake of iron presented to cells as
either Fe2+ or Fe3+. Furthermore, we explored
the potential role of copper for the activity of SFT by depleting cells
in culture with Cu-deficient media (24). Our results demonstrate that
Cu depletion decreases both Tf- and non-Tf-bound Fe uptake by HeLa
cells and that the function of SFT is impaired under these conditions.
Cu depletion was also found to partially inhibit a cell surface
ferrireductase, implicating a role for this activity in SFT-mediated
import of Fe3+. This idea is strongly supported by the
finding that SFT-stimulated Fe2+ uptake is unaffected in
Cu-depleted cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Preparation for Experiments--
HeLa cells
stably expressing a green fluorescent protein (GFP) chimera of SFT,
referred to here as HeLa(SFT), were previously established (23).
HeLa(SFT) and HeLa cells were grown in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 300 g/liter L-glutamine, 10% fetal bovine serum, 50 units/ml penicillin G, and 50 µg/ml streptomycin. For some experiments, cells were cultured under the same
conditions, except that serum was Cu-depleted as described below. For
transport assays, cells were grown to near confluence in 6-well (35-mm)
plates. In preparation for ferrireductase assays, HeLa cells were
washed three times with ice-cold phosphate-buffered saline (PBS),
followed by one additional wash in Hanks' buffer (137 mM
NaCl, 5.36 mM KCl, 1.3 mM CaCl2,
410 µM MgSO4, 490 µM MgCl2, 337 µM
Na2HPO4, 440 µM
KH2PO4, 4.17 mM NaHCO3,
and 5.55 mM dextrose).
Iron Uptake Measurements--
55FeCl3
was purchased from NEN Life Science Products (>3 mCi/mg).
55Fe-nitrilotriacetic acid (NTA) was prepared by complexing
55Fe with a 4-fold molar excess of NTA in 20 mM
HEPES-Tris, pH 6.0, 100 mM NaCl; this mixture was then
adjusted to pH 7 with NaOH essentially as described by Teichmann and
Stremmel (25). To start uptake assays, HeLa cells were incubated in
serum-free DMEM for 1 h, and then fresh serum-free DMEM was added
with specified concentrations of 55FeNTA. Transport assays
were performed at 37 or 4 °C for times indicated in the figure
legends. To quench uptake, cells were rapidly placed on ice, washed
three times with ice-cold PBS, and then incubated on ice with 1000-fold
molar excess unlabeled FeNTA for 20 min to remove nonspecifically bound
iron (26). Six hundred µl of PBS containing 1 mM EDTA
were added to each well to lift cells, and the amount of
cell-associated radioactivity was measured by scintillation counting of
duplicate 200-µl aliquots of this cell suspension. A 40-µl aliquot
of the cell suspension was solubilized with Triton X-100 (0.1% final
concentration), and protein content was determined by the Bradford
assay using bovine serum albumin as a standard (27). Uptake was
calculated as femtomoles of 55Fe per µg of cell protein.
For some experiments, cells were incubated at 37 or 4 °C with
55Fe3+ in the presence of freshly dissolved
ascorbate at a 1:20 molar ratio (final concentrations were 1 µM 55FeCl3 and 20 µM ascorbate). Accumulation of 55Fe from Tf
was measured as previously detailed (23). HeLa cells were incubated
with 40 nM [55Fe]Tf at 37 °C for 3 h
and then washed in ice-cold PBS, followed by a 45-min incubation on ice
with 1 µM unlabeled Tf to remove surface-bound
radioactivity. After three additional washes with ice-cold PBS, cells
from each well were harvested in 600 µl of PBS containing 1 mM EDTA. The amount of radioactivity in duplicate 200-µl
aliquots was measured, and the protein concentration was determined as
described above.
Serum Cu Depletion and Superoxide Dismutase (SOD) Activity
Measurements--
Cu depletion of serum was carried out as described
by Tong et al. (24). Briefly, serum was dialyzed at 4 °C
against two changes (12 h each) of 30 mM
TriethylenetetramineTM (TRIEN) in PBS. To eliminate TRIEN,
the serum was further dialyzed against three changes of PBS. The serum
protein concentration was found to be the same before and after
dialysis. To replete the dialyzed serum, CuCl2 was added at
a final concentration of 2 µM.
To determine the Cu status of HeLa cells, Cu,Zn-SOD activity was
measured following the protocol of Percival (28). Cells were washed
three times in ice-cold PBS and then suspended in 0.5 mM
EDTA, pH 8, containing 0.1 mM phenylmethylsulfonyl fluoride and 0.5 mg/liter pepstatin A (108 cells/ml). After being
frozen and thawed twice to ensure disruption of cell membranes, a
cytosolic fraction was collected upon centrifugation at 100,000 × g for 45 min at 4 °C. SOD assays were performed by incubating 50-µl aliquots of the cytosol with 250 µl of pyrogallol reagent (1 mM pyrogallol in 10 mM HCl and 1 mM diethylenetriaminepentaacetic acid) and 500 µl of 50 mM Tris-HCl, pH 8.2. After a 3-min incubation at room
temperature, the reaction products were measured spectrophotometrically at 340 nm. The activity of Cu,Zn-SOD was determined by subtracting the
activity due to the mangano form of SOD found in cytosol treated with
2.0 mM KCN. Enzyme activity was expressed as
A340/106 cells.
Ferrireductase Assays--
Extracellular ferrireductase activity
was monitored by the cell-mediated production of membrane-impermeant
ferrocyanide from ferricyanide (5, 29). In brief, HeLa cells were
washed in ice-cold PBS three times, followed by a final wash with
Hanks' buffer and then incubated in Hanks' buffer with
K3Fe(CN)6 at 37 or 4 °C. To stop
ferricyanide reduction, the cells were rapidly chilled on ice; to
measure the amount of ferrocyanide produced, a 700-µl aliquot of the
cell-conditioned media was mixed with, in order, 100 µl of 3 M sodium acetate, pH 6.4, 100 µl of 0.2 M
citric acid, 50 µl of 3.34 mg/ml bathophenanthroline sulfonate, and
50 µl of 3.3 mM FeCl3 prepared in 0.1 M acetic acid. After this reaction was allowed to proceed
at room temperature for 15-20 min, the absorbance at 535 nm was
measured. The difference of values obtained between incubations at 37 and 4 °C was taken to represent the cell surface ferrireductase
activity (A535/mg of protein).
Western Blot Analysis--
HeLa cells from a confluent 10-cm
plate were collected in PBS containing 1 mM EDTA and washed
three times in ice-cold PBS. Cells were resuspended in 100 µl of 25 mM HEPES, pH 7.4, 85 mM sucrose, 100 mM KCl, and 20 µM EGTA, frozen in liquid
nitrogen, and thawed slowly at room temperature to disrupt cells.
Nuclei and cell debris were removed by centrifugation at 850 × g for 10 min. The postnuclear supernatant was then collected
and subjected to ultracentrifugation using a Sorval RP100AT3 rotor at
95,000 rpm for 15 min at 4 °C to collect the membrane fractions for
SDS-PAGE. After electrophoretic transfer to nitrocellulose, Western
blots were incubated for 1 h in a blocking solution containing 10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20, and 5% dried milk. Antiserum recognizing the predicted fourth
extramembranous loop of SFT (Glu149-Met224) was
raised by injection of a glutathione S-transferase fusion peptide into rabbits. After removal of glutathione
S-transferase antibodies using glutathione-conjugated
Sepharose-4B saturated with glutathione S-transferase, this
antiserum was added in a 1:200 dilution in 10 mM Tris-Cl,
pH 8.0, 150 mM NaCl, 0.05% Tween 20, and 5% dried milk.
Following overnight incubation at 4 °C, the blot was washed three
times with 10 mM Tris-Cl, pH 8.0, 150 mM NaCl,
0.05% Tween 20 and then incubated for 1 h at room temperature with alkaline phosphatase-linked goat anti-rabbit IgG (1:10,000 dilution). The blot was then copiously washed with 10 mM
Tris, pH 8.0, 150 mM NaCl and incubated with nitro blue
tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate for color
development. The alkaline phosphatase reaction was quenched by placing
the blot in PBS containing 2 mM EDTA.
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RESULTS |
SFT Stimulates Tf-independent Iron Uptake--
SFT, a
"stimulator of Fe transport," was recently cloned by functional
expression using Xenopus oocytes (23). Although it has been
previously shown that SFT can stimulate Tf-mediated Fe transport by
mammalian cells, the fact that SFT was identified by expression of
Tf-independent transport activity indicates its involvement in
non-Tf-bound Fe uptake across the plasma membrane. To characterize
further the latter activity, iron uptake by HeLa cells stably
expressing an SFT-GFP chimera, HeLa(SFT), was measured. The results of
Fig. 1 show the time course of
non-Tf-bound Fe uptake measured for both non-transfected HeLa cells
(squares) and HeLa(SFT) (circles).
55Fe uptake by HeLa(SFT) cells is approximately 50%
greater than the activity of control cells, demonstrating the
stimulation of Tf-independent transport by SFT. As previously reported
(26, 30), the transport of 55Fe is time- and
temperature-dependent. At 37 °C, uptake is saturable, whereas at 4 °C, cell-associated radioactivity is less than 5% of
the total cell-associated radioactivity measured at 37 °C.

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Fig. 1.
Time course of iron uptake. HeLa(SFT)
(circles) and control HeLa 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 (fmol/µg of protein) taken up by HeLa cells 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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To better define properties of SFT-mediated Fe transport, a series of
inhibition experiments were carried out (Table
I). FeNH4 citrate blocks
uptake, indicating that the observed transport activity is specific for
Fe rather than its chelating ligand (NTA). Excess
diethylenetriaminepentaacetic acid (1 mM), a
membrane-impermeant Fe3+-specific chelator, also completely
blocks 55Fe uptake by both HeLa(SFT) and control cells.
Interestingly, an Fe2+-specific chelator, ferrozine, also
suppresses 55Fe accumulation by 60-70%, suggesting that
SFT-mediated transport may not be specific for Fe3+.
Treatment of cells with 50 µM chloroquine, a diffusable
weak base that inhibits the dissociation of Fe from internalized Tf to
block iron assimilation (31, 32), has no effect on SFT-mediated uptake
of non-Tf bound Fe. This result suggests that SFT either functions at
the plasma membrane or within endosomal compartments in a
pH-independent manner, although the time course of uptake (Fig. 1) and
the observed saturability of transport strongly argue for the
involvement of a cell surface carrier in this process. Finally, it has
been previously reported that Cd2+ inhibits Tf-independent
Fe transport by HeLa (30) and K562 cells (26); the presence of 1 mM Cd2+ appears to partially antagonize
SFT-mediated iron transport in a similar fashion.
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Table I
Properties of Fe transport by SFT
Assimilation of non-Tf-bound iron was measured for cells incubated with
1 µM 55FeNTA for 30 min. Specific uptake was
taken as the difference in cell-associated radioactivity determined at
37 and 4 °C. Transport activity in the presence of the indicated
inhibitors was calculated as percent of uptake determined for untreated
cells. To study the effects of cadmium, the cells were preincubated
with 1 mM CdSO4 for 30 min prior to addition of
55FeNTA. Shown is the average of data obtained in three
independent experiments.
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To characterize further the kinetic properties of non-Tf-bound Fe
uptake by HeLa(SFT) cells, initial rates of transport were measured as
a function of FeNTA concentration. The results of these experiments
(Fig. 2, panel A) reveal the
typical Michaelis-Menten curve, indicating that the transport process
is mediated by a limited number of carriers. A double-reciprocal plot
of initial transport rates, v0, is shown in
panel B (Fig. 2). HeLa(SFT) (circles) and control
cells (squares) have nearly identical values for the apparent Km of uptake: 5.1 ± 1.0 versus 5.6 ± 1.1 µM (n = 4). These values are consistent with previous studies of Tf-independent
Fe uptake by HeLa cells (30). As discussed by Sturrock et
al. (30), this kinetic parameter is an "apparent Km" since it essentially reflects the property of
iron accumulation by cells and not necessarily transport. Nevertheless, expression of SFT increases the Vmax of
transport from 7.0 ± 1.2 to 14.7 ± 4.2 pmol/min/mg protein
for HeLa(SFT); this increase would be expected if more transport sites
were expressed in the stably transfected cells.

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Fig. 2.
Kinetic analysis of iron uptake mediated by
SFT. HeLa(SFT) (circles) and control HeLa cells
(squares) were incubated with different concentrations of
55FeNTA for 15 min. Iron uptake measurements were carried
out as detailed for Fig. 1 and Table I. Panel A, presents
the Michaelis-Menten plot of HeLa(SFT) data obtained from four
different experiments (±S.D.); v0 (pmol
55Fe/min/mg protein) is shown as a function of [Fe]
(µM). Panel B, shows the double-reciprocal
plot of data obtained for HeLa(SFT) cells (open circles) and
HeLa cells (open squares) from a single experiment (±S.E.).
Similar results were obtained on three separate occasions.
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Western blot experiments reveal that the stably transfected cells do
indeed express ~2-fold greater amounts of SFT/SFT-GFP. To raise
antibodies against SFT, a glutathione S-transferase fusion peptide was prepared containing residues Glu149 to
Met224, representing the integral membrane protein's
"loop 4" or L4 domain. This segment is predominantly hydrophilic
and therefore predicted to be extramembranous (23). Fig.
3 demonstrates that the antiserum raised
against the L4 peptide recognizes both SFT and SFT-GFP. Endogenous HeLa
cell SFT migrates as an 85-87-kDa species; this is the predicted mass
of SFT homodimers that have been previously shown to remain associated
on SDS-polyacrylamide gels even under reducing conditions (23).
Interestingly, SFT-GFP also appears to form dimers that migrate as a
145-kDa species, consistent with the calculated mass for a homodimer of
the chimera, but it is curious that mixed "dimers" are not
observed. A plausible interpretation of this result is that
heterodimers may be intrinsically unstable and therefore degraded by
the HeLa cells.

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Fig. 3.
Western blot analysis of SFT present in HeLa
and HeLa(SFT) cells. Antiserum against the L4 domain of SFT was
raised as detailed under "Experimental Procedures." Membrane
fractions (100 µg of protein) from HeLa(SFT) and nontransfected
control cells were electrophoresed on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose. After blocking, the Western blot was
incubated with a 1:200 dilution of primary antibody overnight and then
washed and incubated with secondary alkaline phosphate-conjugated anti-rabbit antibody (1:10,000) prior to color development. SFT and
SFT-GFP are denoted by right arrows; standards (rabbit
muscle phosphorylase b, 97 kDa, and bovine serum albumin, 68 kDa) are denoted by left arrows.
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Expression of SFT Does Not Stimulate HeLa Cell
Ferrireduction--
Combined, the results discussed above indicate a
strong correlation between the level of SFT expressed by HeLa cells and
the Vmax of iron uptake, implicating a direct
role for SFT as an Fe carrier. This idea is consistent with the
characterized properties of SFT-mediated Fe uptake (Table I) that
closely resemble features previously described for Tf-independent
uptake (26). One cannot completely rule out the possibility, however,
that SFT enhances Fe uptake through some rather indirect but
stoichiometric interaction. A candidate role for SFT might be that of a
ferrireductase to reduce Fe3+ to Fe2+ prior to
translocation across the bilayer. Indeed, multiple studies have
revealed the requisite function of such a reductase activity in the
uptake of both Tf-bound (3, 4) and non-Tf-bound iron (5-8).
To investigate whether expression of SFT alters the intrinsic cell
surface ferrireductase activity of HeLa cells (6), cell-mediated production of ferrocyanide from ferricyanide was measured (Fig. 4). Neither the rate nor the extent of
ferrireduction is affected comparing the activity of HeLa(SFT) cells
(circles) with the endogenous function of nontransfected
cells (squares). This evidence excludes a functional role
for SFT in stimulating cell surface ferrireductase activity. Our
previous studies have shown that ferricyanide (the substrate used in
the ferrireductase assay) is a competitive inhibitor of non-Tf-bound
Fe3+ transport (5). Thus, these data suggest that SFT
stimulates uptake by acting directly as or on the carrier component of
this transport system rather than its associated ferrireductase
activity and, moreover, that the action of the ferrireductase
endogenous to HeLa cells is not rate-limiting for Tf-independent
uptake.

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Fig. 4.
SFT expression does not alter intrinsic
activity of HeLa cell surface ferrireductase. Ferrireductase
activity was measured as described under "Experimental Procedures."
Briefly, cells were incubated with 10 µM
K3Fe(CN)3 at 37 or 4 °C in Hanks' buffer. At times shown, 700 µl from the cell assay medium was mixed with 100 µl of 3 M sodium acetate, pH 6.4, 100 µl of 0.2 M citric acid, 50 µl of bathophenanthroline sulfonate,
and 50 µl of 3.3 mM FeCl3 prepared in 0.1 M acetic acid to assay for ferrocyanide production. Results
shown are the difference in absorption at 535 nm measured at 37 and
4 °C for duplicate samples (±S.E.). The time course of
ferrireduction catalyzed by HeLa(SFT) (circles) and control cells (squares) is presented with similar results obtained
in three separate experiments.
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Cu Depletion Inhibits Tf- and Non-Tf-bound Fe Uptake Mediated by
SFT--
Numerous studies have established a connection between copper
and iron metabolism (2, 15, 33). For example, yeast cells defective in
Cu transport display diminished Fe uptake activity (34). In studies of
cultured human cells, Tf-mediated Fe uptake was also found to be
reduced by experimentally induced Cu depletion (24). To investigate
whether Cu plays a role in SFT function, fetal bovine serum was
depleted by dialysis against the chelator TRIEN. After 4 days in
culture with Cu-depleted serum, HeLa cell SOD activity was decreased by
60-70%, consistent with previous results reported by Percival (28).
Cells grown under this condition have markedly reduced levels of
Tf-dependent and -independent Fe uptake activity (Fig.
5). Notably, although HeLa(SFT) cells have 51 ± 17% greater Tf-mediated uptake activity (panel
A) and 38 ± 11% greater non-Tf Fe transport (panel
B), Cu depletion reduces both activities to the same level as
non-transfected control HeLa cells. Thus, SFT apparently cannot
functionally stimulate Fe uptake in Cu-depleted cells. The decrease in
Fe assimilation by both pathways is marked as follows: Tf-mediated
uptake is diminished by 70-80%, whereas Tf-independent Fe transport
drops by ~50%. The fact that the effects of Cu depletion on HeLa
cell Tf-mediated uptake are more pronounced may indicate subtle
differences between Tf-dependent and -independent Fe
transport mechanisms. Studies in yeast have implicated the existence of
multiple iron uptake systems in high eukaryotes (2, 9-12). It is
possible that certain Fe transport mechanism(s) in mammalian cells,
other than SFT-mediated uptake, maintain normal function despite the Cu
deficiency. For example, the human equivalent of the rodent divalent
cation transporter DCT1/nRAMP2 may participate in HeLa cell
Tf-independent Fe uptake, and its activity may not be affected in the
Cu-depleted state.

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Fig. 5.
Effects of Cu depletion on iron uptake.
To lower cellular Cu levels, serum was dialyzed against 30 mM TRIEN in PBS and added to culture media supplemented
with or without 2 µM CuCl2. After 4 days of
culture in the Cu-depleted media, cells displayed Cu,Zn-SOD levels that
were 60-70% reduced (not shown). Both HeLa(SFT) and control HeLa
cells were cultured in the Cu-depleted (solid bars) or
Cu-replete medium (open bars). Tf-mediated Fe uptake (panel A) and Tf-independent Fe uptake (panel B)
activities were measured as described under "Experimental
Procedures." 55Fe uptake was calculated as percent
control measured for cells cultured under Cu replete conditions. Shown
are the mean values (±S.D.) from three independent experiments.
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Cu Depletion Reduces HeLa Cell Surface Ferrireductase
Activity--
Because ferrireduction has been implicated in the
membrane translocation of Fe released from Tf within endosomes (3, 4) as well as the import of non-Tf-bound Fe at the cell surface (5-8), one explanation for the effects of Cu depletion is that ferrireductase activity may be impaired, thus inhibiting Fe assimilation from both
pathways. To examine the influence of Cu depletion on ferrireduction, cell-mediated reduction of ferri- to ferrocyanide was monitored as
described above. The ferrireductase activities for HeLa and HeLa(SFT)
cells cultured in either Cu-depleted medium (solid bars) or
Cu-repleted medium (open bars) are presented in Fig.
6. Expression of SFT does not
significantly affect total cell surface ferrireductase activity
consistent with the data shown in Fig. 4. However, depletion of
cellular Cu levels diminishes the cell surface reductase activity by
~60%; for comparison, under these same conditions uptake of non-Tf-bound Fe falls by ~50% (Fig. 5B). Hence, in the
Cu-depleted state, cell surface ferrireductase activity appears to
become rate-limiting for transport. A reasonable conclusion drawn from these results is that the influence of Cu on SFT function in non-Tf Fe
uptake may be due to decreased cell surface ferrireductase activity.

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Fig. 6.
Cu depletion reduces HeLa cell surface
ferrireductase activity. Ferrireductase activity was measured as
described for Fig. 4. Both HeLa and HeLa(SFT) cells were cultured under
Cu-replete (open bars) or Cu-depleted conditions
(solid bars) for 4 days prior to the measurements of
ferrireductase activities. SOD activity was inhibited by 62% for
HeLa(SFT) and by 67% for HeLa cells on this occasion. Shown are the
average of duplicate samples (±S.E.), and the data are typical of
results obtained on three separate occasions.
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SFT Stimulates Fe2+ Uptake and This Activity Is
Unaffected by Cu Depletion--
According to the envisioned mechanism
of iron transport, the ferrireductase acting to convert
Fe3+ to Fe2+ would act upstream of an
Fe2+ carrier that translocates the cation across the
membrane bilayer. If the influence of Cu on Fe uptake can be attributed
to the diminished ferrireductase activity observed for depleted cells
as discussed above (Fig. 6), a prediction would be that the transport
of Fe2+ remains unaffected by these conditions. Related to
this hypothesis is the idea that SFT function, which is impaired by Cu
depletion (Fig. 5) and which does not modulate ferrireductase activity
(Fig. 4), may stimulate Fe uptake by acting as or on the putative
Fe2+ carrier mechanism.
To test whether SFT mediates Fe2+ uptake, transport assays
were performed with 55FeCl3 prepared in buffer
containing freshly dissolved ascorbate (1:20 molar ratio) to
effectively reduce the radioactive iron. The time course of uptake
presented in Fig. 7 (panel A)
demonstrates that HeLa(SFT) cells (circles) have enhanced
transport activity relative to nontransfected cells
(squares). HeLa(SFT) cells display levels of uptake 50%
greater than control cells, similar to the results described earlier
(Fig. 1). Although these data do not necessarily discriminate that
Fe2+ is the actual cationic form translocated across the
bilayer, our combined results do demonstrate that SFT functionally
stimulates Fe assimilation regardless of whether cells are presented
with ferric or ferrous forms. This activity is distinct from that
characterized for the rat intestinal iron transporter DCT1, which
appears to recognize and mediate uptake of divalent cations alone
(20).

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Fig. 7.
SFT stimulates Fe2+ uptake.
Panel A presents the time course of Fe2+ uptake
by HeLa(SFT) (circles) and control cells
(squares) incubated at 37 °C (filled symbols)
or 4 °C (open symbols) with 1 µM
55FeCl3 in the presence of freshly dissolved
ascorbate at a 1:20 molar ratio. At the indicated times, cells were
placed on ice and washed several times with ice-cold PBS to remove
nonspecifically bound radioactivity, and cell-associated cpm were
measured as described in Fig. 1. Panel B compares the
accumulation of 55Fe for HeLa(SFT) and control cells
cultured in either Cu-depleted (solid bars) or Cu replete
(open bars) medium for 4 days. Fe2+ uptake
assays were carried out as for panel A with a 2-h
incubation. Shown are the mean values (±S.D.) obtained from three
different experiments.
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Since our results indicate that cell surface ferrireductase activity is
the potential Fe3+ transport factor affected by Cu
depletion, Fe2+ uptake assays were also performed with
Cu-depleted HeLa(SFT) and control cells. Panel B (Fig. 7)
further demonstrates the stimulation of Fe uptake by SFT (compare
open bars). Importantly, nearly identical levels of
transport are maintained for both HeLa(SFT) and control cells that have
been Cu-depleted (compare open bars with solid bars). Thus, the ability of HeLa cells to take up iron presented in the ferrous form is insensitive to Cu depletion; moreover, stimulation of this transport activity by SFT is also unaffected. These
results indicate that a HeLa cell ferrireductase activity that is
impaired by depletion of cellular Cu levels must function upstream of
SFT to enable its apparent stimulation of Fe3+ uptake and
that this activity, which presumably mediates the reduction of
Fe3+ to Fe2+, is not required for the apparent
stimulation of Fe2+ uptake by SFT.
 |
DISCUSSION |
The stimulation of non-Tf-bound Fe transport by SFT has all of the
features previously described for Tf-independent transport by K562 (26)
and HeLa (30) cells. Uptake is time- and
temperature-dependent, inhibited by Cd2+,
blocked by Fe3+ and Fe2+ chelators, and appears
specific for the cation rather than its anionic ligand. Moreover, SFT
function in Tf-independent transport also appears to depend on cell
surface ferrireductase activity, as previously characterized for uptake
of ferric iron (5-8). The observations that HeLa(SFT) cells display a
Vmax two times greater than non-transfected HeLa
cells and that levels of SFT expression are approximately doubled in
the stably transfected cell line directly support the hypothesis that
SFT itself is a membrane carrier for iron. Indeed, SFT is an integral
membrane protein localized to the plasma membrane and endosomal domains (23); its expression stimulates both Tf-dependent and
-independent Fe assimilation (see Fig. 5), and it contains an
REXXE domain putatively involved in Fe binding (10).
Mutation of these critical glutamic acid residues results in the loss
of function of SFT (23), further implicating a direct role for SFT in
the translocation of Fe across membrane bilayers. Despite the strength
of this combined evidence, however, it is important to keep in mind the
consideration that the enhanced non-Tf-bound Fe uptake by HeLa(SFT)
cells could be a secondary consequence of SFT expression. Recent
controversies surrounding the precise role for cystic fibrosis
transmembrane conductance regulator in ATP transport reflect similar
concerns (35). Until rigorous evidence is obtained through
reconstitution studies, the precise role of SFT in the transmembrane
transport of Fe may remain elusive (see below).
The complexities associated with membrane transport of Fe have been
revealed by genetic studies in yeast. Multiple carrier mechanisms have
been defined (9-12), similar to recent reports investigating iron
uptake in mammalian systems (20, 22, 23). FET4, for example, appears to
play a direct role in the uptake of Fe2+ (12); this
activity resembles the function of the rodent DCT1/nRAMP2 transporter
in intestinal iron uptake (20, 22). It is likely that the latter
transporter may play a role in endosomal transport of Fe as well since
it has been discovered that the Belgrade rat, which has genetic defects
in the release of Tf-bound Fe from endosomes (36), harbors the exact
same mutation in DCT1/nRAMP2 as the mk mouse (37). One
notable difference is the ability of DCT1/nRAMP2 to mediate uptake of
other divalent cations (20). The idea that a common carrier exists to
translocate iron and other metals across the plasma membrane is
consistent with observations made for uptake by reticulocytes (38),
HeLa cells (30), fibroblasts (30, 39), and hepatocytes (8, 40) but
contrasts with our studies of K562 cells that suggest this
erythroleukemia cell line has distinct transport properties (26). The
specificity for Fe by FET4 not only resembles the characteristics of a
second yeast iron transport mechanism involving FET3 and FTR1 but also
parallels the properties we have defined for SFT, which was identified
by screening a K562 cell cDNA library (23). Both FET4 and FET3/FTR1 depend on the function of FRE1, a yeast ferrireductase, to transport Fe3+ (13, 14). Similarly, our investigation demonstrates a
strong dependence on HeLa cell surface ferrireductase activity for
SFT's stimulation of non-Tf-bound Fe3+ uptake. This
correlation is revealed by the finding that Cu depletion impairs
ferrireductase function and that expression of SFT is unable to
compensate for the loss in Fe3+ uptake under these
conditions (Fig. 5).
Certain facets of the yeast FTR1/FET3 iron uptake system are comparable
to elements associated with SFT function. FTR1 and SFT are both
integral membrane proteins containing six transmembrane-spanning domains and are of similar size (~40 kDa). Both of these putative carriers have REXXE motifs that Stearman et al.
(10) speculate may be involved in iron binding. In contrast, FET4 lacks
the appearance of similar motifs within its primary sequence and has a
greater mass of 63 kDa (11). The activities of both FTR1 and SFT
in the transport of ferric iron also appear to depend on Cu. However, our investigation reveals clear mechanistic distinctions between yeast
and man in the relationship of Cu in cellular iron uptake.
The importance of Cu in yeast Fe transport is believed to be associated
with its activity as a cofactor for FET3. FET3 was identified early in
the search for membrane carriers of iron (9); in a concurrent search
for iron transport proteins, Dancis et al. (34) uncovered
the necessity for a copper transport protein, CTR1. The connection
between FET3 and CTR1 was revealed by the findings that FET3 was a
multicopper oxidase (9) and that although
ctr1
fet3 yeast strains were unable to take
up Fe2+, activity could be restored to
ctr1
yeast grown in high copper media (34). Later studies also defined the
requirement for an intracellular Cu transporter, CCC2 (41). Finally,
the role of FTR1 in FET3-mediated iron uptake was defined by Klausner
and colleagues (10) when they characterized yeast transport mutants that phenotypically resembled fet3 mutants but had defects
that could not be compensated for by addition of excess Cu to the
growth medium. FTR1 appears to be required for FET3 expression and
loading with copper (10); elimination of the FTR1 gene results in a block in FET3 biosynthesis and expression at the cell surface, whereas
FET3 deletion mutants reciprocally cannot express FTR1 at the plasma
membrane. The idea that FTR1 is an iron transporter is supported by the
finding that mutants in its REXXE domain cannot take up iron
despite the fact that FET3 oxidase activity is unimpaired (10).
The multifactorial nature of Fe3+ uptake by yeast,
requiring FRE1, FET4, or FTR1/FET3, as well as the Cu-transporting
proteins CTR1 and CCC2, emphasizes the concerns raised earlier
regarding the exact role of SFT in mammalian non-Tf-bound Fe transport. Individually, each of these yeast genes appears to modulate iron assimilation and could serve as stimulators of Fe transport. To this
end, a complete description of yeast Fe import also remains elusive
since iron is required for growth, and fet3fet4 deletion strains, predicted to be defective in both of the pathways described above, are still capable of survival (2, 12).
Similar to the mechanism of Fe uptake in yeast, Cu depletion of HeLa
cells also results in defective Tf- and non-Tf-bound iron assimilation
(Fig. 5), yet our studies demonstrate that at least for the latter
activity, impaired cell surface ferrireductase function can account for
this loss (Fig. 6). Although it is possible that a ferroxidase activity
analogous to FET3 could be compromised by Cu deficiency, under our
assay conditions both basal and SFT-stimulated Fe2+ uptake
remains unperturbed. Because oxidation of Fe2+ by FET3 is
apparently required for FTR1 function in yeast (10), we conclude that
if a similar activity is involved in SFT-mediated iron uptake, it is
unlikely to be dependent on cellular Cu status.
This investigation yields two related findings regarding the function
of SFTs: 1) SFT stimulates the import of iron presented as either
Fe2+ or Fe3+, and 2) it is unlikely that SFT
simply acts as a ferrireductase to stimulate Fe uptake. Because our
results clearly demonstrate that Cu depletion inhibits ferric iron
uptake and that ferrireduction is impaired under these conditions, we
conclude that SFT-mediated Fe transport is downstream of the cell
surface ferrireductase activity, but whether its activity stimulates
the membrane translocation of ferrous iron alone is uncertain. As
discussed above, FRE1/2 ferrireductase activity is required for yeast
iron uptake when presented in the ferric form (13, 14), yet the
activity of the FET3 oxidase is also necessary for the role of FTR1s in
transport; these observations prompted Stearman et al. (10)
to suggest that FTR1 may bind both Fe2+ and
Fe3+ during its mechanism of transmembrane transport.
Importantly, the yeast reductase FRE1 has been identified to be a
b-type cytochrome similar to the NADPH oxidase
gp91phox. Shatwell et al. (42) have reported
biophysical characteristics of FRE1 indicating that it is a heme
protein; this is consistent with early investigations of Lesuisse and
Labbe (43) in which they describe the lack of ferrireductase activity
in heme-deficient yeast strains. In this context, analogies in the role
of copper for ferric iron uptake between yeast and man possibly may be
drawn since Cu deficiency is known to impair heme biosynthesis (see Ref. 33 and references therein). Therefore, one may speculate that the
influence of Cu depletion on HeLa cell Fe uptake may result from
defective heme synthesis leading to the loss of a cytochrome
b-like cell surface ferrireductase. Further experimentation is required to fully elucidate the role of Cu in mammalian iron assimilation and its exact function in SFT-mediated transport.