Research Article
4365
Subcellular localization of iron regulatory proteins to
Golgi and ER membranes
Stephanie M. Patton1, Domingo J. Piñero2, Nodar Surguladze1, John Beard3 and James R. Connor1,*
1
Department of Neurosurgery, G.M. Leader Family Laboratory for Alzheimer’s Disease Research, Pennsylvania State University College of
Medicine, Hershey, PA 17033, USA
2
New York University, Department of Nutrition and Food Studies, 35 West 4th Street, 10th floor, New York, NY 10012-1172, USA
3
Department of Nutritional Sciences, Pennsylvania State University, S-126 Henderson Building, University Park, PA 16802, USA
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 5 July 2005
Journal of Cell Science 118, 4365-4373 Published by The Company of Biologists 2005
doi:10.1242/jcs.02570
Summary
Interaction between iron regulatory proteins and iron
responsive elements on certain mRNAs is at the core of
regulation of intracellular iron homeostasis. Previous
results suggested that in cultured cells iron regulatory
proteins (IRPs) exist in cytosolic and microsomal
subcellular locations and that this distribution is affected
by cellular iron status. In this study, we tested the
hypothesis that the membrane-associated fractions of iron
regulatory proteins are specifically in the endoplasmic
reticulum and Golgi membranes. Confocal microscopy
revealed that IRP1 could be co-localized to the endoplasmic
reticulum and the Golgi apparatus. To examine the
intracellular distribution of IRPs biochemically, we used
rats fed normal or iron-deficient diets. As expected, the
IRPs were found predominantly in the cytosolic fraction.
However, subfractionation of crude microsomal
preparations revealed IRP1 in the Golgi apparatus. In
animals fed an iron-deficient diet, IRP1 was found in the
Introduction
Iron is a crucial nutrient for all cells but excess iron can result
in cellular damage through the production of free radicals via
the Fenton reaction (Gutteridge and Halliwell, 1989). In
addition, iron deficiency in rodents causes dramatic
behavioral and motor effects (Beard and Connor, 2003;
Glover and Jacobs, 1972; Hunt et al., 1994; Youdim et al.,
1989). Consequently, it is essential for cells to maintain
intracellular iron homeostasis. The model for cellular iron
homeostasis has become a classic example of posttranscriptional regulation of protein synthesis. In this model,
mRNA of critical proteins involved in the movement, storage
and utilization of iron contain iron response elements (IREs).
These are specific sequences that, by interacting with
cytoplasmic iron regulatory proteins (IRP1 and IRP2),
regulate the fate of the mRNAs (Crichton et al., 2002). Also,
iron levels in the cell regulate IRP1 and IRP2 via different
mechanisms. IRP1 activity changes from IRE binding to
cytosolic aconitase with increasing levels of iron, while IRP2
is degraded through the proteosomal pathway (Guo et al.,
1994; Samaniego et al., 1994). Control of the interaction
between the IREs and the IRPs has been studied under
Golgi apparatus and the endoplasmic reticulum. To
identify the mechanisms and factors involved in the
localization of iron regulatory proteins in the cytosol and
membrane fractions, cells were treated with a phorbol
ester, a protein kinase C inhibitor (chelerythrine),
␤, and 1,2-bishydrogen peroxide, interleukin-1␤
(o-aminophenoxy)-ethane-N,N,-N⬘⬘N⬘⬘-tetraacetic
acid
tetraacetoxy-methyl ester. The results indicate that ironregulatory-protein-binding activity in the membrane
fraction can be altered by cell stress or iron status and that
phosphorylation plays a role in the translocation. As a
result of this study we propose a novel model for
intracellular distribution of IRPs and identify differences
between the two iron regulatory proteins.
Key words: Iron regulatory proteins, Endoplasmic reticulum, Golgi
apparatus, Localization
different conditions and has been described in detail
elsewhere (Drapier et al., 1993; Eisenstein, 2000; Hanson and
Leibold, 1998; Pantopoulos and Hentze, 1995; Schalinske
and Eisenstein, 1996).
Functionally, cytoplasmic IRPs exist in the cell in three
major pools: (1) bound to an IRE, (2) free and available to
bind to an IRE and (3) free but unavailable to bind IRE (for
example, as part of the cytoplasmic aconitase activity pool).
Variations in the levels of intracellular iron produce a change
in the IRP/IRE binding activity and, as a consequence, a
change in the levels of the proteins involved in iron
homeostasis, such as ferritin and transferrin receptor
(Eisenstein, 2000). We have previously reported that IRPs are
associated with an intracellular membrane fraction (Pinero et
al., 2001). In this study, we explore in greater detail the
intracellular distribution of IRPs using both a microscopic and
biochemical approach. We identify the ER and Golgi
membranes as the sources of the IRP activity in the
microsomal membrane fraction and reveal that only IRP1 can
be detected. Finally, the localization of IRP1 to the membranes
involves phosphorylation and is responsive to cellular iron
status and agents of stress.
4366
Journal of Cell Science 118 (19)
Journal of Cell Science
Materials and Methods
Microscopic analysis
In order to visualize the distribution of IRPs in cells, we used
immunocytochemistry and epitope tagging (c-myc). The microscopic
analysis was performed on the following cell lines: NIH 3T3 and
SW1088. Both cell lines were purchased from American Type Culture
Collection (ATCC; Rockville, MD, USA).
The initial immunocytochemical studies were performed using
antibodies against IRP1 and IRP2 that were provided by Betty Leibold
(University of Utah, USA). The epitope-tagged antibody (c-myc) was
purchased from Ambion.
For co-localization studies, ~5⫻104 cells were plated on poly-Dlysine-treated glass slides. Cells were allowed to attach for 1 hour at
37°C under a humid 5% CO2 atmosphere, then the slides were
submerged in either Dulbecco’s modified Eagle’s medium (DMEM,
Gibco, Rockville, MD, USA), DMEM supplemented with ferric
ammonium citrate (100 mg/l), or DMEM supplemented with
desferrioxamine (DFO) (100 ␮M) and cultured at 37°C under a humid
5% CO2 atmosphere. After 72 hours, the slides were removed, washed
once with Hank’s buffered salts solution and twice with PBS and fixed
with 4% paraformaldehyde for 30 minutes. Slides were blocked as
described above and then incubated with primary anti-rat IRP1 (Alpha
Diagnostics, International; San Antonio, TX, USA), the ER marker
anti-human calnexin (AF18; Abcam, Cambridge, MA, USA), or the
Golgi marker anti-human 58K Golgi protein (58K-9; Abcam; in 5%
normal goat serum) for 90 minutes at room temperature, as per the
manufacturer’s protocols. Free antibodies were removed by three
washes with PBS and slides were then incubated with Alexa Fluor
555-conjugated goat anti-mouse (organelle markers) and Alexa Fluor
488-conjugated goat anti-rabbit secondary antibody (for IRP1) and
DAPI for a further 60 minutes. The slides were washed, mounted and
covered with a coverslip. Negative controls for antibody staining were
prepared as described above, except that the primary antibody was
omitted. Confocal microscopy was performed using a Leica TCS
confocal laser scanning unit equipped with a DMR inverted
microscope and a 63/1.4 objective. An argon/krypton laser was used
to generate light at 488 and 555 nm for Alexa excitation and 360 nm
for DAPI excitation. A high-pass filter with wavelength cut-off at 500
nm was used to recover Alexa fluorescence and a low-pass filter with
wavelength cutoff at 460 nm was used to detect DAPI fluorescence.
Each fluorophore was excited sequentially to insure the lowest
interference between the detector channels.
Fractionation of SW1088 cells for isolation of ER-rich fractions
The protocol for isolation of ER-rich fractions was adapted from the
methods of Zhang et al. (Zhang et al., 1998). Briefly, twenty 150 cm2
culture flasks were washed in Hank’s buffered salts and once in the
homogenization medium (0.25 M sucrose, 1 mM EDTA, 10 mM
Hepes-NaOH, pH 7.4 with protease inhibitor cocktail). The cells were
suspended in homogenization medium and harvested by scraping. The
cells were centrifuged at 2.5 g for 3 minutes, resuspended in
homogenization medium that contained protease inhibitors and then
disrupted by Dounce homogenization. The homogenate was
centrifuged at 3000 g for 10 minutes to remove cell debris and the
resulting supernatant was centrifuged again at 100,000 g for 40
minutes. The pellet was washed with homogenizing medium and
resuspended in the same medium. A 12 ml linear Optiprep gradient
(0-20%) was prepared and the vesicle solution was layered on top of
the gradient and this was centrifuged at 200,000 g for 16 hours; 1 ml
fractions were collected. NADPH-cytochrome C reductase and lactate
dehydrogenase assays were performed on all fractions to determine
ER and cytosolic activity, respectively (Graham, 1993).
Western blot analysis
To demonstrate the distribution of IRPs in SW1088 microsomal
fractions, 6% SDS-PAGE was used; 5 ␮g of total protein was loaded
for IRP1 analysis and 2.5 ␮g of total protein was loaded for IRP2
analysis. The proteins were transferred to PVDF membranes and
incubated with 5% powdered milk in Tris-buffered saline-Tween 20
overnight at 4°C. The primary antibody was diluted 1:1000 and
incubated with the membrane for 1 hour. Following washes in Trisbuffered saline-Tween 20 (TBS-Tween), the membrane was incubated
in secondary antibody that had been diluted 1:5000. The IRP1 primary
antibody was the same as used for immunohistochemistry. The IRP2
primary antibody (clone 4G11) was a gift from Wolff Kirsch (Loma
Linda University, Loma Linda, CA, USA). The secondary antibody
used for IRP1 was a rabbit anti-chicken IgG conjugated to horseradish
peroxidase. The secondary antibody used for IRP2 was rabbit antimouse IgG conjugated to horseradish peroxidase. The distribution
of protein on the membrane was visualized using a KPL
chemiluminescence kit.
Fractionation of rat livers for isolation and separation of ER
and Golgi
Sprague-Dawley rats were fed either control or iron-deficient diets
prepared as described previously (Pinero et al., 2000) for 6 weeks,
after which the animals were sacrificed, and the livers were removed,
weighed and minced. Homogenization medium (0.25 M sucrose, 1
mM EDTA, 10 mM Hepes-NaOH, pH 7.4) was added and the livers
were Dounce homogenized. The homogenates were centrifuged at
4000 g for 5 minutes. The microsomal pellets were obtained by
centrifugation at 140,000 g for 1 hour. The microsomal pellets were
resuspended in 1 ml of homogenization medium and each was toploaded onto a 2.5%-30% pre-formed discontinuous Optiprep gradient.
The gradients were centrifuged at 200,000 g for 2.5 hours and 1 ml
fractions were isolated by tube puncture.
Western blot analysis of fractions containing IRP, ER and Golgi
To demonstrate the distribution of IRPs in rat liver microsomal
fractions, 4%-20% gradient SDS-PAGE was used; 10 ␮g of total
protein was loaded for IRP1 analysis and 5 ␮g of total protein was
loaded for ER and Golgi analysis. The proteins were transferred to
PVDF membrane and incubated with 5% powdered milk in TBSTween overnight at 4°C. The following primary antibodies were used
(anti-IRP1, Alpha Diagnostics, International; San Antonio, TX; and
anti-calnexin and anti-Golgi 58K protein, Abcam). Both anti-IRP1
and anti-calnexin antibodies were diluted 1:1000 and anti-Golgi 58K
protein was diluted 1:5000 and incubated with the membrane for 1
hour. Following washes in TBS-Tween, the membrane was incubated
in secondary antibody that had been diluted 1:5000. The secondary
antibody used for IRP1 and calnexin was a goat anti-rabbit IgG
conjugated to horseradish peroxidase and the secondary antibody used
for Golgi 58K protein was rabbit anti-mouse IgG conjugated to
horseradish peroxidase. The distribution of protein on the membrane
was visualized using a KPL chemiluminescence kit. All analyses were
quantitated using the Fuji LAS-3000 system.
Cell culture
The following sets of experiments were performed to evaluate the
intracellular distribution of IRP binding activity after activation of
IRPs. Human astrocytoma cells (SW1088) and human embryonic
kidney cells (HEK293) were purchased from ATCC. The cells were
grown in a 5% CO2 atmosphere at 37°C in DMEM supplemented
with 10% fetal bovine serum (Biocell, Rancho Dominguez, CA,
USA), 4 mmol/l glutamine (Sigma, St Louis, MO, USA), 1000 U/ml
penicillin G and 100 ␮g/ml streptomycin sulfate and 250 ng/ml
amphotericin B. All the experiments were performed when the cells
reached 70-80% confluence. The cells were treated with 0.2 ␮M
phorbol 12-myristate 13-acetate (PMA; Biomol, Plymouth Meeting,
Subcellular localization of iron regulatory proteins
4367
Journal of Cell Science
PA, USA) (for 16 hours), a phorbol ester shown to induce IRP activity
in HL-60 cells (Schalinske and Eisenstein, 1996), interleukin-1
(Sigma) (for 16 hours), hydrogen peroxide (for 30 minutes),
chelerythrine (Biomol; 2.0 ␮M, for 16 hours) an inhibitor of protein
kinase C, as well as the intracellular calcium chelator BAPTAAM (1,2-bis-(o-aminophenoxy)-ethane-N,N,-N⬘N⬘-tetraacetic acid
tetraacetoxy-methyl ester; Biomol) (for 30 minutes). Untreated cells,
as well as cells treated with DMSO at the same concentration used
for the cells treated with PMA or BAPTA-AM, served as controls.
Additionally, cells were also treated for 16 hours with either ferric
ammonium citrate (FAC; 100 mg/l; Sigma) or the iron chelator
deferoxamine (DFO, 100 ␮mol/l; Sigma) to demonstrate their
responsiveness to iron addition or chelation. When the cells were
treated with BAPTA-AM, the medium was changed after 30 minutes
and replaced with either control medium or FAC- or DFO-containing
medium for the next 16 hours. The results were consistent for all cell
types used.
Separation of cytosolic and membrane fraction in cell lines
After completion of the experiments, the cells were washed with
Hank’s solution (Sigma), treated with trypsin until detachment, and
collected. To separate membrane-bound organelles from cytosol, the
cells were washed and homogenized in lysis buffer (10 mm/l Hepes
pH 7.4, 40 mmol/l KCl, 5% glycerol, 3 mmol/l MgCl2, 0.1 mmol/l
EDTA) containing a protease inhibitor cocktail (Sigma) and Prime
RNase inhibitor (Eppendorf Scientific, Westbury, NY, USA).
Homogenization was carried out using a sonicator with a microtip
(Branson Sonifier 250, Branson Inc., Danbury, CT, USA), on pulse
mode. The homogenates were centrifuged at 4,000 g for 5 minutes at
4°C to sediment the nuclei. The resulting supernatant was centrifuged
at 145,000 g for 1 hour at 4°C and the cytosolic fraction was collected.
The pellet was washed twice in 1 ml lysis buffer, resuspended in 100
␮l of lysis buffer and either processed or stored at –70°C. 50
␮l of this solution were treated with Triton X-100 (final
concentration 0.5%) at 4°C for 1 hour, centrifuged at 20,000
g for 5 minutes at 4°C, and the supernatant collected. This
supernatant will be referred to here as membrane fraction.
Fig. 1. Cellular distribution of IRP1 and IRP2. (A,B) Distribution
of (A) IRP1 using an IRP1 antibody (red) and (B) IRP2 using an
IRP2 antibody (red), in NIH3T3 cells. The cell nuclei are
visualized with DAPI (blue). Both A and B reveal a cytosolic
distribution for IRP1 and IRP2 but IRP2 distribution is more
punctate than IRP1. (C,D) Distribution of (C) c-myc-tagged IRP1
and (D) c-myc-tagged IRP2, in NIH3T3 cells. C and D are
representative cells that show the cytosolic distribution of both
IRP1 and IRP2 (clear area is the cell nucleus). IRP1 is localized
more in the perinuclear region whereas IRP2 localization extends
further toward the perimeter of the cell.
RNA band shift assay
A synthetic RNA transcript containing the IRE was generated
from the oligonucleotide template 5⬘-ttatgctgagtgatatccctctcctaggacgaagttgtcacgaacctgcctagg-3⬘ with T7 polymerase in
the presence of [32P]CTP. Binding reactions were carried
out as described previously (Leibold and Munro, 1988).
Briefly, equal amounts of protein for the cytoplasmic and
membrane fractions were incubated with the synthetic
radiolabeled IRE probe. To measure total IRP binding
activity, the samples were first treated with 2% ␤mercaptoethanol (BME). The RNA-protein complexes were
separated on a 6% native polyacrylamide gel and visualized
by autoradiography. The autoradiograms were scanned and
the resulting digital image subjected to densitometric band
analysis using the Labworks Analysis Software (UVP Inc.,
Upland, CA, USA). The results of the RNA band shift assays
were expressed as active IRP binding from each treatment as
a ratio of control.
Results
Immunocytochemical analysis indicates that the
intracellular distribution of IRP1 and IRP2 is similar
Neither IRP1 nor IRP2 was detected in the nucleus and
both were predominantly located in the perikaryal
cytoplasm. IRP2 extended from the perikaryal
Fig. 2. Co-localization of IRP1 with the ER marker calnexin in SW1088
cells. (A-C) Representative cells selected from D (arrow). A-C are confocal
images of the same cells showing the intracellular distribution of IRP1 (green
in A), the ER marker calnexin (red in B) and the merged image (C). The
yellow indicates strong overlap in the perinuclear region.
4368
Journal of Cell Science 118 (19)
cytoplasm into cellular processes more frequently
than IRP1. IRP2 immunoreaction product also had
a more punctate appearance than IRP1. A similar
observation was made with the c-myc tagged IRP
proteins (Fig. 1C,D).
Journal of Cell Science
Immunocytochemical analysis indicates that
native IRP1 co-localizes with both ER and
Golgi markers in SW1088 cells
In order to further explore the perikaryal location
of IRP1, confocal microscopy was used to
analyze SW1088 cells that had been stained for
IRP1, ER and Golgi distribution. Fig. 2 also
demonstrates
a
perikaryal
cytoplasmic
distribution of IRP1 as well as the co-localization
with anti-calnexin antibody. Fig. 3 demonstrates
the co-localization of IRP1 with the Golgi
antibody anti-58K Golgi protein. Fig. 4
demonstrates that localization of IRP1 to the ER
in microscopic studies is unaffected by changes
in iron status.
Fig. 3. Co-localization of IRP1 with the Golgi marker 58K Golgi protein in
SW1088 cells. A-C are representative cells selected from D (arrow). A-C are
confocal images of the same cells showing the intracellular distribution of IRP1
(green in A), the Golgi marker Golgi 58K protein (red in B) and the merged
image (C). The yellow indicates strong overlap in the perinuclear region.
Native IRP1 immunoreactivity is co-localized
with ER activity in SW1088 microsomal
fractions
On the basis of the microscopy results, a crude
microsomal SW1088 cell fraction was obtained
and sub-fractionated using density centrifugation
to further assess the location of IRPs. IRP1, but not
Fig. 4. Co-localization of IRP1 with the ER marker calnexin in iron-depleted or iron-loaded SW1088 cells. These are representative confocal
images of SW1088 cells that were exposed to iron (A-D) or to the iron chelator DFO (E-H) for 72 hours prior to fixation. Following fixation the
cells were stained with DAPI (blue) to visualize the nucleus and with an IRP1 antibody (green in B and F) or the ER marker calnexin (red in C
and G). The merged images are shown in D and H. The co-localization of IRP1 and the ER are indicated by yellow. Treatment with iron or an
iron chelator did not result in changes in IRP1 distribution that could be identified microscopically.
Subcellular localization of iron regulatory proteins
Co-localization of IRP1 to ER marker enzyme NADPHcytochrome C reductase
Fractionation of Rat Liver (Iron Deficient)
60,000,000
0.06
0.00001
0.05
0.000008
0.04
0.000006
0.03
0.000004
0.02
NADPH cytochrome C
reductase activity
0.000012
0.07
0.000002
0.01
0
7
8
9
10
11
3,500,000
40,000,000
3,000,000
30,000,000
2,500,000
2,000,000
20,000,000
1,500,000
1,000,000
10,000,000
500,000
0
0
5
4,000,000
IRP1
0
1
12
Absorbance-Background (AUBG) (IRP1)
0.000014
0.08
4,500,000
ER
50,000,000
ER Absorbance-Background
(AU-BG) (ER and Golgi)
0.09
IRP1 immunoreactivity (OD
x mm)
5,000,000
Golgi
0.000016
0.1
4369
2
3
4
5
6
7
8
9 10 11 12
Fraction number
Fraction number
Fig. 5. Distribution of ER in density gradient fractions in relation to
IRP1 immunoreactivity. This graph shows the distribution of IRP1
and ER immunoreactivity in control rat livers. The primary y-axis
shows the IRP1 and ER distribution in the density gradient fractions.
The secondary y-axis shows the ER marker, cytochrome C-NADPH
reductase, which was used to plot the distribution of ER in fractions
obtained from density gradient centrifugation. The fraction number is
shown on the x-axis. Enzyme activity in each fraction is plotted as a
function of total protein. 5 ␮g of each fraction was subjected to
western blot analysis to determine IRP1 immunoreactivity. These
data confirm the localization of IRP1 immunoreactivity in ER
containing fractions. These experiments were performed in duplicate
and representative data are shown.
Fig. 7. Representative IRP1 subcellular distribution from irondepleted rat livers. This graph shows the distribution of IRP1, ER
and Golgi immunoreactivity in liver fractions from rats fed a lowiron diet. The y-axis shows the mean pixel density for IRP1, ER and
Golgi in the density gradient fractions. The fraction number is shown
on the x-axis. Total protein (10 ␮g) was added to detect IRP1 and 5
␮g of protein was added to detect both ER and Golgi by western blot
analysis. These data indicate that under iron-depleted conditions, the
IRP1 immunoreactivity is found in ER-containing fractions as well
as fractions that contain Golgi immunoreactivity. These experiments
were run in triplicate and representative data are shown.
IRP2, was detected in fractions containing ER
membrane (based on NADPH cytochrome C reductase
assay), however, it was apparent that the distribution of
IRP1 was biphasic (Fig. 5). Cytosolic contamination of
the ER fraction was determined by lactate
dehydrogenase assay to be less than 2%.
Fractionation of Rat Liver (Control)
2,500,000
12,000,000
Golgi
10,000,000
ER
2,000,000
IRP1
8,000,000
1,500,000
6,000,000
1,000,000
4,000,000
500,000
2,000,000
0
Absorbance-Background
(AU-BG) (IRP1 and Golgi)
ER Absorbance-Background
(AU-BG) (ER)
Journal of Cell Science
IRP1 immunoreactivity
NADPH cytochrome C reductase activity
0
1
2
3
4 5
6
7
8 9 10 11 12 13 14 15
Fraction number
Fig. 6. Representative IRP1 subcellular distribution from control rat livers.
This graph shows the distribution of IRP1, ER and Golgi immunoreactivity
in liver fractions from rats fed a normal diet. The y-axis shows the mean
pixel density for IRP1, ER and Golgi in the density gradient fractions. The
fraction number is shown on the x-axis. Total protein (10 ␮g) was added to
detect IRP1 and 5 ␮g of protein was added to detect both ER and Golgi by
western blot analysis. These data demonstrate that under control conditions,
a large proportion of IRP1 immunoreactivity is found in cytosol-containing
fractions. Additionally, under control conditions, a small proportion of IRP1
immunoreactivity is also found in Golgi-containing fractions. These
experiments were run in triplicate and representative data are shown.
Native IRP1 immunoreactivity is co-localized with
both ER and Golgi in iron-deficient rat liver
microsomal fractions
To further study the biphasic distribution of IRP1 that
was observed in the SW1088 fractionation experiments
in a model system that would provide greater sample
size we isolated liver membrane fractions by density
gradient analysis from rats fed control and irondeficient diets. As shown in Fig. 6 immunoblot analysis
revealed that IRP1 did not fractionate with ER in control
rat livers, however, a relatively small amount of IRP1
was detected in the Golgi-containing fractions. The
largest amount of IRP1 was found in those fractions that
were less dense than the Golgi. These less dense
fractions were determined by LDH activity to have
higher cytosolic content, indicating that under control
conditions, the majority of IRP1 remains in the
cytosolic fraction. As shown in Fig. 7, immunoblot
analysis revealed more IRP1 in fractions containing the
Golgi marker in iron-depleted rat livers than in livers
from rats on control diets. Additionally, a small fraction
of IRP1 was found in the ER-containing fractions. IRP2
was absent in the membrane fractions from both the
4370
Journal of Cell Science 118 (19)
IRP-IRE interaction with calcium chelation
IRP-IRE Interaction in HEK-293
Cells
14.0
*
12.0
CN
FAC
DFO
Bapta
B-FAC
*
*
*
B - DFO
Ratio of Control
4.00
CN
FAC
DFO
H202
*
IL-1
3.50
3.00
*
2.50
*
*
2.00
1.50
*
*
*
*
Ratio of control
4.50
10.0
8.0
*
*
*
*
6.0
4.0
1.00
*
0.50
*
2.0
0.00
CYTOSOL
MEMBRANE
Cy t o s o l
Membrane
Fig. 10. Electrophoretic mobility shift assays (EMSAs) for IRPs in
SW1088 cells in response to iron exposure (FAC), iron chelation
(DFO), calcium chelation (BAPTA), and BAPTA concomitant with
FAC (B-FAC) or DFO (B-DFO). IRP/IRE interaction in SW1088
cells decreases upon FAC treatment with or without concomitant
addition of the calcium chelator, BAPTA in both the cytosolic and
membrane fractions. The IRP/IRE interaction increases upon
treatment with DFO alone, BAPTA alone or DFO/BAPTA together
in both the cytosolic and membrane fractions. IRP localization to the
membrane fraction occurs with iron and calcium chelation. *P<0.05.
control and iron-deficient rat livers, but was present in the
cytosolic fractions of both (data not shown).
Factors affecting IRE-binding activity in the microsomal
fraction
Two cell types were chosen for this study, HEK293 cells and
SW1088 to demonstrate that the membrane-associated IRPs
were not unique to a single cell type. Fig. 8 shows the changes
in IRE-binding activity for both the cytosolic fraction and the
Effect of PMA or CET on IRP-IRE
Interaction
Ratio of Control
Journal of Cell Science
0.0
Fig. 8. Electrophoretic mobility shift assays (EMSAs) for IRPs in
HEK-293 cells in response to iron loading (FAC), iron chelation
(DFO), treatment with hydrogen peroxide (100 ␮M for 30 minutes),
and IL-1␤ (10 ng/ml for 16 hours). The IRP/IRE interaction
decreases with iron and IL-1␤ treatments in both the cytosolic and
membrane fractions, and IRP/IRE interaction increases with DFO
and hydrogen peroxide treatments in both the cytosolic and
membrane fractions. The data suggest IRPs localize to the cytosolic
fraction with iron treatment and localize to the membrane fraction
with H2O2 treatment. *P<0.05.
3
2.5
2
1.5
1
0.5
0
*
CN
*
*
PMA
*
CET
CYTOSOL
MEMBRANE
Fig. 9. Electrophoretic mobility shift assays (EMSAs) for IRPs in
SW1088 cells in response to phorbol 12-myristate 13-acetate (PMA,
0.2 ␮M) and chelerythrine (CET, 2.0 ␮M). IRP/IRE interaction in
SW1088 cells decreases upon treatment with PMA in both the
cytosolic and membrane fractions and increases upon treatment with
CET in both the cytosolic and membrane fractions. The data suggest
IRP localization to the cytosolic fraction occurs with protein kinase
C inhibition. *P<0.05.
membrane fractions after treatment with ferric ammonium
citrate (FAC), DFO, H2O2 or IL-1␤ in HEK293 cells.
Treatment with FAC produced a decrease in the IRE binding
activity, as did treatment with IL-1␤. DFO and H2O2 treatment
resulted in an increase in IRE-binding activity. The treatment
with iron or H2O2 produced a larger response in the membrane
than in the cytosolic fraction, while the response to treatment
with IL-1␤ was greater in the cytosolic fraction.
A similar IRE binding profile to that observed in HEK293
cells was seen in SW1088 cells. However, following iron
manipulation, the responses were amplified in SW1088 cells
(Figs 8 and 10). Therefore SW1088 cells were chosen for
further investigation into the possible mechanisms involved in
the differential localization of IRPs in the two subcellular
fractions.
Because IRPs reportedly have sites for phosphorylation
(Schalinske et al., 1997), SW1088 cells were treated with PMA
(an activator of protein kinase C) or chelerythrine (an inhibitor
of protein kinase C) to determine the role of phosphorylation
on IRP activity in the membrane fraction. Treatment with PMA
resulted in a decrease in IRE binding while treatment with
chelerythrine resulted in an increase in IRE binding (Fig. 9).
Because protein kinase C is a calcium-dependent protein, we
examined the role of calcium as a regulator of IRP activity in
the membrane fraction using the intracellular calcium chelator
BAPTA-AM. When the SW1088 cells were treated with
BAPTA-AM, there was an increase in the IRE-binding activity
in both the cytosolic and the membrane fraction similar to the
one produced by the treatment with DFO. However, when the
cells were treated with iron or DFO after treatment with
BAPTA-AM, the IRE binding activity was similar to the ones
that received iron or DFO without BAPTA-AM (Fig. 10).
Journal of Cell Science
Subcellular localization of iron regulatory proteins
Discussion
Our laboratory previously reported that IRE-binding activity in
NIH3T3 cells and brain was present in both membrane
(microsomal) and cytosolic fractions. Additionally, an in vitro
analysis suggested that iron chelation resulted in an increase in
the IRP binding activity in both the cytosolic and microsomal
fractions and an increase in the amount of IRP1 in the
microsomal fraction (Pinero et al., 2001). The purpose of the
current study was to more specifically identify the microsomal
fraction and begin to elucidate the intracellular events
associated with the putative redistribution of IRP1 between the
cytosolic and membrane compartments. In this study we
used multiple microscopic and biochemical approaches to
demonstrate the subcellular compartmentalization of IRPs. The
microscopic studies reveal that both IRP1 and IRP2 are
localized in the cytosol with IRP1 having a more perinuclear
distribution than IRP2. Immunocytochemistry was performed
on cells in culture as an initial attempt to determine the
subcellular localization of IRP1. Under control conditions,
IRP1 was co-localized with both the ER and Golgi. Iron
loading or iron depletion of the cultured cells did not result in
observable changes in the amount of IRP1 that co-localized
with the ER or Golgi markers. In our previous study in which
we examined the subcellular distribution of IRP in both rat
brains and various cell lines, under varying iron conditions, we
determined that about 10% of the IRP/IRE binding activity is
membrane associated (Pinero et al., 2001). Changes in this
relatively small population of IRP1 in the membrane
compartment may be below the detectable level
microscopically.
To further explore this apparent association between IRP1,
ER and Golgi, a biochemical approach was utilized. The
microsomal membrane fraction from the SW1088 cells was
isolated and further separated into ER-containing fractions,
which revealed that IRP1 co-localized with the ER-activity
marker NADPH-cytochrome C reductase. This finding is
consistent with the microscopical analysis and confirmed a
novel subcellular location for IRP1. The IRP1 distribution
appeared biphasic, which suggested that IRP1 might be
associated with other membrane-bound organelles in addition
to the ER, supporting the microscopical evidence that IRP1 is
also associated with the Golgi. The presence of IRP2 in the ER
fraction in SW1088 cells could not be detected with either
immunocytochemical or biochemical methods.
To obtain sufficient material to perform additional
subcellular fractionation analyses and to demonstrate that the
subcompartmentalization of IRP1 is not unique to cells in
culture we examined livers from rats fed control or irondeficient diets (Han et al., 2003). This model system also
enabled us to determine if iron status affected the intracellular
distribution of IRP1, and allowed us to postulate that the IRP1
distribution may affect mRNA stabilization. In the livers of the
rats fed a control diet, IRP1 is predominantly associated with
the cytosolic fraction, as is well established (Pinero et al.,
2001), but is also clearly present in the Golgi apparatus. This
novel finding is consistent with the microscopical analysis of
SW1088 cells. Unlike the microscopical analysis of IRP1
distribution in the cell culture studies, no IRP1 was found in
the ER fraction of the control rat livers. However, the ER
fraction from iron-deficient rats did contain a relatively small
4371
amount of IRP1 in addition to IRP1 in the Golgi membrane
fraction.
The association of IRP1 with intracellular membranes is a
novel finding. The function of IRP1 is well established. We
are not proposing that there is a new function for the IRPs in
these subcellular compartments, but rather a novel concept in
their processing and how they function. For example, the
Golgi apparatus could be a site where IRP1 obtains an ironsulfur cluster. The iron-sulfur cluster assembly proteins, IscU1
and IscS, are reportedly localized in the cytoplasm suggesting
that the machinery for Fe-S cluster assembly may also be
present in the cytosol (Tong and Rouault, 2000). Roy et al.
have identified CFD1, a highly conserved P-loop ATPase that
is critical for Fe-S cluster biogenesis and have reported that
the subcellular distribution of CFD1 is 90% cytosolic, and
10% membrane (Roy et al., 2003). The subcellular
localization of CFD1 mirrors the IRP1 subcellular localization
that was reported here. It has been proposed that there is an
intracellular iron trafficking system that is comparable to
the copper trafficking system (Tong and Rouault, 2000).
Expounding on this proposition, IRP1 may obtain a metal
cluster in the Golgi apparatus similar to the process identified
for ceruloplasmin. Copper is transported to the Golgi by the
Cu-binding chaperone protein, HAH1 and incorporated into
ceruloplasmin via a copper-transporting ATPase (Cox and
Moore, 2002).
Because of the novel location of the IRPs in the membrane
fraction, we performed gel-shift assays to determine if the
activity of the IRPs in this fraction were activated similarly to
those in the cytosol. We performed IRE-binding assays
following iron chelation, iron supplementation, treatment with
interleukin-1␤ and treatment with hydrogen peroxide, and each
of the IRP/IRE interactions were similar to those reported for
other cell types in the cytosol (Pantopoulos and Hentze, 1995).
HEK293 cells and SW1088 cells, however, have a different
level of response to manipulation of iron status. Therefore most
of the cell culture experiments were performed in the SW1088
cell line.
Eisenstein and co-workers have previously reported that
phosphorylation may play a role in IRP/IRE interaction
(Eisenstein et al., 1993; Schalinske and Eisenstein, 1996).
Activation of protein kinase C resulted in increased IRP/IRE
interaction in HL-60 cells (Schalinske and Eisenstein, 1996).
In the SW1088 cells, protein kinase C activation decreased
IRP/IRE interaction in the cytosolic and membrane fractions.
Inhibition of protein kinase C with chelethyrine resulted in an
increase in IRP/IRE binding in the cytosolic fraction. These
results demonstrate consistency within the two fractions of the
SW1088 cells and suggest that the role of protein kinase C in
IRP/IRE interaction could be cell-specific.
IRPs in HL-60 cells are activated by phosphorylation via
protein kinase C, which is Ca2+-dependent (Schalinske and
Eisenstein, 1996). We examined the potential role of calcium
in IRP activation in the membrane fraction using the calcium
chelator, BAPTA-AM. SW1088 cells that were treated with
BAPTA-AM alone had an increase in IRP/IRE interaction in
both the cytosolic and membrane fractions, and an increase in
the amount of IRP binding in the microsomal fraction. The
increase in IRP/IRE interaction was similar to that observed in
SW1088 cells treated with DFO alone. The finding that
concomitant addition of BAPTA-AM with DFO to SW1088
Journal of Cell Science
4372
Journal of Cell Science 118 (19)
cells did not result in an increase in IRP/IRE interaction
beyond that observed for either treatment alone
suggests that both chelators individually elicit a
maximum effect on IRPs available to bind IREs. FAC
treatment of SW1088 cells concomitant with BAPTAAM provided results similar to those observed with
FAC treatment alone, suggesting that FAC treatment is
able to overcome the influence of calcium chelation in
IRP/IRE interaction.
In a number of IRP binding assays, we noted that
there was a difference in the relative amounts of binding
activity in the cytosol or membrane fraction following
treatment. This difference in amount of total IRP
binding activity following treatment was first observed
in our earlier study on NIH3T3 cells in the membrane
fraction following treatment with an iron chelator
(Pinero et al., 2001) and was notable in this study
following treatment with CET, hydrogen peroxide,
DFO and BAPTA. These data raise the possibility that
IRPs may translocate between the cytosolic and
membrane fractions unbound to IREs. The evidence
Fig. 11. Proposed mechanism for IRP1 interaction with the ER and Golgi
that the IRPs in the ER and Golgi membranes may not
membrane fractions. This schematic demonstrates the intracellular
be simply a co-migration with IREs on the mRNAs that
distributions of IRP1 discovered in this study. IRP1 is found in Golgi
membranes in addition to the cytosol. Under conditions of low iron and in
were bound in the cytosol is that IRP1 activity could
cells in culture, IRP1 is also found in the ER membranes. We propose that
be detected with the addition of exogenous probe
under low iron conditions IRP1 is incorporated into the ER membrane by a
indicating that unbound but available IRPs are present
hydrophobic tail immediately after translation. Within the ER, IRP1 can
in the membrane fraction. Treatment of the
serve to further stabilize transferrin receptor (TfR) mRNA by binding to
homogenates with 2% BME increases the amount of
IREs that were not bound in the cytoplasm. Once the TfR mRNA has been
IRP activity, further indicating a resident population of
translated, the IRPs in the ER membrane move to the Golgi membrane
IRP in the membrane fraction.
where they can receive an Fe-S cluster. The acquisition of an Fe-S cluster is
The aforementioned findings do not preclude the
not required for IRP1 to be released from the Golgi membrane to the cytosol
possibility that some IRP1 could co-migrate from the
or be recycled to the ER membrane.
cytosol to the membrane fraction while bound to IREs.
Indeed, as shown in Fig. 11, we propose that IRP1
initially binds to the Tf receptor mRNA in the cytosol to
found in association with intracellular membranes but also
stabilize the mRNA, and then under iron-deficient conditions,
supports our inability to detect IRP2 in these fractions. Tailthe additional IREs are bound when the mRNA reaches the ER,
anchored proteins are a group of integral membrane proteins
offering additional stabilization once the mRNA reaches its site
that are retained in a phospholipid bilayer via a single Cof translation. Tf receptor mRNA was chosen for this model
terminal hydrophobic domain (Borgese et al., 2003). These
because this mRNA is synthesized on the rough ER.
proteins are unlike other integral membrane proteins because
Furthermore, the mechanism of additional stabilization for the
they localize to intracellular membranes post-translationally.
Tf receptor mRNA from IRP1 in the ER membrane is
The protein sequences of tail-anchored proteins do not contain
particularly appealing because only two of the five IREs in the
a signal recognition site, which would direct the partially
Tf receptor mRNA must be bound to promote mRNA
synthesized protein to the ER. The N-terminus of tail-anchored
stabilization (Erlitzki et al., 2002). Once the translation of the
proteins, such as cytochrome b(5) and heme oxygenase, are
IRE-containing mRNA has occurred, we propose that the IRP1
synthesized and folded on free polysomes in the cytosol prior
remains attached to the ER membrane and cycles to the cisto the insertion of the hydrophobic domain into a membrane
Golgi membrane where it is released into the cytosol. Insertion
(D’Arrigo et al., 1993). Because IRP1 is a cytosolic protein it
of an iron-sulfur cluster may not be a requirement for release
is also synthesized on free polysomes. Wattenberg and Lithgow
of IRP1 from the Golgi membrane into the cytosol. It is also
(Wattenberg and Lithgow, 2001) propose that tail anchoring of
possible that the IRP1 in the Golgi membrane could be
proteins serve the purpose of localizing certain enzyme
retrieved back to the ER membrane particularly in situations
activities close to the surface of various intracellular
when intracellular iron status is low.
membranes, which assists in the enzymes’ catalytic activity.
A major question in our proposal is the mechanism by which
This is consistent with the novel concept that we propose,
IRP1 would become integrally associated with the ER and
namely, that IRP1 can insert in the intracellular membrane to
Golgi membrane. An analysis of the protein sequence of IRP1
facilitate stabilization of IRE containing mRNAs. It is
using the TMHMM (Tied Mixture Hidden Markov Model)
noteworthy for the new concepts for IRP1 in this paper that
Server 2.0 revealed a putative hydrophobic tail in human IRP1
cytochrome b(5) and heme oxygenase require insertion of a
that is not present in human IRP2, which may facilitate
metal complex for activity similar to IRP1 and all have a
insertion of IRP1 into a membrane (Krogh et al., 2001). This
hydrophobic tail that allows transient insertion into the Golgi
observation not only supports our hypothesis that IRP1 is
membrane.
Subcellular localization of iron regulatory proteins
In summary, we have shown that there are distinct
subcellular populations of IRPs. These subcellular
populations include, but are not limited to, the cytosol, the
ER and the Golgi membrane. Additionally, we have
determined that IRP1, but not IRP2, can be localized to Golgi
apparatus and ER membrane containing fractions. This latter
observation has resulted in the presentation of a novel concept
for the activity of IRP1 in cells that further distinguishes IRP1
from IRP2.
This research was supported by a grant from the National Institutes
of Health: National Institute of Diabetes and Digestive and Kidney
Diseases (1 P01 DK53430-04) and Tobacco Settlement Funds from
the State of Pennsylvania.
Journal of Cell Science
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