From the Department of Nutritional Sciences, University of Missouri, Columbia, Missouri 65211
Received for publication, February 26, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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The ZIP superfamily of transporters
plays important roles in metal ion uptake in diverse organisms. There
are 12 ZIP-encoding genes in humans, and we hypothesize that many of
these proteins are zinc transporters. In this study, we addressed the
role of one human ZIP gene, hZIP1, in zinc transport.
First, we examined 65Zn uptake activity in K562
erythroleukemia cells overexpressing hZIP1. These cells
accumulated more zinc than control cells because of increased zinc
influx. Moreover, consistent with its role in zinc uptake, hZIP1
protein was localized to the plasma membrane. Our results also
demonstrated that hZIP1 is responsible for the endogenous zinc uptake
activity in K562 cells. hZIP1 is expressed in untransfected
K562 cells, and the increase in mRNA levels found in
hZIP1-overexpressing cells correlated with the
increased zinc uptake activity. Furthermore,
hZIP1-dependent 65Zn uptake was biochemically
indistinguishable from the endogenous activity. Finally, inhibition
of endogenous hZIP1 expression with antisense
oligonucleotides caused a marked decrease in endogenous 65Zn uptake activity. The observation that hZIP1 is the
major zinc transporter in K562 cells, coupled with its expression in
many normal cell types, indicates that hZIP1 plays an important role in
zinc uptake in human tissues.
Zinc is an essential nutrient to all organisms because it is a
required catalytic and/or structural cofactor for hundreds of
zinc-dependent enzymes and other proteins such as
transcription factors. Despite its importance, we knew little until
recently about how eukaryotic cells take up zinc from their
environment. The molecular insight into zinc uptake came from the
identification of zinc transporters in fungi and plants. In yeast, the
Zrt1 and Zrt2 proteins are zinc transporters involved in moving zinc
from the extracellular medium across the plasma membrane into the
cytoplasm (1, 2). In plants, the Arabidopsis IRT1
protein transports iron from the soil into the root and is also capable
of zinc, manganese, and cadmium transport (3-5). Zrt1, Zrt2, and IRT1 are the founding members of a superfamily of transporters referred to
as the ZIP family (for Zrt/IRT-like
proteins) (6). Other ZIP transporters have also been
implicated in zinc uptake, including the Arabidopsis
proteins ZIP1, ZIP2, ZIP3, and ZIP4 (7). The Znt1 protein of
Thlaspi caerulescens has been shown to be involved in
zinc uptake as well (8). Thus, many ZIP proteins are involved in zinc
uptake. A notable exception is Zrt3 of Saccharomyces
cerevisiae, a ZIP protein that transports stored zinc from the
lumen of the vacuole into the cytoplasm (9). Thus, ZIP proteins can act in zinc uptake or intracellular zinc transport.
Members of the ZIP family are found at all phylogenetic levels,
including archaebacteria, eubacteria, and eukaryotes. There are
currently ~85 members reported in the sequence data bases, and these
fall into four subfamilies based on their amino acid similarities (10).
Most members are predicted to have eight transmembrane domains and
share a predicted topology where the amino and carboxyl termini are
extracytoplasmic. The greatest degree of conservation is found in
transmembrane domains IV-VIII. Transmembrane domains IV and V are
particularly amphipathic and contain conserved and functionally
critical histidine residues flanked by equally important polar or
charged amino acids (5). These residues are thought to line an aqueous
cavity in the transporter through which the substrate moves (11).
Notably, ZIP proteins do not contain ATP-binding sites or ATPase
domains. Therefore, these proteins must function through either
secondary active transport or facilitated diffusion.
There are 12 known ZIP members in the human genome, and five members
have been found in the mouse (10). Three of the human proteins,
hZIP1,1 hZIP2, and hZIP3, are
very closely related to the fungal and plant proteins known to be zinc
uptake transporters. The recent studies of fungal and plant ZIP
transporters indicated that the ZIP superfamily plays remarkably
conserved roles in metal ion transport and especially zinc uptake.
These observations suggested that the mammalian ZIP proteins play
similar roles. To test this hypothesis, we expressed the hZIP2 protein
in human K562 erythroleukemia cells and showed that hZIP2 localizes to
the plasma membrane (12). Moreover, hZIP2 expression resulted in a
novel zinc uptake activity not found in these cells. Thus, hZIP2 is a
metal ion transporter capable of zinc uptake. In this report, we
continue our characterization of mammalian ZIP transporters by
functional expression of the hZIP1 protein. Our results demonstrate
that hZIP1, like hZIP2, is a zinc transporter. We also found that hZIP1
is the endogenous zinc uptake transporter normally found in K562 cells.
Given the ubiquitous expression of hZIP1 in human tissues, we propose
that hZIP1 is the major zinc transporter for many human cell types.
DNA Manipulations--
An hZIP1 cDNA clone was
isolated from a prostate library using high throughput PCR screening
(Genome Systems Inc.) and sequenced in its entirety. The
hZIP1 ORF was amplified by PCR using the primers
5'-GGCCCAAGCTTGGGATGGGGCCCTGGGGAGAGCC-3' and
5'-AAGGAAAAAAGCGGCCGCCTAGATTTGGATGAAGAGCAG-3' and cloned into the
HindIII and NotI sites of the pRc-CMV mammalian expression vector (Invitrogen). hZIP1 was epitope-tagged at its amino
terminus with one copy of the hemagglutinin antigen (HA) by PCR to
generate CMV-HA-hZIP1. This fragment was digested with HindIII and XbaI, cloned into the pRc-CMV vector,
and confirmed by sequencing.
Cell Culture Methods--
K562 erythroleukemia cells (ATCC
CCL-243) were grown in complete RPMI 1640 medium (Life Technologies,
Inc.) supplemented with 1% glutamine and 10% fetal bovine serum
(Sigma). Cells were cultured in 10-cm3 dishes, incubated in
humidified 5% CO2 isothermal incubators, and transfected
by electroporation (Invitrogen) using 3 × 106 cells
and 20 µg of purified plasmid DNA unless noted otherwise. Stable
transfected cell lines were generated by the limiting dilution method.
The stable cell lines were subsequently maintained in 350 µg/ml G418.
Cell numbers were determined with a hemocytometer, and cultures were
examined weekly for mycoplasma contamination using Hoechst dye and
fluorescence microscopy.
65Zn Uptake Assays and Atomic Absorption
Spectroscopy--
Cells were grown to 50% confluence, harvested by
centrifugation, and washed once in cold uptake buffer (15 mM HEPES, 100 mM glucose, and 150 mM KCl, pH 7.0). The cells were resuspended in uptake
buffer and incubated for 10 min in a shaking 37 °C water bath. The
cells were then mixed with an equal volume of prewarmed uptake buffer
containing the specified concentration of
65ZnCl2 (Amersham Pharmacia Biotech) and
incubated for 15 min unless indicated otherwise. Assays were stopped by
adding an equal volume of ice-cold uptake buffer supplemented with 1 mM EDTA (stop buffer). Cells were collected by filtration
on glass-fiber filters (Schleicher & Schüll) and washed three
times in stop buffer (~10 ml of total wash volume). Cell-associated
radioactivity was measured with a Packard Auto-Gamma 5650 Northern Blot Analysis--
Total mRNA was isolated from
K562 cells using Trizol reagent (Life Technologies, Inc.). Twenty µg
of mRNA was run on a 2% agarose gel containing 6% formaldehyde
and 1× MOPS at 55 V for 5 h. The gel was transferred by capillary
electrophoresis to nylon membranes (Biotrans, ICN) in 10× SSC for
16 h. The blot was baked at 80 °C for 30 min and then
prehybridized at 42 °C for 1 h in Super-Hyb solution (Sigma).
32P-Labeled probes were generated using the random priming
method (14). The denatured probe was added to the prehybridization solution and incubated at 42 °C for 16 h. The blot was then
washed three times with 2× SSC for 5 min, twice with 1× SSC and 1%
SDS for 15 min, and twice with 0.1× SSC and 0.1% SDS for 15 min at 42 °C prior to autoradiography.
Indirect Immunofluorescence--
Indirect immunofluorescence was
performed as previously described (12) except for the use of a laser
scanning confocal microscope.
Inhibition of hZIP1 Expression with Antisense
Oligonucleotides--
Antisense oligonucleotides were designed to bind
to potentially single-stranded regions of the hZIP1 mRNA
as determined with folding prediction algorithms (15). Six
hZIP1-directed oligonucleotides were synthesized, and one
oligonucleotide of random sequence was used as a control for
nonspecific effects (see Table I). The oligonucleotides contained three
phosphorothioate bonds at each end to slow their degradation in cells
(16). K562 cells were grown to 50% confluence, harvested, and washed
three times with PBS. The cells were resuspended in PBS and transfected
with 5 or 15 µg of the oligonucleotides and 10 µg of pEGFP-N1
(CLONTECH) by electroporation. To compensate for
the often low transfection efficiency of K562 cells, we used flow
cytometry to enrich for transfected cells based on GFP fluorescence.
Furthermore, the oligonucleotides were tagged at their 5'-ends with
fluorescein to aid cell sorting. The transfected cells were added to
prewarmed complete medium, grown for 24 h, and sorted by
fluorescence-activated cell sorting (FACS) on a Becton Dickinson FACS
Vantage TurboSort flow cytometer. After sorting,
GFP/fluorescein-positive cells were grown for an additional 24 h
before mRNA was isolated or 65Zn uptake assays were performed.
Semiquantitative RT-PCR Analysis--
Total mRNA was
isolated from K562 cells using Trizol reagent. cDNA was synthesized
with 0.75 µg of total RNA using reverse transcriptase and random
primers (GeneAmp® RNA PCR, PerkinElmer Life Sciences).
hZIP1 and glyceraldehyde-3-phosphate dehydrogenase fragments
were amplified from the cDNA templates using 35 cycles. These
conditions were found to be in the quantitative range for detection of
products based on input template concentration. The hZIP1
amplification primers used were 5'-TACAAGGAGCAGTCAGGGCCGTCA-3' and 5'-CTAGATTTGGATGAAGAGCTGGGCAGT-3', and the
glyceraldehyde-3-phosphate dehydrogenase primers used were
5'-CCACCATGGAGAAGGCTGGGGCTC-3' and 5'-AGTGATGGCATGGACTGTG-GTCAT-3'.
Products were analyzed by agarose gel electrophoresis with ethidium
bromide staining and photographed under UV light with a Eastman Kodak
CCD camera. No products were detected without reverse transcriptase
treatment, indicating the lack of contaminating genomic DNA.
Assessment of hZIP1 Expression in Human Tissues--
The
presence and level of hZIP1 mRNA in different human
tissues were determined using Rapid ScanTM gene expression
panels (Origene Technologies, Boston, MA). Each panel contains
first-strand cDNA prepared from different human tissues that have
been normalized to Characterization of the hZIP1 Zinc Transporter--
A previous
study showed that hZIP1 is expressed in human prostate cells
(17). Therefore, we isolated an hZIP1 cDNA clone from a
prostate cDNA library. This clone was judged to contain the
full-length hZIP1 ORF because of the presence of an 18-base poly(A) tail at its 3'-end and an in-frame stop codon in the
5'-untranslated region upstream of the presumptive initiation codon.
Given that most of the characterized eukaryotic ZIP proteins, including
hZIP2, have been implicated in zinc transport, we hypothesized that
hZIP1 is also a zinc transporter. To test the ability of hZIP1 to
transport zinc, the hZIP1 ORF was cloned into the mammalian
expression vector pRc-CMV, allowing expression from the CMV promoter.
This plasmid was transfected into human K562 erythroleukemia cells, and
stable G418-resistant clonal cell lines were isolated. The
hZIP1-expressing transfectants (hereafter designated as
CMV-hZIP1) and stable vector-only transfectants (CMV) were assayed for
accumulation of 65Zn. These assays were performed in uptake
buffer. As found previously (12), K562 cells had an endogenous zinc
uptake activity when assayed under these conditions (Fig.
1A). Consistent with the ability of hZIP1 to transport zinc, CMV-hZIP1 cells accumulated almost
2-fold more 65Zn over a 60-min period than did the
endogenous activity in CMV control cells. No zinc accumulation was seen
in either cell type at 4 °C, indicating that zinc accumulation by
both systems was temperature-dependent and therefore likely to
be transporter-mediated rather than due to cell-surface binding.
To determine if the higher zinc accumulation observed in CMV-hZIP1
cells was due to increased zinc influx rather than decreased zinc
efflux, we assayed initial rates of 65Zn uptake over a
shorter time period (0-60 s) (Fig. 1B). hZIP1-expressing cells accumulated zinc at an initial rate of ~6
pmol/min/106 cells, whereas in CMV transformants, that rate
was only 2 pmol/min/106 cells. Thus, expression of hZIP1
increased the initial rate of zinc influx 3-fold over control cells. As
we reported previously (12), the endogenous zinc uptake activity in
K562 cells is concentration-dependent and saturable. This
system shows Michaelis-Menten kinetics, with an apparent
Km for zinc of 3.5 µM and a
Vmax of 11 pmol/min/106 cells (Fig.
1C). We determined the concentration dependence of hZIP1
activity by measuring the 65Zn uptake rate over a range of
zinc concentrations in CMV-hZIP1 cells and subtracting the endogenous
activity from those values. hZIP1-dependent uptake activity
was also concentration-dependent and saturable. The
apparent Km for 65Zn uptake in CMV-hZIP1
cells was 3 µM, and the Vmax was
23 pmol/min/106 cells. Therefore, expression of hZIP1 in
K562 cells caused an increase in 65Zn uptake activity that
was time-, temperature-, and concentration-dependent and
saturable. These results are consistent with our hypothesis that hZIP1
is a zinc transporter in human cells.
The experiments in Fig. 1 (A-C) indicate that hZIP1 is
capable of transporting zinc in a buffer lacking any zinc-binding
agents. This buffer condition is very different from what mammalian
cells encounter in vivo. Although the concentration of total
zinc in blood plasma is normally 10-20 µM, most of that
zinc is bound to proteins such albumin and
If hZIP1 is a transporter protein involved in zinc uptake across the
plasma membrane, it should be localized on the plasma membrane.
However, a previous report (19) described preliminary studies
localizing a GFP-hZIP1 fusion protein to an intracellular compartment.
These studies lacked evidence that the fusion protein retained
function, leaving open the possibility that the GFP moiety may disrupt
the protein's normal localization. To reexamine this question of hZIP1
localization, we tagged the amino terminus of the hZIP1 gene
with one copy of the HA antigen and generated stable CMV-HA-hZIP1
transfectants. The epitope-tagged hZIP1 protein was functional as shown
by a 2-fold increase in 65Zn accumulation over vector
controls (data not shown). Localization of the HA-hZIP1 protein was
determined by indirect immunofluorescence. No fluorescence was observed
in cells expressing the untagged hZIP1 protein (Fig.
2, A and C). In
contrast, cells expressing the tagged allele showed a bright rim of
fluorescence at the cell periphery only (Fig. 2, B and
D). These data demonstrated that functional hZIP1 protein is
located on the plasma membrane. Similar results were obtained with
permeabilized and non-permeabilized cells (data not shown), indicating
that the amino terminus of the protein is extracellular, as predicted
(12).
hZIP1 Is the Endogenous Zinc Uptake System in K562 Cells--
It
was intriguing that the Km of the endogenous zinc
uptake system in K562 cells was similar to that observed for both
hZIP1- and hZIP2-dependent activities (this report and Ref. 12). We showed previously that hZIP2 is not expressed in K562 cells and
that its uptake activity is clearly distinguishable from the endogenous
system (12). These observations suggested that perhaps hZIP1 is
responsible for endogenous zinc uptake in K562 cells. To test this
hypothesis, we assayed for hZIP1 mRNA expression in K562
cells by Northern blot analysis and found that it was expressed in
these cells (Fig. 3A).
Furthermore, cells expressing hZIP1 from the CMV promoter
had ~2-fold more hZIP1 mRNA compared with controls.
This 2-fold higher level of expression was also confirmed by RT-PCR
(Fig. 4D) and was consistent
with the 2-fold increase in zinc accumulation in CMV-hZIP1 cells (Fig. 1). As a further test, we determined if hZIP1 activity was
distinguishable from the endogenous system. First, we compared the
sensitivity of hZIP1, hZIP2, and the endogenous system to inhibition by
other metals. Zinc uptake activity was measured in the absence or
presence of a 6-fold molar excess of various divalent cations (Fig.
3B). Mg2+ had no effect on any system. Although
hZIP2 was strongly inhibited by Co2+ and Mn2+,
both hZIP1 and the endogenous system were unaffected. Similarly, Cu2+ and Fe2+ greatly inhibited hZIP2 activity,
but had lesser effects on hZIP1 and the endogenous system. Although
hZIP1 and the endogenous system were inhibited by Ni2+,
hZIP2 activity was not altered. Thus, both hZIP1 and the endogenous uptake activity share remarkably similar profiles of inhibition by
other metal ions that are distinct from those of hZIP2.
We demonstrated previously that
HCO
As a further test of the role of hZIP1 as the endogenous zinc uptake
system in K562 cells, we used antisense oligonucleotides to inhibit
hZIP1 mRNA accumulation. When transfected into cells, antisense oligonucleotides can bind to target mRNA and stimulate its degradation by providing a substrate for RNase H (16). Six antisense oligonucleotides were designed to hybridize to regions in the
5'- and 3'-untranslated regions of hZIP1 mRNA (Table
I). Five µg of each oligonucleotide was
mixed with 10 µg of a GFP expression plasmid and transfected into
K562 cells. After 24 h, successfully transfected GFP-expressing
cells were isolated from the population by FACS. The efficiency of this
sorting was aided by tagging the 5'-end of each oligonucleotide with
fluorescein. Untransfected cells showed a peak below 10 fluorescence
units (Fig. 4A). The profile of an unsorted population
following transfection is shown in Fig. 4B. The sorting
procedure enriched for transfected cells (gate M2) to almost
100% (data not shown). The transfected cells isolated in this
manner were
Transfection with GFP alone had no effect on zinc uptake activity, nor
did cotransfection with 5 µg of a control oligonucleotide of randomly
chosen sequence (see C in Fig. 4D).
Anti-hZIP1 oligonucleotides O2, O4, and O5 had no effect on
zinc uptake activity or hZIP1 expression (data not shown).
However, 5 µg of anti-hZIP1 oligonucleotide O1, O3, or O6
decreased zinc uptake by 30-40%. A mixture of 5 µg each of
oligonucleotides O1, O3, and O6 caused a 90% decrease in zinc uptake.
This effect was not due to nonspecific toxicity of the oligonucleotide
treatment because 15 µg of the control oligonucleotide had no effect
on zinc uptake activity. To determine if endogenous hZIP1
expression was inhibited by oligonucleotide treatment, total RNA was
isolated from these cells and analyzed by quantitative RT-PCR. Neither
5 nor 15 µg of control oligonucleotide affected the hZIP1
mRNA level. Oligonucleotides O1, O3, and O6 and the mixture were
all found to decrease hZIP1 mRNA levels, with no effect
on a control mRNA, glyceraldehyde-3-phosphate dehydrogenase. Thus,
oligonucleotides that inhibited accumulation of endogenous hZIP1 mRNA also inhibited zinc uptake activity. These
results strongly suggest that hZIP1 is the endogenous zinc transporter in K562 cells.
Inhibition of zinc uptake by the mixture of oligonucleotides reduced
zinc uptake activity to only 10% of normal levels. This result
suggests that hZIP1 is the major zinc transporter in at least one
mammalian cell type, K562 erythroleukemia cells. To determine what
tissues may use hZIP1 as a zinc transporter in vivo, we
assayed hZIP1 mRNA expression in various human tissues by semiquantitative RT-PCR. cDNAs prepared from a number of
different tissues contained detectable levels of hZIP1
mRNA (Fig. 5). These included small
intestine, kidney, liver, pancreas, and prostate, i.e.
tissues known to be important in zinc metabolism. Thus, we conclude
that hZIP1 is widely expressed in mammalian tissues. 10-Fold
dilution of these cDNA samples caused hZIP1 mRNA to
be undetectable in all tissue types under these conditions (data not
shown). This result indicates that hZIP1 is expressed at
similar levels in all of these tissues. The ubiquitous expression of
hZIP1 in human tissues suggests that the hZIP1 protein plays
an important housekeeping function in many cell types.
In a recent report, we used functional expression in K562 cells to
characterize the biochemical properties of the hZIP2 zinc transporter
(12). In the course of that study, we concluded that hZIP2 is not
responsible for the endogenous zinc uptake activity in K562 cells. This
conclusion was based on (a) the lack of detectable hZIP2 mRNA in K562 cells and (b) the clear
differences in the biochemical properties of the hZIP2 and endogenous
zinc transport systems. For example,
HCO Perhaps of even greater significance is our demonstration that hZIP1 is
the endogenous zinc transporter in K562 cells. This conclusion is based
on several independent observations. First, we found that
hZIP1 is normally expressed in these cells, and a 2-fold
increase in mRNA level generated by expression from the CMV
promoter correlated closely with a 2-fold increase in zinc uptake
activity. Second, the endogenous uptake system and hZIP1 were
indistinguishable in a number of different tests. For example, these
systems have similar apparent Km values and are sensitive to inhibition by an array of metal ions to almost precisely the same degree. Finally, we found that inhibition of endogenous hZIP1 expression in K562 cells with antisense
oligonucleotides also inhibited zinc uptake activity. Although
antisense oligonucleotides have been reported to give artifactual
results (16), our control experiments indicate that the decrease in
expression is not due to toxicity or a general decrease in mRNA
levels and requires hZIP1-specific sequences to be
effective. It is interesting to note that the mixed
hZIP1 antisense oligonucleotide treatment reduced
uptake activity to 10% of normal levels. These data argue that other
zinc transporters, if present in K562 cells, play minor roles in zinc uptake.
As an alternative test of the ability of hZIP1 to transport zinc, we
expressed the protein in a yeast zrt1 zrt2 mutant that is
defective for zinc uptake. Although this approach was successful for
the characterization of many plant ZIP proteins (4, 7, 8), hZIP1
expression in yeast failed to complement the zrt1 zrt2
mutant, and there was no detectable increase in zinc uptake activity
(data not shown). A similar lack of effect was observed when hZIP2 was
expressed in yeast (12), suggesting that the mammalian members of the
family are not functional in the yeast cellular environment. Unlike
hZIP2, however, hZIP1-expressing yeast cells did show a
zinc-dependent phenotype; those cells were hypersensitive
to the growth inhibitory effects of high zinc, suggesting that the
protein was produced, but perhaps improperly localized in the cell.
Although we have not examined this effect of hZIP1 expression in yeast
further, the zinc dependence of the phenotype provides additional
support to the hypothesis that hZIP1 is a zinc transporter.
The similarity of hZIP1 and hZIP2 to IRT1, an Fe2+
transporter in Arabidopsis, suggests that the human proteins
may also serve as iron transporters. This hypothesis is supported by
the observation that iron can inhibit zinc uptake by these
proteins. However, we have assayed Fe2+ uptake in
cells overexpressing either hZIP1 or hZIP2 and found no
effect.2 These results
indicate that the human zinc transporters are not also involved in iron uptake.
The mechanism of transport used by ZIP proteins is still unresolved.
This is largely because varied results have been obtained when the
properties of different transporters have been analyzed. In yeast, for
example, both Zrt1 and Zrt2 are dependent on energy for zinc transport
(1, 2). In contrast, neither hZIP1 nor hZIP2 (12) requires ATP for
activity. This conclusion is based on the observation that metabolic
inhibitors that reduce ATP levels to below 10% of normal levels (data
not shown) had no effect on uptake activity of either of these
proteins. We determined that zinc uptake by hZIP2 is stimulated by
increased HCO An important unanswered question is what role these proteins play in
zinc transport in vivo. hZIP2 expression has been detected only in prostate (12) and uterine (20) epithelial cells, suggesting that this protein plays a very specialized tissue-specific function. In
marked contrast, hZIP1 is expressed in all 24 human tissues we
examined. This observation, coupled with our results from K562 cells,
suggests that hZIP1 may be the major endogenous zinc uptake transporter
in many cells in the body. This conclusion is supported by Costello
et al. (17), who previously provided evidence that hZIP1 is
responsible for zinc uptake in prostate cells; treatment of those cells
with prolactin and testosterone causes an increase in both
hZIP1 mRNA levels and zinc uptake activity. Finally,
Lioumi et al. (19) observed expression of hZIP1
mRNA in intestinal enterocytes. This location of expression
suggests that hZIP1 may be involved in the uptake of dietary zinc from
the intestine. It should be noted that hZIP1 was designated
ZIRTL (for Zrt/IRT transporter-like) by these authors. In an
earlier report, Costello et al. (17) named this gene
hZIP1, and we have retained that nomenclature here.
One paradox that arose from our studies of hZIP1 and hZIP2 is that
these transporters have a surprisingly low affinity for their
substrate. Both transporters have a Km value of ~3
µM for free Zn2+ ions. Similar
Km values have been reported for zinc transporters
in a large number of mammalian cell types (21). The paradox arises when
we consider the free Zn2+ concentration in mammalian serum.
Although the total zinc concentration of serum is ~20
µM, very little metal is present in an unbound form (18).
In serum, ~75% Zn2+ is bound to albumin, and 20% is
bound to With the characterization of hZIP1 and hZIP2, our understanding of zinc
homeostasis in mammalian cells is greatly improving. A second family of
zinc transporters has also been identified in mammals. This family is
called the CDF (for cation diffusion facilitator) family, and like the ZIP proteins, members of
this group have been implicated in zinc transport in organisms of all phylogenetic levels (10, 23). One mammalian CDF protein, ZnT-1, is a
zinc efflux protein that transports zinc out of the cell (22). ZnT-1
may play a role in removing excess zinc from cells and may also serve
to transport zinc across the basolateral membrane of the intestinal
enterocyte during zinc absorption (24, 25). A second member of the CDF
family, ZnT-2, compartmentalizes intracellular zinc in the late
endosome of the cell (26, 27). This zinc sequestration reduces the
toxicity of intracellular zinc. Cellular zinc status is likely to be
controlled by regulation of many of these transporters. Expression of
both ZnT-1 and ZnT-2 has been shown to be induced in zinc-treated cells
(28) or in animals fed zinc-rich diets (24, 29). The zinc-responsive
transcription factor MTF-1 was found to regulate ZnT-1 (28), and
it seems likely that MTF-1 also regulates ZnT-2 expression. Thus, zinc treatment increases the cell's capacity to both export and sequester excess zinc. It is not yet clear if the uptake transporters are also
regulated in response to zinc status. Many ZIP genes in yeast and
plants are expressed at higher levels under zinc-limiting conditions
(1, 7, 9). In yeast, this up-regulation was shown to occur at a
transcriptional level and is mediated by the zinc-responsive Zap1
transcription factor (30). It is therefore intriguing that
hZIP1 mRNA levels decrease in prostate-derived PC-3
cells treated with higher than normal levels of zinc (17). We are
currently examining the regulation of hZIP1 and
hZIP2 in response to zinc availability to determine if such
a mechanism contributes to mammalian zinc homeostasis. Furthermore, the
potential roles of the other human ZIP genes remain to be addressed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-counter.
Chloride salt stock solutions (100 mM) of cadmium, copper,
cobalt, magnesium, manganese, and nickel were prepared in distilled
water. A ZnCl2 stock was prepared at 100 mM in
0.02 N HCl, and an FeCl3 stock was prepared at
50 mM in 0.1 N HCl. Sodium ascorbate (1 mM) was used to reduce Fe3+ to Fe2+
where indicated. Ascorbate treatment alone did not alter zinc uptake
activity (data not shown). Sodium bicarbonate stocks (1 M)
were prepared fresh before use. For experiments with complete medium as
the uptake buffer, the 65Zn was added to the medium and
incubated at 20 °C for 24 h before use to ensure equilibration
of the 65Zn with medium components. The statistical
significance of differences in values was determined using STATVIEW
software (Abacus Concepts, Inc., Berkeley, CA) and subjected to one-way
analysis of variance followed by Scheffe's test. Michaelis-Menten
kinetic constants were determined using Cleland Kinetic software
(13). For atomic absorption spectroscopy, cells were harvested at 50%
confluence and washed twice with equal volumes of PBS with or without 1 mM EDTA. The cells were resuspended in 6 M
nitric acid and incubated at 95 °C for 24 h. Acid-digested
samples were then assayed for zinc content with a Varian Spectra AA-30
atomic absorption spectrophotometer, and the final values were
normalized to cell number.
-actin. The cDNAs are provided serially
diluted over a 4-log range to allow semiquantitative assessment of
expression levels. hZIP1 primers were used to amplify a
581-base pair fragment from within the hZIP1 ORF. The
products were analyzed by agarose gel electrophoresis, stained with
ethidium bromide, and photographed under UV light with the CCD camera.
-Actin primers were used to confirm similar levels of input cDNA in each PCR sample.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of hZIP1 zinc uptake in K562
cells. A, zinc accumulation was assayed in CMV-hZIP1
transfectants (triangles) or vector-only CMV cells
(circles) with 3 µM 65Zn at
37 °C (closed symbols) or 4 °C (open
symbols). B, zinc accumulation was measured in
CMV-hZIP1 and CMV cells with 3 µM 65Zn at
37 °C from 0 to 60 s. C, concentration dependence of
zinc uptake was determined over a range of substrate concentrations.
hZIP1-dependent activity (dashed
line) was estimated by subtracting the CMV value from the
corresponding CMV-hZIP1 rates. D, CMV (open
bars) and CMV-hZIP1 (closed bars) cells were
grown in complete medium to 50% confluence, harvested, and washed with
PBS ± 1 mM EDTA prior to analysis by atomic
absorption spectroscopy. E, zinc accumulation was determined
in complete medium at 37 °C with 3 µM 65Zn
added as a tracer. F, zinc accumulation was determined in
complete medium over 15 min with the indicated concentrations of
65Zn. Each point represents the mean in a representative
experiment (n = 3), and the error bars
indicate ±1 S.D.
2-macroglobulin. A reasonable estimate of the free
Zn2+ concentration in plasma is ~0.2 nM (18),
and the relatively high Km value of hZIP1 activity
called into question the relevance of this transporter in zinc
acquisition in vivo. Therefore, it was important to
determine whether hZIP1 could function under more physiological
conditions. To address this question, we first examined the effects of
hZIP1 expression on zinc accumulation by cells grown in complete
culture medium containing 10% fetal bovine serum. The total zinc
content of this medium was determined by atomic absorption spectroscopy
to be 11 µM, and the albumin concentration was ~60
µM. Cell-associated zinc levels were measured in CMV and
CMV-hZIP1 cells grown to 50% confluence in complete medium. When
extracellular zinc was removed prior to analysis by washing the cells
with PBS, CMV-hZIP1 cells were found to have accumulated ~60% more
zinc than control cells (Fig. 1D). When PBS plus 1 mM EDTA was used as a more stringent wash buffer for the
removal of surface-bound zinc, cell-associated zinc was reduced in both
cell types. However, hZIP1-expressing cells consistently accumulated
more zinc than control cells. This conclusion was also supported by the
analysis of uptake rates using complete medium as the assay buffer. The
zinc uptake rate in CMV-hZIP1 cells was ~2-fold higher than in
controls (Fig. 1E). As expected, these rates were
20-30-fold lower than in synthetic buffer, probably due to decreased
substrate availability in medium containing zinc-binding agents such as
albumin. This conclusion was supported by the inability to saturate the
transport process in complete medium by adding zinc up to 60 µM (Fig. 1F). Taken together, these results
indicate that hZIP1 can function as a zinc transporter under
physiological conditions as simulated by complete medium.
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Fig. 2.
hZIP1 is localized to the plasma
membrane. CMV-hZIP1 (A and C) and
CMV-HA-hZIP1 (B and D) cells were analyzed by
indirect immunofluorescence using anti-HA antibody with Nomarski optics
(A and B) and confocal fluorescence (C
and D) microscopy.
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Fig. 3.
hZIP1-dependent and endogenous
zinc uptake activities are biochemically indistinguishable.
A, Northern blot analysis of hZIP1 and -actin
mRNAs from untransfected K562, CMV, and CMV-hZIP1 cells.
B, inhibition of endogenous (open
bars), hZIP1-dependent (closed bars),
or hZIP2-dependent (hatched bars) zinc uptake
determined with 65Zn (3 µM) and the indicated
metal ion (20 µM). All results are reported as the
percent of the untreated controls, and the hZIP1- and
hZIP2-dependent activities were determined as described in
the legend to Fig. 1C. C, effects of
HCO
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Fig. 4.
Antisense oligonucleotide inhibition of hZIP1
expression and endogenous zinc uptake activity. A,
shown are the results from FACS analysis of untransfected K562 cells.
B, shown are the results from FACS analysis of K562 cells
24 h after transfection with 10 µg of GFP-expressing pEGFP-N1.
C, viability of cells obtained following cell sorting was
determined by front and side light scattering. D, the
upper panel shows the rate of 65Zn
uptake determined for the indicated cells with 3 µM
65Zn over 15 min at 37 °C. Each point represents the
mean in a representative experiment (n = 3), and the
error bars indicate ±1 S.D. The middle
panel shows the results of quantitative RT-PCR analysis of
hZIP1 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNAs. C is the control
oligonucleotide. The lower panel shows the
results of RT-PCR analysis of controls to demonstrate the quantitative
nature of the method. CMV-hZIP1 cells had ~2-fold more
hZIP1 mRNA than CMV cells, and template concentrations
from 0.12 to 2 µg generated a corresponding increase in
product.
,
N
80% viable as judged by their front and side
light-scattering properties (Fig. 4C, gate R1). After sorting, the cells were cultured in complete medium for an
additional 24 h prior to zinc uptake assays and mRNA analysis by RT-PCR.
hZIP1-directed antisense oligonucleotides
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Fig. 5.
Expression of hZIP1 mRNA
in several human tissues as assessed by RT-PCR using Rapid
ScanTM gene expression panels. cDNA templates are
as follows; lane 1, brain; lane 2, heart;
lane 3, kidney; lane 4, spleen; lane
5, liver; lane 6, colon; lane 7, lung;
lane 8, small intestine; lane 9, muscle;
lane 10, stomach; lane 11, testis; lane
12, placenta; lane 13, salivary gland; lane
14, thyroid; lane 15, adrenal gland; lane
16, pancreas; lane 17, ovary; lane 18,
uterus; lane 19, prostate; lane 20, skin;
lane 21, leukocytes; lane 22, bone marrow;
lane 23, fetal brain; lane 24, fetal liver.
-Actin was also assayed to ensure approximately equal template
concentrations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin. Much of the remaining zinc is
complexed with amino acids such as histidine and cysteine. Because of
the high chelation capacity of serum, the free Zn2+
concentration in serum is calculated to be in the low nanomolar range.
Given this extremely low concentration of substrate, it was initially
unclear how these transporters could contribute to zinc accumulation by
mammalian cells under physiological conditions. The solution to this
paradox comes from considering the capacity of these transporters
relative to the zinc requirements of the cell. Steady-state cell
accumulation of zinc is ~100 pmol/106 cells (Fig.
1D), which is equivalent to 1 × 108 atoms
of zinc/cell. This value is similar to those obtained by others (22).
With a doubling time of 24 h, the uptake rate required to maintain
this level of zinc in growing cells is ~0.1 pmol/min/106
cells, i.e. a value almost identical to the uptake rate
observed using complete medium as the assay buffer (Fig. 1E)
and far lower than the rate (11 pmol/min/106 cells)
measured in buffer (Fig. 1C). Thus, our studies demonstrated that the capacity (i.e. Vmax) for
zinc uptake is so high relative to the cellular demand for zinc that
sufficient levels can be obtained despite the chelation capacity of
serum and the apparent low affinity of the transporters.
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ACKNOWLEDGEMENTS |
---|
We thank Louise Barnett, Jessica Wagner, and Jon Broomhead for technical assistance and the members of the Eide laboratory for many helpful discussions.
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FOOTNOTES |
---|
* This work was supported by a grant from the University of Missouri Research Board and by a subcontract from National Institutes of Health Grant CA79903 (to principal investigator R. B. Franklin, University of Maryland).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO 65211. Tel.: 573-882-9686; Fax: 573-882-0185; E-mail:
eided@missouri.edu.
Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M101772200
2 L. A. Gaither, unpublished observation.
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ABBREVIATIONS |
---|
The abbreviations used are: hZIP, human ZIP; RT-PCR, reverse transcription-polymerase chain reaction; ORF, open reading frame; CMV, cytomegalovirus; HA, hemagglutinin; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Zhao, H.,
and Eide, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
2454-2458 |
2. |
Zhao, H.,
and Eide, D.
(1996)
J. Biol. Chem.
271,
23203-23210 |
3. |
Eide, D.,
Broderius, M.,
Fett, J.,
and Guerinot, M. L.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5624-5628 |
4. | Korshunova, Y. O., Eide, D., Clark, W. G., Guerinot, M. L., and Pakrasi, H. B. (1999) Plant Mol. Biol. 40, 37-44[Medline] [Order article via Infotrieve] |
5. |
Rogers, E. E.,
Eide, D. J.,
and Guerinot, M. L.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12356-12360 |
6. | Guerinot, M. L. (2000) Biochim. Biophys. Acta 1465, 190-198[Medline] [Order article via Infotrieve] |
7. |
Grotz, N.,
Fox, T.,
Connolly, E.,
Park, W.,
Guerinot, M. L.,
and Eide, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7220-7224 |
8. |
Pence, N. S.,
Larsen, P. B.,
Ebbs, S. D.,
Letham, D. L.,
Lasat, M. M.,
Garvin, D. F.,
Eide, D.,
and Kochian, L. V.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4956-4960 |
9. |
MacDiarmid, C. W.,
Gaither, L. A.,
and Eide, D.
(2000)
EMBO J.
19,
2845-2855 |
10. | Gaither, L. A., and Eide, D. J. (2001) Biometals, in press |
11. | Eng, B. H., Guerinot, M. L., Eide, D., and Saier, M. H. (1998) J. Membr. Biol. 166, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Gaither, L. A.,
and Eide, D. J.
(2000)
J. Biol. Chem.
275,
5560-5564 |
13. | Cleland, W. W. (1979) Methods Enzymol. 43, 103-138 |
14. | Feinberg, A. P., and Vogelstein, B. (1984) Anal. Biochem. 137, 266-267[Medline] [Order article via Infotrieve] |
15. | Zucker, M., Matthews, D. H., and Turner, D. H. (1999) in RNA Biochemistry and Bio/Technology (Barciszewski, J. , and Clark, B. F. C., eds) , Kluwer Academic Publishers, Norwell, MA |
16. | Branch, A. D. (1998) Trends Biochem. Sci. 23, 45-50[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Costello, L. C.,
Liu, Y.,
Zou, J.,
and Franklin, R. B.
(1999)
J. Biol. Chem.
274,
17499-17504 |
18. |
Magneson, G. R.,
Puvathingal, J. M.,
and Ray, W. J.
(1987)
J. Biol. Chem.
262,
11140-11148 |
19. | Lioumi, M., Ferguson, C. A., Sharpe, P. T., Freeman, T., Marenholz, I., Mischke, D., Heizmann, C., and Ragoussis, J. (1999) Genomics 62, 272-280[CrossRef][Medline] [Order article via Infotrieve] |
20. | Yamaguchi, S. (1995) Kokubyo Gakkai Zasshi 62, 78-93[Medline] [Order article via Infotrieve] |
21. |
Reyes, J. G.
(1996)
Am. J. Physiol.
270,
C401-C410 |
22. | Palmiter, R. D., and Findley, S. D. (1995) EMBO J. 14, 639-649[Abstract] |
23. | Paulsen, I. T., and Saier, M. H. (1997) J. Membr. Biol. 156, 99-103[CrossRef][Medline] [Order article via Infotrieve] |
24. |
McMahon, R. J.,
and Cousins, R. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
4841-4846 |
25. |
McMahon, R. J.,
and Cousins, R. J.
(1998)
J. Nutr.
128,
667-670 |
26. | Palmiter, R. D., Cole, T. B., and Findley, S. D. (1996) EMBO J. 15, 1784-1791[Abstract] |
27. | Kobayashi, T., Beuchat, M., Lindsay, M., Frias, S., Palmiter, R. D., Sakuraba, H., Parton, R. G., and Gruenberg, J. (1999) Nat. Cell Biol. 1, 113-118[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Langmade, S. J.,
Ravindra, R.,
Daniels, P. J.,
and Andrews, G. K.
(2000)
J. Biol. Chem.
275,
34803-34809 |
29. |
Liuzzi, J. P.,
Blanchard, R. K.,
and Cousins, R. J.
(2001)
J. Nutr.
131,
46-52 |
30. | Zhao, H., and Eide, D. J. (1997) Mol. Cell. Biol. 17, 5044-5052[Abstract] |