Copper-stimulated Endocytosis and Degradation of the
Human Copper Transporter, hCtr1*
Michael J.
Petris
§,
Kathryn
Smith
,
Jaekwon
Lee¶, and
Dennis J.
Thiele¶
From the
Department of Nutritional Sciences,
University of Missouri, Columbia, Missouri 65211 and the
¶ Department of Biological Chemistry, University of Michigan
Medical School, Ann Arbor, Michigan 48109
Received for publication, September 16, 2002, and in revised form, December 2, 2002
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ABSTRACT |
Copper uptake at the plasma membrane and
subsequent delivery to copper-dependent enzymes is
essential for many cellular processes, including mitochondrial
oxidative phosphorylation, free radical detoxification,
pigmentation, neurotransmitter synthesis, and iron metabolism. However,
intracellular levels of this nutrient must be controlled because it is
potentially toxic in excess concentrations. The hCtr1 protein functions
in high affinity copper uptake at the plasma membrane of human cells.
In this study, we demonstrate that levels of the hCtr1 protein at the
plasma membrane of HEK293 cells were reduced when cells were exposed to
elevated copper. This decrease in surface hCtr1 levels was associated
with an increased rate of endocytosis, and low micromolar
concentrations of copper were sufficient to stimulate this process.
Inhibitors of clathrin-dependent endocytosis prevented the
trafficking of hCtr1 from the plasma membrane, and newly internalized
hCtr1 and transferrin were co-localized. Significantly, elevated copper
concentrations also resulted in the degradation of the hCtr1 protein.
Our findings suggest that hCtr1-mediated copper uptake into mammalian
cells is regulated by a post-translational mechanism involving
copper-stimulated endocytosis and degradation of the transporter.
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INTRODUCTION |
Copper is an essential nutrient for all organisms because it is
required by a variety of enzymes that are involved in critical areas of
metabolism (1). Because copper is also potentially toxic when it
accumulates beyond cellular needs, intracellular levels of this
nutrient must be precisely regulated. In the yeast, Saccharomyces
cerevisiae, yCtr1 and yCtr3, are plasma membrane-associated high
affinity copper transporters (2, 3). Recent studies have identified the
human and mouse high affinity copper uptake proteins, hCtr1 and mCtr1
(4, 5). Like the yCtr1 protein, the mammalian homologues possess three
predicted membrane spanning regions and form oligomeric complexes,
possibly to facilitate formation of a membrane pore for copper uptake
(6-8). Deletion of the mCtr1 gene in mice results in
embryonic lethality, suggesting mCtr1-mediated copper uptake is
essential during mammalian embryogenesis (9, 10). In mouse embryonic
cell lines in which both mCtr1 alleles have been disrupted,
we have recently shown that a myc-tagged human Ctr1 protein functions
in copper uptake and delivery to the copper-dependent
enzymes cytochrome c oxidase, superoxide dismutase, and
tyrosinase (11). In the cultured human embryonic kidney cell line,
HEK293,1 the hCtr1-myc
protein is predominantly located at the plasma membrane and functions
in copper uptake in a time-, temperature-, pH-, and
K+-dependent manner (8). Recent studies suggest
that hCtr1 is predominantly in perinuclear vesicles in some cell lines,
whereas in others it is at the plasma membrane (7, 8). The basis for
this variability in hCtr1 localization in different cell lines is unknown.
In yeast, the expression of yCtr1 is up-regulated via the Mac1p
transcription factor when extracellular copper levels are low (12-14).
In addition to this transcriptional regulation of yCtr1 expression, the
levels of the protein at the plasma membrane are post-translationally
regulated by copper availability. When yeast cells are exposed to low
micromolar levels of copper, yCtr1 is stimulated to endocytose from the
plasma membrane to cytoplasmic vesicles (15). This could serve to
rapidly prevent excessive copper uptake via yCtr1 and reduce the
potential for copper toxicity, or alternatively, provide a vesicular
route for yCtr1-mediated copper uptake. At higher concentrations of
copper (~10 µM) the yCtr1 protein is degraded at the
plasma membrane via a mechanism that is independent of endocytosis
(15). Despite progress in understanding the biochemical aspects of
hCtr1-mediated copper transport in human cells, it is unknown whether
the function of hCtr1 is regulated by copper availability. Unlike
yeast, there is currently no evidence for copper-regulated
transcription of mammalian Ctr1 (5). Therefore, we tested
whether copper levels in the growth media regulate hCtr1 distribution
in cultured mammalian cells. Using HEK293 and Chinese hamster ovary
(CHO) cell lines transfected with a functional myc-tagged hCtr1
protein, we showed that the level of hCtr1 protein at the plasma
membrane was reduced when cells were exposed to elevated copper in the
media. The reduced levels of hCtr1 at the plasma membrane were
associated with an increased rate of hCtr1 endocytosis, which was
specifically inducible by low micromolar copper levels. Higher copper
levels resulted in the degradation of hCtr1. Our findings suggest that
copper-stimulated endocytosis and degradation of the hCtr1 protein
could play a key role in regulating copper entry across the plasma
membrane of mammalian cells.
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EXPERIMENTAL PROCEDURES |
Reagents, Cell Lines, and Antibodies--
The human embryonic
kidney (HEK293) cell lines expressing either the myc-tagged hCtr1
protein, or pcDNA3.1 vector, were isolated by transfection using
the LipofectAMINE 2000 reagent (Invitrogen) as previously described
(8). The functional c-myc epitope-tagged hCtr1 expression construct in
pcDNA3.1(+) vector and pTK-Hyg (Clontech) were
cotransfected in HEK293 cells. Hygromycin-resistant cell lines were
established by the selection of transfected cells with the
supplementation of 100 µg/ml hygromycin B (Invitrogen) in the culture
medium. hCtr1 expression was analyzed by Western blotting using the
anti-myc antibody and by measuring stimulation of 64Cu
uptake, as described previously (8). CHO cells were transiently transfected using LipofectAMINE 2000 using the pcDNA3.1 plasmid containing the myc-tagged hCtr1 cDNA as previously described (8). All cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 units/ml
penicillin and streptomycin in a 5% CO2, 37 °C
incubator. Monoclonal anti-myc antibody, 9E10, streptavidin-horseradish
peroxidase (HRP) conjugate, and anti-mouse antibodies conjugated to HRP
were purchased from Roche Molecular Biochemicals. Alexa 488 anti-mouse
antibodies, transferrin-biotin conjugate, and transferrin-Texas Red
were purchased from Molecular Probes. Chlorpromazine, cycloheximide,
and methyl-
-cyclodextrin were purchased from Sigma.
Immunofluorescence Microscopy to Assess Surface and Intracellular
Pools of hCtr1 Protein--
Cells were grown in 24-well trays for
48 h on sterile glass coverslips. In some experiments, copper was
added to the media at the indicated concentrations and times. To detect
the total pool of hCtr1-myc protein, cells were washed twice with 1 ml
of ice-cold PBS and fixed for 10 min at 25 °C using 4%
paraformaldehyde. The cells were then permeabilized with 0.1% Triton
X-100 in PBS for 5 min, blocked for 1 h with 1% bovine serum
albumin and 3% skim milk in PBS, and then probed with the anti-myc
antibodies (10 µg/ml) followed by Alexa 488 anti-mouse antibodies
(1/1000). To label only the surface pool of hCtr1-myc, the Triton X-100 permeabilization step was omitted and fixed cells were blocked and
probed with the anti-myc and Alexa 488 antibodies as described above.
The endocytosis of hCtr1-myc was assessed by detecting the uptake of
anti-myc antibodies added to the media of Hek/hCtr1-myc cells. Cells
were pre-grown for 48 h in basal media and then incubated for
either 2 or 5 min in basal- or copper-supplemented media containing 10 µg/ml anti-myc antibodies at 37 °C. Cells were transferred to ice
to prevent further trafficking of hCtr1-myc, washed twice with ice-cold
PBS, and the surface-bound antibodies were removed by three washes for
2 min with acidic buffer on ice (100 mM glycine, 20 mM magnesium acetate, 50 mM potassium chloride,
pH 2.2). After a further two washes with PBS on ice, cells were fixed,
permeabilized, and processed for immunofluorescence as described above.
In double labeling endocytosis experiments, 100 µg/ml Texas
Red-conjugated transferrin and 10 µg/ml anti-myc antibodies were
added to the media of cells for 2 min and processed for
immunofluorescence microscopy as described above. Confocal
immunofluorescence microscopy was performed with a ×60 oil objective
and an IX70 Olympus microscope fitted with a Bioradiance 2000 laser
(Bio-Rad).
Detection of hCtr1 Protein Levels at the Plasma
Membrane--
The pool of hCtr1-myc at the plasma membrane was
assessed by measuring the levels of anti-myc antibodies bound to the
surface of Hek/hCtr1-myc cells. Hek/hCtr1-myc cells were cultured for 48 h in 6-well trays, washed twice with PBS on ice, and fixed for
10 min in 4% paraformaldehyde without subsequent permeabilization steps. Cells were then blocked using 3% skim milk in PBS, and incubated with the anti-myc antibody (1/500) for 30 min at room temperature. Cells were washed five times in PBS to remove unbound antibodies, and then lysed by sonication in SDS buffer solution containing 62 mM Tris-Cl (pH 6.8), 2% SDS, 100 mM dithiothreitol, and protease inhibitor mixture (Roche
Molecular Diagnostics). Cell lysates containing the solubilized
anti-myc antibodies that were bound to the hCtr1-myc protein at the
plasma membrane were separated using 4-20% SDS-PAGE, transferred to
nitrocellulose membranes, and the anti-myc antibodies were then
detected using anti-mouse HRP antibodies (1/5000) by chemiluminescence
(Roche Molecular Biochemicals). Tubulin protein levels were detected on
parallel immunoblots using anti-tubulin antibodies (1:40,000; Sigma).
Assay of hCtr1 Endocytosis--
The endocytosis of hCtr1-myc was
determined by measuring the uptake of anti-myc antibodies added to the
cultured media of Hek/hCtr1-myc cells. Cells were pregrown in 6-well
trays for 48 h in basal media, and then incubated for the
indicated times at 37 °C in basal- or copper-supplemented media
containing 10 µg/ml anti-myc antibodies. Cells were washed twice with
PBS on ice, and surface-bound antibodies were removed by three washes
in ice-cold acidic buffer (above). Cells were harvested by scraping
into ice-cold PBS and pelleted by centrifugation at 1000 × g. The cell pellets were solubilized in SDS buffer (above),
and 20 µg of lysates containing internalized anti-myc antibodies were
separated using 4-20% SDS-PAGE, transferred to nitrocellulose
membranes, and detected by chemiluminescence using anti-mouse HRP
antibodies, as described above. In some experiments, 100 µg/ml
biotinylated transferrin was added to the media instead of anti-myc
antibodies and the internalized transferrin was detected using
streptavidin-peroxidase on Western blots.
Immunoblot Analysis of hCtr1 Protein--
Immunoblot detection
of hCtr1-myc protein was essentially as described previously (8). Cells
cultured in 25-cm2 flasks were scraped into ice-cold PBS
and pelleted by centrifugation. After several washes in ice-cold PBS,
the cells were lysed for 20 min on ice in buffer containing 1% Triton
X-100, 1 mM EDTA, 100 mM dithiothreitol, and
protease inhibitor mixture (Roche Molecular Biochemicals). Samples were
centrifuged for 10 min at 16,000 × g and 20 µg of
protein lysates were separated using 4-20% SDS-PAGE, transferred to
nitrocellulose membranes, and detected by chemiluminescence using
anti-myc antibodies (1:1000) followed by anti-mouse HRP antibodies
(1/5000).
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RESULTS |
Elevated Copper Concentrations Reduce hCtr1 Levels at the Plasma
Membrane--
This study was aimed at determining whether the levels
and distribution of the hCtr1 copper transporter is subject to
regulation by copper. We previously engineered a human Ctr1 cDNA
with an amino-terminal myc epitope (hCtr1-myc) in the expression
plasmid pcDNA 3.1, and demonstrated this tagged protein is a
functional copper transporter and located at the plasma membrane in
transfected HEK293 cells (8). Our initial studies investigated the
localization of hCtr1-myc transiently expressed in CHO cells cultured
in growth media containing elevated copper. Immunofluorescence
microscopy using the anti-myc antibody revealed strong labeling of
hCtr1-myc at the plasma membrane of CHO cells grown in basal media,
with some punctate vesicular labeling in the cytoplasm (Fig.
1A). However, in cells exposed
to elevated copper there was a striking change in localization to a
punctate vesicular distribution throughout the cytoplasm, and a marked
reduction in levels of hCtr1-myc at the plasma membrane (Fig.
1B). These observations suggested that the location of
hCtr1-myc in CHO cells was altered by elevated copper levels. We then
investigated the effect of copper on the location of hCtr1-myc in the
HEK293 cell line, Hek/hCtr1-myc, which stably expresses the hCtr1-myc
protein (8). The plasma membrane location for hCtr1-myc in
Hek/hCtr1-myc cells grown in basal medium was confirmed (Fig.
1C). However, similar to that observed for CHO cells, when
Hek/hCtr1-myc cells were exposed to elevated copper levels the
hCtr1-myc protein was localized in a perinuclear distribution, and
there was little apparent staining at the cell periphery (Fig.
1D). There was no immunofluoresence signal detected in CHO
or HEK293 cells transfected with the pcDNA3.1 vector alone (data
not shown). These data suggest that elevated copper concentrations
reduced the levels of hCtr1-myc at the plasma membrane in both HEK293
and CHO cells.

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Fig. 1.
Elevated copper alters the localization of
hCtr1. Immunofluorescence microscopy analysis of hCtr1-myc
distribution in CHO and HEK293 cells. CHO cells transiently transfected
with the hCtr1-myc expression plasmid (A and B),
or HEK293 cells stably expressing myc-tagged hCtr1 protein
(Hek/hCtr1-myc) (C-F), were cultured in basal media
(A, C, and E) or media containing 100 µM copper for 16 h (B, D, and
F). The total pool of hCtr1-myc was detected using anti-myc
antibodies after fixing cells with 4% paraformaldeyde and then
permeabilizing by treatment with 0.1% Triton X-100 prior to detection
using anti-myc antibodies (A-D). The surface pool of
hCtr1-myc protein is shown for untreated- and copper-treated
Hek/hCtr1-myc cells that were fixed with 4% paraformaldeyde without
permeabilization (E and F).
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Previous studies have demonstrated that the amino terminus of hCtr1 is
extracellular (6, 16), and this topology allowed us to take advantage
of the amino-terminal position of the myc epitope tag to directly test
whether elevated copper reduces hCtr1 levels at the plasma membrane.
Hek/hCtr1-myc cells were grown in basal media or media containing 100 µM copper for 16 h, and immunofluorescence
microscopy was carried out with the anti-myc antibodies using intact
paraformaldehyde-fixed cells that had not been permeabilized. Strong
labeling of hCtr1-myc was observed at the surface of intact
Hek/hCtr1-myc cells grown in basal medium (Fig. 1E),
however, there was no surface labeling of hCtr-myc in copper-treated
cells (Fig. 1F). The integrity of the plasma membrane was
confirmed after fixation by the absence of labeling with antibodies
against the intracellular protein, protein-disulfide isomerase (data
not shown). As an independent assay, immunoblots were used to assess
the reduced levels of hCtr1-myc protein at the plasma membrane of
copper-treated Hek/hCtr1-myc cells. This was achieved by measuring the
levels of anti-myc antibodies that were recovered from the surface of
intact Hek/hCtr1-myc cells after these cells were probed with anti-myc
antibodies. Hek/hCtr1-myc cells were grown in basal- or
copper-supplemented media, fixed with paraformaldehyde, and the intact
cells were then probed with the anti-myc antibodies to label hCtr1-myc
protein at the plasma membrane. After extensive washing of cells with
PBS to remove unbound antibodies, the cells were lysed with SDS buffer
and the solubilized anti-myc antibodies were detected by SDS-PAGE and Western blotting with anti-mouse antibodies conjugated to HRP. As a
control, the affinity purified anti-myc antibodies were run on the same
gel and detected as a single 100-kDa band (Fig.
2A, lane 1). The
100-kDa band was strongly detected in the sample from Hek/hCtr1-myc
cells grown in basal medium, demonstrating abundant levels of anti-myc
antibodies on the surface because of the myc epitope of hCtr1 (Fig.
2A, lane 2). Markedly reduced levels of anti-myc
antibodies were recovered from the surface of cells grown in elevated
copper (Fig. 2A, lane 3). There were no
detectable anti-myc antibodies bound to the surface of untreated- or
copper-treated HEK293 cells stably transfected with the empty pcDNA3.1 vector (Fig. 2A, lanes 4 and
5). These findings independently confirmed the
immunofluorescence microscopy results and suggested that elevated
copper reduces the level of hCtr1-myc protein at the plasma membrane.
We then explored the kinetics of hCtr1 clearance from the plasma
membrane following exposure of Hek/hCtr1-myc cells to elevated copper.
A clear reduction in the levels of surface-bound anti-myc antibodies
occurred after a 10-min exposure to elevated copper, and decreased over
the course of 180 min (Fig. 2B). This decrease in surface
hCtr1-myc expression occurred independently of de novo
protein synthesis, because we observed the same effect in cells
pretreated with the translation inhibitor, cycloheximide, for 30 min
followed by exposure to media containing both copper and
cycloheximide (data not shown).

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Fig. 2.
Elevated copper reduces surface expression of
hCtr1. A, immunoblot analysis of anti-myc antibodies
bound to the surface of Hek/hCtr1-myc cells (lanes 2 and
3) or HEK293 cells stably transfected with pcDNA3.1
vector (lanes 4 and 5) that were cultured for
16 h in basal media ( Cu) or 100 µM
copper (+Cu). Cells were fixed using paraformaldehyde,
blocked, and then probed with anti-myc antibodies. The surface-bound
antibodies were solubilized with SDS buffer, separated using SDS-PAGE,
and detected by immunoblotting with anti-mouse antibodies conjugated to
HRP as described under "Experimental Procedures." Purified anti-myc
9E10 antibodies were run as a control (lane 1), and tubulin
protein was detected to indicate protein loading. B,
immunoblot analysis of anti-myc antibodies bound to the surface of
Hek/hCtr1-myc cells after the cells were cultured in media containing
100 µM copper for the indicated times, as per
A and under "Experimental Procedures."
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Copper-stimulated Endocytosis of hCtr1--
The rapidity with
which surface levels of hCtr1 were reduced in response to elevated
copper prompted us to test whether this is accomplished through
copper-stimulated endocytosis of hCtr1. We exploited the extracellular
location of the myc epitope of hCtr1-myc to assess endocytosis of the
protein. Using the same strategy previously employed to assess
endocytosis of the Menkes disease copper transporter, ATP7A (17), we
surmised that if hCtr1-myc endocytosed from the plasma membrane,
anti-myc antibodies added to the growth media would bind to hCtr1-myc
and be internalized by Hek/hCtr1-myc cells. Initially,
immunofluorescence microscopy was used demonstrate that the surface
pool of hCtr1-myc could be labeled by the anti-myc antibodies added to
the media of living Hek/hCtr1-myc cells that were cooled on ice to
inhibit endocytosis (Fig. 3A).
There was no labeling by HEK293 cells transfected with the pcDNA3.1
vector (data not shown), indicating that the anti-myc antibodies were
specifically bound to the extracellular myc epitope of the hCtr1-myc
protein in Hek/hCtr-myc cells. We found that several washes with cold
acidic buffer removed the surface-bound antibody (Fig. 3B).
This washing step was important in establishing conditions that would
remove surface antibodies and permit the visualization of internalized
antibodies in subsequent endocytosis experiments. To test the effect of
copper on the endocytosis of hCtr1-myc, cells were pre-grown for
48 h in basal media and then incubated for 2 or 5 min in basal or
elevated copper media containing anti-myc antibodies to allow uptake of
the antibodies via hCtr1-myc endocytosis. After these antibody uptake
time periods were completed, cells were rapidly cooled on ice and the
surface-bound anti-myc antibodies were removed by washing cells with
ice-cold acidic buffer. The protected internalized antibodies were then
detected by confocal immunofluorescence microscopy. After 2 min there
was no apparent uptake of anti-myc antibodies by cells in basal media (Fig. 3C). However, after 2 min in elevated copper, anti-myc
antibodies were detected in vesicular compartments close to the cell
periphery (Fig. 3D). Interestingly, by 5 min in basal media,
the anti-myc antibodies had accumulated to low levels in peripheral
compartments (Fig. 3E). This indicated that endocytosis of
hCtr1-myc had occurred in the absence of copper supplementation.
However, after 5 min in elevated copper the staining of intracellular
anti-myc antibodies was more intense than in basal media and there was
a substantial concentration in the perinuclear region (Fig.
3F). There was no internalization of anti-myc antibodies by
untreated or copper-treated HEK293 cells transfected with the
pcDNA3.1 vector (data not shown), suggesting that the uptake of
anti-myc antibodies in the Hek/hCtr1-myc cells occurred through
specific binding to hCtr1-myc protein. These data suggest that the
hCtr1-myc protein undergoes endocytosis in basal media, and that
elevated copper stimulates this process.

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Fig. 3.
Elevated copper stimulates endocytosis of
hCtr1-myc. The hCtr1-myc protein at the plasma membrane of living
Hek/hCtr1-myc cells was labeled with anti-myc antibodies on ice. Cells
were then washed with either PBS (A) or acidic buffer
(B) prior to fixation, permeabilization, and detection by
immunofluorescence microscopy. Note the removal of surface-associated
antibodies with acidic buffer. C-F, assessment of hCtr1-myc
endocytosis through the uptake of extracellular anti-myc antibodies.
Hek/hCtr1-myc cells pre-grown in basal media were exposed for 2 (C and D) or 5 min (E and
F) to anti-myc antibodies in basal media (C and
E) or media containing 100 µM copper
(D and F). Cells were then washed with acidic
buffer to remove surface antibodies and the intracellular antibodies
were then detected by immunofluorescence microscopy.
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To more quantitatively demonstrate copper-induced endocytosis of
hCtr1-myc, we measured levels of internalized anti-myc antibodies by
immunoblotting experiments. As we had done earlier for the immunofluorescence experiments, we initially confirmed that washing cells with acidic buffer removes surface-bound anti-myc antibodies. Hek/hCtr1-myc cells were pre-grown for 48 h in basal media, cooled on ice, and then incubated for 10 min with the anti-myc antibodies to
label hCtr1-myc protein at the plasma membrane. Cells were either
washed with PBS or acidic buffer on ice, lysed in SDS buffer, and the
anti-myc antibodies were detected on Western blots. The 100-kDa band
corresponding to the anti-myc antibody was detected in the samples
derived from PBS-washed cells (Fig. 4,
lane 1), however, this was absent from cells that were
washed with acidic buffer (Fig. 4, lane 2). This indicated
that surface-bound anti-myc antibodies were removed by this acidic
buffer treatment. To explore the effect of elevated copper on the
endocytosis of hCtr1-myc, the Hek/hCtr1-myc cells were pre-grown for
48 h in basal media and then incubated in either basal or 100 µM copper media containing the anti-myc antibodies. Cells
were transferred to ice to prevent further trafficking, washed
extensively with acidic buffer to remove surface-bound anti-myc
antibodies, and cell lysates were prepared to allow intracellular
anti-myc antibodies to be detected on immunoblots. After a 2-min uptake
period, internalized anti-myc antibodies were detected in
copper-treated cells (Fig. 4, lane 4), however, no signal
was detected in cells exposed to basal media (Fig. 4, lane
3). However, after 5 min of uptake the anti-myc antibodies were
weakly detected in cells exposed to basal media (Fig. 4, lane
5), but the levels were 20-fold lower than in the copper-treated
cells, as determined by densitometry measurements of band intensity
(Fig. 4, lane 6). This elevated uptake of anti-myc antibodies in copper-treated cells relative to untreated cells was also
apparent at the 10-min time point (Fig. 4, lanes 7 and 8). As we found earlier in the immunofluorescence studies,
there was no internalization of anti-myc antibodies in HEK293 cells stably transfected with the pcDNA3.1 vector alone (data not shown). To address the specificity of copper-induced hCtr1-myc endocytosis, the
effects of copper treatment on the uptake of transferrin was evaluated
in Hek/hCtr1-myc cells. Elevated copper did not alter the uptake of
transferrin (Fig. 4, lanes 9 and 10), indicating that the increased uptake of anti-myc antibodies in copper-treated cells was not because of a general increase in endocytosis from the
plasma membrane. These data suggest that elevated copper specifically stimulates the endocytosis of hCtr1-myc from the plasma membrane, and
are consistent with earlier results showing a copper-induced decrease
in surface levels of hCtr1.

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Fig. 4.
Time course analysis of copper-stimulated
endocytosis of hCtr1-myc by immunoblot detection. Hek/hCtr1-myc
cells were labeled on ice with anti-myc antibodies, washed with either
PBS (lane 1) or acidic buffer (lane 2), and the
antibodies were detected using immunoblotting experiments. This result
demonstrated the removal of surface-bound anti-myc antibodies by the
acidic buffer washes. hCtr1-myc endocytosis was determined by assessing
the uptake of anti-myc antibodies at 37 °C. Cells were incubated in
the presence of 10 µg/ml anti-myc antibodies added to basal media
( Cu) or media containing 100 µM copper
(+Cu) for the indicated times. Surface antibodies were
removed by washing cells in cold acidic buffer and intracellular
antibodies were then detected by SDS-PAGE and immunoblotting using
anti-mouse antibodies conjugated to HRP (lanes 3-8). Note
the reduced uptake of antibodies from basal media relative to elevated
copper. In parallel experiments, the uptake of biotinylated transferrin
over 10 min from basal media ( Cu) or 100 µM
copper media (+Cu) was assessed by immunoblotting
(lanes 9 and 10). Tubulin levels are shown in the
lower panel to indicate protein loading for all
samples.
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Co-localization of Newly Internalized hCtr1-myc and
Transferrin--
To begin to understand the pathway of hCtr1-myc
endocytosis, we determined if newly internalized anti-myc antibodies
co-localized with transferrin, a marker of early endosomes originating
from clathrin-mediated endocytosis (18). Hek/hCtr1-myc cells were exposed to elevated copper media containing both the anti-myc antibodies and human transferrin conjugated to the Texas Red
fluorophore. After a 2-min incubation at 37 °C, the cells were
washed with cold acidic buffer to remove any anti-myc antibodies and
transferrin bound to the cell surface. Cells were then fixed,
permeabilized, and the internal anti-myc antibodies were detected with
anti-mouse Alexa 488 antibodies. The internalized transferrin (Fig.
5A, red) and
anti-myc antibodies (Fig. 5B, green) were
co-localized within vesicular compartments, as shown by the
predominantly yellow labeling in the overlay image (Fig.
5C). These findings suggested that newly internalized
hCtr1-myc and transferrin co-localize with early endosomes.

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Fig. 5.
Newly internalized hCtr1-myc and transferrin
are co-localized. Hek/hCtr1-myc cells were incubated for 2 min in
media containing 100 µM copper, the anti-myc antibodies,
and transferrin conjugated to the Texas Red fluorophore. Cells were
then washed with acidic buffer to remove antibodies and transferrin
from the plasma membrane, fixed, permeabilized, and intracellular
antibodies were detected with Alexa 488 anti-mouse antibodies.
Cytoplasmic vesicles containing internalized transferrin (A,
red), and anti-myc antibodies (B,
green), were extensively co-localized as indicated by
yellow compartments in the merged image
(C).
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Copper-induced Internalization of hCtr1-myc Is
Clathrin-dependent--
Because transferrin is
internalized via clathrin-mediated endocytosis, the finding that newly
internalized transferrin and hCtr1-myc were co-localized prompted us to
test whether the internalization of hCtr1-myc was
clathrin-dependent. Chlorpromazine and
methyl-
-cyclodextrin (MCD) have been used extensively to inhibit
endocytosis of surface proteins. Chlorpromazine is a cationic
amphiphilic drug that inhibits the assembly of the clathrin adaptor
protein, AP2, on clathrin-coated pits (19), whereas MCD is a more
general inhibitor of endocytosis through its ability to extract
cholesterol from the plasma membrane and inhibit formation of
clathrin-coated pits and caveolae (20). Hek/hCtr1-myc cells were
incubated for 20 min with either chlopromazine or MCD, prior to a 2-h
incubation in the presence of the inhibitor plus elevated copper.
Immunofluorescence microscopy was then used to assess the intracellular
location of hCtr1-myc protein. The expected internalization of
hCtr1-myc protein to cytoplasmic vesicles in copper-treated cells was
observed in the absence of endocytosis inhibitors (Fig.
6B), as shown earlier (Fig.
1D). Chlorpromazine and MCD treatments did not alter the
plasma membrane location of hCtr1-myc in cells in basal media (Fig. 6,
C and E). However, the copper-induced
internalization of hCtr1-myc protein from the plasma membrane was
inhibited in cells treated with chlorpromazine or MCD (Fig. 6,
D and F). In control experiments, the uptake of transferrin was inhibited by both chlorpromazine and MCD (data not
shown). These observations suggest that hCtr1 is internalized by
clathrin-dependent endocytosis.

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Fig. 6.
Chlorpromazine and MCD inhibit copper-induced
hCtr1 endocytosis and increase surface levels of hCtr1 in basal
media. A-F, Hek/hCtr1-myc cells were pretreated for 20 min with chlorpromazine (5 µg/ml) or MCD (10 mM), and
then incubated for 2 h in either basal media containing the
endocytic inhibitor or 100 µM copper (+Cu)
containing the endocytic inhibitor. Control Hek/hCtr1-myc cells were
incubated for 2 h in basal media (A) or 100 µM copper (B) without endocytosis inhibitors.
The location of hCtr1-myc was assessed by immunofluorescence microscopy
using anti-myc antibodies. G, analysis of surface levels of
hCtr1-myc protein in Hek/hCtr1-myc cells treated with chlorpromazine or
MCD. Hek/hCtr1-myc cells were incubated for 2 h in basal media
(lane 1), 5 µg/ml chlopromazine (lane 2), or 10 mM MCD (lane 3). Cells were fixed using
paraformaldehyde, blocked, and then probed with anti-myc antibodies.
The surface-bound antibodies were solubilized with SDS buffer,
separated using SDS-PAGE, and detected by immunoblotting with
anti-mouse antibodies conjugated to HRP.
|
|
Our earlier finding that anti-myc antibodies were internalized by
hCtr1-myc cells in basal media suggested that the hCtr1-myc protein may
constitutively cycle via the plasma membrane and intracellular compartments in the absence of elevated copper. To further explore whether hCtr1-myc constantly cycles via the plasma membrane, we tested
whether endocytosis inhibitors would result in elevated levels of
hCtr1-myc protein at the plasma membrane. To test this hypothesis, we
assessed the surface levels of hCtr1-myc protein after incubating
hCtr1-myc cells in basal media containing chlorpromazine or MCD.
hCtr1-myc cells were incubated for 2 h at 37 °C in basal media
containing either chlorpromazine or MCD, and surface levels of the
hCtr1-myc protein were determined by assessing the recovery of
surface-bound anti-myc antibodies from the surface of intact cells. As
shown in Fig. 6G, the surface levels of hCtr1-myc protein were elevated in cells treated with chlorpromazine and MCD, relative to
untreated cells. These data suggest that although the hCtr1-myc protein
has a steady-state localization at the plasma membrane, the protein
cycles via intracellular compartments in the absence of elevated copper.
Sensitivity and Metal Specificity of Copper-induced hCtr1
Endocytosis--
To further characterize the process of
copper-stimulated hCtr1-myc endocytosis, we determined the sensitivity
of this response. Hek/hCtr1-myc cells were pre-grown in basal media for
48 h, and the uptake of anti-myc antibodies was assessed after 10 min in media containing a range of copper levels. Copper levels as low as 0.5-1.0 µM stimulated the increased internalization
of anti-myc antibodies compared with basal media (Fig.
7A, lanes 3 and
4), and this progressively increased until saturation was
between 5 and 20 µM copper (Fig. 7A,
lanes 6 and 7). To test metal specificity, the
uptake of anti-myc antibodies was investigated in media containing 5 or
20 µM AgNO3, FeCl2, or
ZnCl2 (Fig. 7B). The uptake of anti-myc antibodies was stimulated by 5 and 20 µM silver ions with
similar efficiency to equimolar levels of copper, whereas iron or zinc had relatively little, if any, effect on uptake. Therefore, the stimulation of hCtr1-myc endocytosis is sensitive to low copper concentrations and is somewhat specific for this metal ion.

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Fig. 7.
Sensitivity and metal specificity of
copper-induced hCtr1 endocytosis. A, immunoblots were
used to detect internal levels of anti-myc antibodies endocytosed by
Hek/hCtr1-myc cells after 10 min in either basal media (lane
1) or media containing increased copper concentrations
(lanes 2-8). Tubulin levels are shown in the lower
panel to indicate protein loading for all samples. B,
immunoblots were used to detect intracellular anti-myc antibodies
endocytosed by Hek/hCtr1-myc cells after 10 min in either basal media
(lane 1) or media containing the indicated concentrations of
copper (Cu) (lanes 2 and 3), silver
(Ag) (lanes 4 and 5), iron
(Fe) (lanes 6 and 7), and zinc
(Zn) (lanes 8 and 9). Tubulin levels
are shown to indicate protein loading of samples.
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|
Copper-induced hCtr1 Degradation--
To further explore the role
of copper in regulating the function of hCtr1, we investigated whether
levels of the protein were altered in response to elevated copper in
the growth media. Hek/hCtr1-myc cells were pre-grown in basal media for
48 h and pretreated for 20 min with 100 µg/ml cycloheximide to
inhibit new protein synthesis. Copper was then added to the
cycloheximide-containing media at a range of concentrations for 2 h prior to immunoblot analysis of steady-state hCtr1-myc protein levels
using anti-myc antibodies (Fig. 8). We
and others have previously shown that the hCtr1 protein migrates as a
30-35-kDa monomer and 60-70-kDa dimer by SDS-PAGE (7, 8). These
expected sizes for the hCtr1-myc protein were observed in Hek/hCtr1-myc
cells after probing Western blots with the anti-myc antibody (Fig. 8,
lane 3, arrows). Both the 35- and 70-kDa forms of
the hCtr1-myc proteins were absent in nontransfected HEK293 cells and
HEK293 cells transfected with the expression vector, pcDNA3.1 (Fig.
8, lanes 1 and 2). Despite the presence of two
weakly cross-reacting proteins endogenous within HEK293 cells (Fig. 8,
asterisks), the effect of copper on the levels of hCtr1-myc
protein was readily detected using the anti-myc antibody. A substantial
decrease in levels of hCtr1-myc protein was observed in media
containing 1-5 µM copper (Fig. 8, lanes 4 and
5), which was further exacerbated at copper concentrations
between 5 and 20 µM. Both the 35- and 70-kDa forms of the
hCtr1-myc protein (Fig. 8, lanes 5 and 6) were
decreased by copper treatment. Together with our earlier data, these
findings suggest that copper triggers both the endocytosis and
degradation of the hCtr1 protein.

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Fig. 8.
Elevated copper decreases levels of hCtr1-myc
protein. Western blot analysis of steady-state levels of hCtr1-myc
in Hek/hCtr1-myc cells after exposure to elevated copper. HEK293,
HEK293 cells stably expressing the vector pcDNA 3.1, or
Hek/hCtr1-myc cells were pretreated for 20 min with cycloheximide to
inhibit protein translation. Hek293/hCtr1-myc cells were then exposed
for 2 h to media containing the indicated copper concentrations.
Total protein extracts were prepared from cells, separated by SDS-PAGE,
transferred to nitrocellulose membranes, and hCtr1-myc protein detected
using the affinity purified anti-myc antibody (1:3000) followed by
anti-mouse secondary antibodies conjugated to HRP (1:5000).
Arrows indicate the expected position of monomeric and
dimeric forms of hCtr1-myc protein present only in the
hCtr1-myc-transfected cells. Note the striking reduction in levels of
hCtr1-myc protein in Hek/hCtr1-myc cells exposed to 20 and 100 µM copper levels. Asterisks indicate
nonspecific cross-reacting proteins that are present in both
untransfected and transfected HEK293 cells. Tubulin protein levels in
each sample are shown in the lower panel to indicate protein
loading.
|
|
 |
DISCUSSION |
Copper homeostasis is controlled via a number of finely tuned
mechanisms through uptake, distribution, and excretion. As a continuation of our previous studies defining the biochemical and
physiological function of hCtr1 in copper uptake, we explored whether
hCtr1 levels at the plasma membrane are altered in response to changes
in copper concentration. Analysis of hCtr1-myc expressed in CHO and
HEK293 cell lines demonstrated that the protein was located at the
plasma membrane. Significantly, the surface expression of hCtr1-myc was
decreased when CHO and HEK293 cells were exposed to elevated copper in
the growth media, although the extent of this decrease was more
pronounced in HEK293 cells (Fig. 1). Further analysis using
nonpermeabilized Hek/hCtr1-myc cells demonstrated reduced levels of
hCtr1-myc at the plasma membrane after a 10-min exposure to 100 µM copper. This observation led us to ask whether copper
decreases the surface expression of hCtr1-myc by stimulating hCtr1-myc
endocytosis. The extracellular orientation of the myc epitope in
hCtr1-myc allowed us to assess endocytosis of the protein by measuring
the uptake of anti-myc antibodies from the culture media by
Hek293/hCtr1-myc cells. Using both confocal immunofluorescence microscopy and immunoblots, a time-dependent uptake of
anti-myc antibodies was observed in both untreated and copper-treated
cells. However, the rate of antibody internalization in copper-treated cells was markedly elevated relative to untreated cells. Collectively, these data suggest that elevated copper reduces the levels of hCtr1-myc
at the plasma membrane, and that this is associated with increased
endocytosis of the protein. The finding that silver also strongly
stimulated the endocytosis of hCtr1-myc was significant, as it
suggested that monovalent copper, rather than the divalent ion, is the
primary substrate for this process. It is also noteworthy that Cu(I) is
the primary substrate for copper uptake via hCtr1 (8). The inhibitors
of clathrin-mediated endocytosis, chlorpromazine and
methyl-
-cyclodextrin, prevented the copper-induced relocalization of
hCtr1-myc protein from the plasma membrane to cytoplasmic vesicles. Moreover, newly internalized anti-myc antibodies and transferrin were
co-localized. Taken together, these data support the notion that hCtr1
enters cells via clathrin-mediated endocytosis and is trafficked to
early endosomes. The ability of the hCtr1-myc protein to internalize
anti-myc antibodies from basal media suggested the protein was
endocytosed in the absence of elevated copper. Moreover, the increased
levels of hCtr1-myc protein at the plasma membrane of cells exposed to
endocytosis inhibitors provided further evidence supporting the notion
that hCtr1 is constantly endocytosed in basal media.
The reduced levels of hCtr1-myc protein at the plasma membrane
following copper-stimulated endocytosis is consistent with a
homeostatic control mechanism. Under low copper conditions the abundant
expression of hCtr1 at the plasma membrane would permit high affinity
uptake of copper. However, when extracellular copper concentrations are
elevated, the clearance of hCtr1 from the plasma membrane would prevent
excessive copper uptake and potential copper toxicity. A notable
finding of our study was that elevated copper levels also resulted in
the degradation of the hCtr1 protein. The degradation of the protein
would provide an additional means to down-regulate copper uptake when
copper is present at elevated concentrations, and may also serve to
prevent recycling of internalized hCtr1 protein to the plasma membrane.
Our findings, together with previous findings that the yeast Ctr1
protein also undergoes copper-stimulated endocytosis and degradation,
reveal a remarkably conserved mechanism for regulating copper uptake by
eukaryotic cells that probably originated early during evolution.
A significant finding of our study was that copper supplementation of
~0.5-1.0 µM in the culture media was sufficient to
increase the endocytosis of hCtr1-myc from the plasma membrane.
Interestingly, copper uptake via hCtr1-myc in the Hek/hCtr1-myc cell
line has a Km of 1.71 ± 0.39 µM
(8). Hence, the concentrations of copper that stimulate copper uptake
via hCtr1 are similar to copper levels that enhance its endocytosis.
This observation raises the question of how hCtr1 mediates the uptake
of copper into cells. Does copper pass through the lipid bilayer of the
plasma membrane as a transport substrate of hCtr1, or as a ligand that
enters the cell via endocytosis of a copper-hCtr1 complex? One
possibility is that the binding of copper to an extracellular region of
hCtr1 protein may trigger endocytosis of the copper-hCtr1 complex to an
intracellular compartment from which copper is subsequently transported
into the cytoplasm by hCtr1 or an alternative transporter. This
endocytic model of copper uptake is reminiscent of the uptake of
transferrin-bound iron by the transferrin receptor (21). The
observation that hCtr1 undergoes endocytosis in basal media is
consistent with an endocytic model of copper uptake. Alternatively, copper may enter the cell via two separate hCtr1-dependent
processes: the first via direct hCtr1-mediated transport of copper
across the plasma membrane, and the second via endocytosis of the
copper-hCtr1 complex. This latter mechanism involving endocytosis may
function to supply copper to specific intracellular organelles, either for the purposes of copper storage or for specific metabolic processes.
The copper-stimulated endocytosis of hCtr1 is reminiscent of
copper-stimulated endocytosis that has been reported for the mouse and
chicken homologues of the prion protein (22). Unlike prion protein
endocytosis, which is stimulated by high copper levels (>100
µM), we observed increased hCtr1 endocytosis at low micromolar copper levels. Although this difference in sensitivity clearly distinguishes copper-induced endocytosis of hCtr1 from that of
the prion protein, it remains to be shown whether a common copper-responsive mechanism underlies the endocytosis of both proteins.
It will be important to establish whether the copper-stimulated endocytosis of hCtr1 occurs in all cell types that express the protein.
The localization of the hCtr1 protein appears to be cell type specific.
In HeLa, H441, A549, and HepG2 cell lines, hCtr1 is predominantly
located in perinuclear vesicles, however, the protein is localized at
the plasma membrane in CaCo2, Ht29, and HEK293 cells (7, 8). This
variability in hCtr1 localization may be because of cell type
differences in the rates of hCtr1 trafficking between the cell surface
and intracellular compartments. Indeed, the recent finding that an
inhibitor of endocytosis causes the vesicular pool of hCtr1 to shift to
a plasma membrane distribution in HeLa cells suggests that this
intracellular pool of hCtr1 cycles via the plasma membrane (7).
However, the same study found no evidence that elevated copper affects
the steady state location of hCtr1 in CaCo2 or HeLa cells (7), the
reasons for which are currently unclear. It is conceivable that
copper-stimulated endocytosis of hCtr1 only occurs in certain cell
types. Alternatively, elevated copper may not alter the steady-state
location of hCtr1 in some cell types because only a small fraction of
the total pool of hCtr1 traffics via the plasma membrane, or that
different cell types have distinct regulatory pools of copper available to stimulate hCtr1 trafficking.
In summary, we have identified a post-translational mechanism for
regulating high-affinity copper uptake in mammalian cells involving
copper-stimulated endocytosis and degradation of the hCtr1 copper
transporter. We are currently exploring the mechanisms involved in
sensing copper, the sorting signals involved in triggering hCtr1
endocytosis, and the vesicular compartments through which hCtr1
traffics. The discovery of copper-stimulated endocytosis of hCtr1
broadens our understanding of how cytoplasmic copper levels are
regulated in mammalian cells. Our findings, together with previous
studies, which show that copper triggers the relocalization of the
Menkes and Wilson copper exporters from the
trans-Golgi network (23, 24), suggest that copper-induced
trafficking regulates the function of all three known high affinity
mammalian copper transporters. Understanding how the trafficking of
these transporters is coordinated to maintain copper homeostasis will be an important goal of future studies.
 |
ACKNOWLEDGEMENTS |
We thank Drs. David Eide and Elizabeth Rogers
for critical evaluation of the manuscript and helpful discussions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants DK 59893 (to M. J. P.) and GM62555 (to D. J. T.), the International Copper Association (to D. J. T.), and
American Heart Association Postdoctoral Fellowship 9920536 (to J. L.).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, University of Missouri, Columbia, MO 65211. Tel.: 573-882-9685; Fax: 573-882-0185; E-mail: petrism@missouri.edu.
Published, JBC Papers in Press, December 25, 2002, DOI 10.1074/jbc.M209455200
 |
ABBREVIATIONS |
The abbreviations used are:
HEK293, human
embryonic kidney 293;
CHO, Chinese hamster ovary;
HRP, horseradish
peroxidase;
PBS, phosphate-buffered saline;
MCD, methyl-
-cyclodextrin.
 |
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