From the Western Human Nutrition Research Center,
Agriculture Research Service, United States Department of Agriculture
and the § Department of Nutrition and the Rowe Program in
Genetics, University of California Davis, Davis, California 95616
Received for publication, July 29, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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ZnT7, a novel member of the zinc transporter
(ZnT) family, was identified by searching the expressed sequence tag
(EST) databases with the amino acid sequence of ZnT1. Like the other
ZnT proteins, the protein (387 amino acids) predicted from this gene
contains six transmembrane domains and a histidine-rich loop between
transmembrane domains IV and V. We show that Znt7 is widely
transcribed in mouse tissues with abundant expression in the liver and
small intestine and moderate expression in the kidney, spleen, brain,
and lung. An affinity-purified antibody raised against the amino acids
299-315 of mouse ZnT7 specifically reacted with the proteins with
apparent molecular masses of 85, 43, and 65 kDa in small intestine and lung tissues by Western blot analysis. Immunofluorescence microscope analysis reveals that ZnT7 is localized in the Golgi apparatus and
cytoplasmic vesicles. Exposure of the ZnT7-expressing Chinese hamster
ovary (CHO) cells to zinc causes an accumulation of zinc in the Golgi
apparatus, suggesting that ZnT7 facilitates zinc transport from the
cytoplasm into the Golgi apparatus.
Zinc, a divalent cation, is required for the function of many
zinc-containing proteins, many of which participate in various aspects
of gene regulation, protein synthesis, intracellular protein trafficking, hormone function, and immune function (1). Intracellular zinc concentration is strictly regulated, and intracellular zinc homeostasis is maintained through various mechanisms including zinc
sensing, binding, and sequestrating. Zinc sensing is mediated by
regulating the expression of zinc transport proteins, including those
for zinc influx during zinc deficiency and efflux during zinc excess
(2). The intracellular binding of excess zinc ions is largely
facilitated by metallothioneins (3). Recent studies in zinc transport
proteins, however, have indicated that zinc transporter
(ZnT)1 proteins play critical
roles in maintaining intracellular zinc homeostasis through efflux and
sequestration mechanisms during zinc excesses (4-9).
ZnT proteins are characterized by their ability to decrease the
cytoplasmic zinc concentration by transporting zinc out of cells or
into intracellular compartments. Members of the ZnT family have similar
membrane topology with six transmembrane domains and a histidine-rich
loop between transmembrane domains IV and V, where zinc may be
bound by histidines and subsequently transported across the
membrane. Six ZnT proteins have been cloned and characterized since 1995.
ZnT1 is involved in zinc efflux across the plasma membrane (4). The
Znt1 mRNA is ubiquitously expressed in mice and is up-regulated in cells with elevated intracellular zinc (2, 10). ZnT2,
ZnT3, and ZnT4 are similar to each other. They reside on the vesicular
membranes of cells and facilitate the zinc accumulation in vesicles.
The tissue distributions of ZnT2, ZnT3, and ZnT4 in mice are
overlapped, yet are tissue-specific. ZnT2 is widely expressed but not
detectable in liver, mammary gland, muscle, adipose, thymus, and spleen
(2, 6). ZnT2 expression in the small intestine and kidney is markedly
decreased in rats fed with zinc-deficient food (2). ZnT3 is only
expressed in brain and testis (6). Targeted disruption of the
Znt3 gene in mice leads to depletion of the
histochemically reactive zinc in synaptic vesicles of neurons (11).
ZnT4 is abundantly expressed in brain and mammary glands with low level
expression in other tissues (2, 7, 12, 13). A premature termination
mutation in the Znt4 gene causes insufficient zinc
deposition into milk in the lactating mammary glands of the
lethal milk mice (7). As a consequence, the pups of any
genotype suckled on homozygous lethal milk mothers die of
zinc deficiency before weaning (14-16).
ZnT5 is ubiquitously expressed with abundant expression in pancreas. In
the human pancreas, ZNT5 is associated with the secretory granules
where zinc is enriched in the insulin-containing Studies from our laboratory and others have indicated that there should
exist other specific ZnT proteins in small intestine and liver for
intracellular zinc sequestration, as the reported ZnT2-6 proteins are
either not expressed or weakly expressed in these tissues (2, 4-9,
12). Our research interest in zinc absorption promoted us to search the
mouse EST databases with the amino acid sequences of the ZnT proteins.
Here we describe the identification and functional characterization of
a new member of the ZnT family, ZnT7. Our studies demonstrate that the
ZnT7 protein is abundantly expressed in small intestine and lung and functions in an accumulation of zinc in the Golgi apparatus in the
ZnT7-expressing CHO cells.
Plasmid Construction--
The mouse EST clone (AI036588)
containing the Znt7 cDNA sequence was purchased from
ResGen (Invitrogen). The Znt7 cDNA sequence was
confirmed by sequencing. The entire open reading frame (ORF) sequence
of Znt7 was excised from this clone using the enzymes
XbaI and BamHI and then inserted into the same
sites of pBluescript II SK+ (Stratagene) to generate
pBS/Znt7. The yeast expression plasmid, pY-ZnT7, was
constructed by excision of the ORF of Znt7 from
pBS/Znt7 with the enzymes SacI and
EcoRI and then insertion of it into the same sites of pYES2
(Invitrogen). The mammalian expression plasmid, pZnT7-GFP, was made in
several cloning steps. First, the Znt7 ORF sequence lacking
a stop codon was obtained by PCR using the T7 primer and a primer with
a SacII site incorporated into the stop codon
(5'-TCCTTCCTTCCGCGGCATTGCCGCAAAG-3'). The pY-ZnT7 plasmid was
used as the PCR template. The resulting PCR product was digested with
HindIII and SacII and subsequently inserted
into the same sites of pEGFP-N1 (Clontech). Plasmid
pZnT7-GFP was verified by sequencing (Davis Sequencing, LLC, Davis,
CA). Second, the HindIII and SmalI fragment
containing Znt7 was excised from pZnT7-GFP and cloned into
the HindIII and EcoRV sites of pcDNA3.1(+)Myc/His6(A) (Invitrogen) to generate
pcDNA3.1/ZnT7-Myc. Finally, the pcDNA3.1/ZnT7-Myc plasmid
was digested with AgeI, blunted with Klenow (New England
Biolabs), and further digested with HindIII. The DNA
fragment containing the Znt7 ORF in which the Myc epitope
was fused in frame to the 3'-end of the Znt7 ORF was
purified and inserted into the HindIII and EcoRV
sites of pcDNA5/FRT (Invitrogen). The final construct,
pcDNA5/ZnT7-Myc, was sequenced and used for the generation of a
stable CHO cell line.
Cell Culture and Generation of Stable Cell Lines--
NRK cells
were cultured as described (9). CHO and CHO/FRT (Invitrogen) cells were
maintained in 1:1 DMEM/F12 (Ham) medium with 15 mM Hepes,
2.5 mM L-glutamine, 2 mg/ml pyridoxine
hydrochloride, and 10% fetal bovine serum (FBS) (Invitrogen). The
ZnT7-Myc-expressing cell line and control cell line were generated by
transfecting pcDNA5/ZnT7-Myc or pcDNA5FRT into CHO/FRT cells
along with pOG44 (Flp recombinase) (Invitrogen) using a LipofectAMINE
plus kit (Invitrogen). The stable cell lines were selected by culturing the cells in the medium containing 100 µg/ml hygromycin B
(Invitrogen). hBRIE 380 (a rat intestinal epithelial cell line from G. Aponte, University of California, Berkeley, CA) (18) and WI-38 (a human lung fibroblast cell line from the American Type Culture Collection) were cultured in DMEM (high glucose) and 10% FBS (Invitrogen).
Antibodies--
Rabbit anti-ZnT7 antibody was raised against a
synthetic peptide from amino acids 299-315 of mouse ZnT7
(TPPSLENTLPQCYQRVQ) and affinity-purified (Pierce). The results
from a BLAST search of the SWISSPORT data base indicated that this
peptide is unique. The monoclonal anti-Myc and anti-GM130 antibodies
were purchased from Stressgen and Transduction Laboratories,
respectively. The Alexa 488-conjugated goat anti-rabbit and anti-mouse
antibodies were purchased from Molecular Probes. The rabbit anti-ZnT2
antibody was kindly given by Dr. Shannon Kelleher (University of
California Davis), and the rabbit anti-ZnT4 and anti-ZnT6 were
described by Huang et al. in 2002 (9).
Northern Blot Analysis--
Poly(A)+ RNA was
isolated from tissues including liver, kidney, spleen, heart, brain,
small intestine, and lung of the C57BL/6J strain using the FastTrack
2.0 mRNA isolation system (Invitrogen). The Northern blot was
prepared as described (7). The blot was probed with a
32P-labeled full-length ORF DNA fragment of Znt7
in the ExpressHyb hybridization solution (Clontech)
for 2 h at 60 °C. The blot was rinsed twice with 2× SSC/0.1%
(w/v) SDS solution and was washed twice with 1× SSC/0.1% (w/v) SDS
solution at 60 °C for 15 min. Finally, the blot was washed twice
with 0.1× SSC/0.1% (w/v) SDS solution at 60 °C for 10 min. The
blot was exposed to film with an intensifying screen at Western Blot Analysis--
The tissues, including brain, lung,
liver, kidney, heart, and small intestine, were isolated from C57BL/6J
mice. The total proteins (50 µg) from each of these tissues were
separated on a 4-20% Tris-Glycine Ready Gel (Bio-Rad) and transferred
to a nitrocellulose membrane (Bio-Rad) as described previously (19). The blots were probed with the affinity-purified anti-ZnT7 (1:250 dilution), anti-ZnT4 (1:100), and anti-ZnT6 (1:250) antibodies or
anti-ZnT2 anti-sera (1:1000) in 1× PBS, pH 7.4, containing 0.01%
Tween and 5% non-fat milk powder overnight at 4 °C followed by a
peroxidase-conjugated secondary antibody (1:2500 dilution) (Pierce). The ZnT7, ZnT4, ZnT6, and ZnT2 proteins were visualized using an ECL kit (Amersham Biosciences).
Immunofluourscence Microscopy--
Immunofluorescence analysis
was performed as described (9). hBRIE 380, WI-38, NRK,
pcDNA3.1/ZnT7-Myc transiently transfected NRK, and vector
pcDNA5FRT or pcDNA5/ZnT7-Myc stably transfected CHO cells were
cultured in slide chambers for 48 h, fixed with 4%
paraformaldehyde, and permeabilized with 0.4% saponin (Sigma). Where
indicated, the stably transfected CHO cells were treated with Brefeldin
A (BFA) (5 µg/ml) for the indicated time prior to the fixation. The
cells were stained with the affinity-purified anti-ZnT4 or ZnT6
antibody (1:20 dilution) or with the monoclonal anti-Myc, TGN38, or
GM130 antibody (1:100 dilution) followed by Alexa 488-conjugated goat
anti-rabbit or anti-mouse antibody (1:250 dilution). Photomicrographs
were obtained by a Nikon Eclipse 800 microscope with a digital camera.
Zinquin Staining--
Zinquin staining was performed using
vector pcDNA5/FRT or pcDNA5/ZnT7-Myc stably transfected CHO
cells. Cells were grown in slide chambers for 48 h and then
treated with 0 or 75 µM ZnSO4 for 3 h in
the DMEM/F12 medium containing 10% chelex-treated FBS (Bio-Rad). After
ZnSO4 treatment, cells were rinsed three times with 1×
PBS, pH7.4, and then incubated in the zinc-deficient medium containing
5 µM Zinquin ethyl ester (Dojindo) for 2 h. Where
indicated, the Zinquin-treated cells were further incubated with
Brefeldin A (5 µg/ml) at 37 °C for 30 min and recovered from BFA
treatment at 37 °C for 60 min in the presence of Zinquin. The cells
were washed with 1× PBS, pH7.4, and examined and photographed using a
digital Nikon Eclipse 800 microscope with a C-9051 filter (Nikon).
Identification of Znt7--
Search of the mouse EST databases with
the amino acid sequence of the mouse ZnT1 protein revealed an
overlapping set of the cDNA clones homologous to ZnT1. The mouse
EST clone AI036588 was obtained from ResGen (Invitrogen) and sequenced.
This clone contains a single open reading frame encoding 378 amino
acids with a calculated molecular mass of 42.5 kDa. The predicted amino acid sequence is similar to the members of the ZnT family, ZnT1-6, with the closest homology to the C-terminal portion (351 amino acids)
of ZnT5 (47.5% identity and 61.0% similarity). In addition, the
predicted protein contains six transmembrane domains and a histidine-rich loop between transmembrane domains IV and V, which are
highly conserved among members of the ZnT family (Fig.
1a). Moreover, the
histidine-rich loop of this novel protein contains the most abundant
histindine residues among ZnT proteins (Fig. 1a). A PROSITE
data base scan indicates that this novel protein contains five
potential protein kinase C phosphorylation sites, one casein kinase II
phosphorylation site, one tyrosine kinase phosphorylation site, and one
N-glycosylation site. Given the predicted amino acid
sequence and protein structural similarities to the members of the ZnT
family, we designated this novel gene as Znt7 and the gene
product as ZnT7.
A phylogenetic tree for all identified mouse ZnT family proteins was
generated using the Genetics Computer Group program PILEUP (Fig.
1b). ZnT2, ZnT3, and ZnT4 cluster closely together, as they may all be involved in secretory/synaptic vesicles transport and lysosomal/endosomal zinc storage in different cells. ZnT1, ZnT5, and
ZnT7 are close relatives, with ZnT7 closer to ZnT5 than to ZnT1. ZnT6
is more distant from all other ZnT proteins.
A mouse and human genome data base search revealed that the mouse
Znt7 gene is located on chromosome 3, and the human
ZNT7 gene is on chromosome 1p13.3, a region with homology to
the mouse genome by synteny. Both the mouse and human Znt7
genes contain at least 11 exons, and the genome structures with respect
to the intron and exon boundaries are conserved between them (data not shown). Mouse ZnT7 shares a 96% amino acid identity with human ZNT7.
To date, neither mouse nor human inherited disease with defects in zinc
metabolism has been mapped to either locus.
Expression of Znt7 mRNA--
Expression of Znt7
mRNA in a variety of mouse tissues including liver, kidney, spleen,
heart, brain, small intestine, and lung was examined by Northern blot
analysis. An approximate 1.8-kb mRNA was detected at a high level
in liver, spleen, and small intestine and at a moderate level in
kidney, lung, and brain but barely detected in heart (Fig.
2). The specificity of the detected Znt7 transcripts in the Northern blot analysis was assured
by using poly(A)+ RNA, full-length Znt7 ORF
cDNA probe, and stringent hybridization and wash conditions
together with the fact that the size and the expression pattern of
Znt7 is distinguished from the other Znt message
RNAs (2, 6-9). The mouse Znt7 mRNA is also found in the
libraries of embryo, mammary gland, ovary, uterus, pancreas, salivary
gland, skin, testis, thymus, and tongue (UniGene Cluster Mm.
28490).
Characterization of the ZnT7 Gene Product--
To identify the
ZnT7 gene product and examine the tissue expression patterns
of the ZnT7 protein in mice, a polyclonal antibody against a synthetic
peptide corresponding to the amino acids 299-315 of mouse ZnT7 was
generated, and the resulting antibody was affinity-purified. 50 µg of
total protein extracts from brain, lung, liver, kidney, heart, and
small intestine were subjected for a Western blot assay using the
anti-ZnT7 antibody. The small intestine was split into three segments
of equal length, consisting of the duodenum-jejunum (proximal),
jejunum-ileum (middle), and distal ileum (distal). This anti-ZnT7
antibody recognized a ZnT7-specific band migrating at ~65 kDa in the
lung tissue while it detected one major band of 85 kDa and a minor band
of 43 kDa (Fig. 3) in the proximal segment of small intestine. These protein bands were not seen when
preimmune sera were used (data not shown). A much fainter band of 85 kDa could also be detected in the middle and distal segments of small
intestine after longer exposure (data not shown). ZnT7 appears to be
differentially modified in mouse tissues such as lung and small
intestine, because the apparent molecular masses of ZnT7 on the Western
blot are larger than the calculated molecular mass of 42.5 kDa. The
protein band of 43 kDa in the proximal segment of small intestine may
represent the unmodified ZnT7 protein, because its apparent molecular
mass is consistent with the calculated molecular mass of ZnT7. The ZnT7
protein was not detectable in brain, liver, kidney, and heart on the
Western blots despite the ZnT7 transcripts seen in these tissues by
Northern blot analysis (Figs. 2 and 3), suggesting that the expression
of the ZnT7 protein may be regulated at levels of post-transcription
and translation in those tissues.
The specificity of the affinity-purified anti-ZnT7 antibody for the
ZnT7 protein in the Western blot analysis was first assured by using a
synthetic peptide of mouse ZnT7 that is unique among the ZnT proteins
to generate and purify the antibody. Secondly, the ZnT7 detected in
lung and small intestine was not recognized by preimmune sera (data not
shown). Lastly, the anti-ZnT7 antibody did not react with the
homologous proteins, such as ZnT2, ZnT4, and ZnT6, which are readily
detected by their respective antibodies in the protein extracts
isolated from mouse brain (Fig. 3) (9, 20).
Localization of ZnT7 to the Golgi Apparatus and a Vesicular
Compartment--
Previous studies from our laboratory and others have
demonstrated that there appears to be ZnT proteins dedicated to
specific subcellular compartments for zinc transport in mammalian cells (4-9). Therefore, it is likely that ZnT7 lies in a particular subcellular compartment in mammalian cells. ZnT7 is known to exist in
the small intestinal and lung tissues based on the Western blot
analysis of total proteins from multiple mouse tissues (Fig. 3). We
sought to determine the subcellular localization of ZnT7 in the cells
originated from these tissues. As shown in Fig.
4, the majority of the anti-ZnT7
antibody-stained intracellular proteins clustered at the perinuclear
region in rat small intestinal epithelial cells (hERIE 380) and human
lung fibroblasts (WI-38), suggesting that ZnT7 may localize to the
Golgi apparatus. A punctate staining pattern in the cytoplasm, an
indication of cytoplasmic vesicle localization, was also observed in
the hERIE 380 and WI-38 cells. To further determine the exact
localization of the mouse ZnT7 and differentiate its subcellular
localization from ZnT4 and ZnT6, we compared the subcellular staining
patterns of a ZnT7-Myc fusion protein with those of ZnT4, ZnT6, and two
Golgi markers, GM130 (cis-Golgi matrix protein) (21) and
TGN38 (trans-Golgi network protein) (22), in the NRK cells.
In this experiment, ZnT7-Myc in which the Myc epitope was tagged at the
C-terminal end of the mouse ZnT7 protein was transiently expressed in
the NRK cells, and ZnT7-Myc was detected using a monoclonal anti-Myc
antibody (Stressgen). We chose to use ZnT7-Myc and NRK cells for
comparison of the subcellular staining patterns, because the expression
of endogenous ZnT7 in the NRK cells was limited, and the distributions of ZnT4 and ZnT6 as well as the Golgi markers had been well documented (Refs. 9, 12, 21, 22, and data not shown). The immunofluorescence microscopy assay revealed that the ZnT7-Myc fusion protein manifested a
predominant perinuclear staining with some punctate structures scattered throughout the cells in the transiently transfected NRK
cells, which resembled the staining patterns of the endogenous ZnT7
observed in the hBRIE 380 and WI-38 cells (Fig. 4). No fluorescence was
detected in the vector-expressing NRK cells (Fig. 4). The perinuclear
staining was similar to that of GM130 (21) and TGN38 (22), strongly
suggesting that ZnT7-Myc is associated with Golgi apparatus (Fig. 4).
In addition, the punctate staining of ZnT7-Myc in the cytoplasm of NRK
cells is reminiscent of that of ZnT4 and ZnT6 (Fig. 4). However, the
punctate particles of ZnT7-Myc is rather homogenous in size as compared
with that of ZnT4, which shows a heterogenous punctate staining, and
that of ZnT6, which displays a punctate/tubular staining throughout the
cytoplasm and underlying the plasma membrane (Fig. 4). The differences
in the sizes, shapes, and locations of stained cytoplasmic particles may suggest that ZnT7, ZnT4, and ZnT6 are localized in different vesicular compartments.
Accumulation of Zinc in the Golgi Apparatus by ZnT7--
Given
that the ZnT proteins, including ZnT2-6, appear to function in
transport of the cytoplasmic zinc into a variety of intracellular organelles, we hypothesized that ZnT7 may serve as a zinc transporter to accumulate zinc in the Golgi apparatus. A CHO cell line stably expressing the ZnT7-Myc fusion protein was generated to functionally characterize ZnT7 in mammalian cells using the Invitrogen Flp-In System. As shown in Fig. 5a,
the ZnT7-Myc fusion protein was detected at the perinuclear region of
the CHO cells expressing ZnT7-Myc by the anti-ZnT7 (section
B) as well as anti-Myc (section C)
antibodies where it was represented by the overlapping area
(section D). The localization of ZnT7-Myc in the Golgi
apparatus of the ZnT7-Myc-expressing CHO cells was further conformed by
the treatment of the cells with Brefeldin A. BFA has been known to
disrupt the Golgi apparatus, leading to either redistribution of
cis/trans-Golgi proteins into the endoplasmic
reticulum or accumulation of TGN proteins around the microtubule
organizing center (MTOC). The effect of BFA on the distribution of a
particular protein is often used in determining its subcellular
localization. Treatment of the ZnT7-Myc-expressing CHO cells with 5 µg/ml BFA resulted in diffusion of the perinuclear staining into the
cytoplasm (Fig. 5b, section F). The
redistribution of ZnT7-Myc is reminiscent to that of GM130 known to
reside in the Golgi apparatus (Fig. 5b, section
I). Furthermore, incubation of the BFA-treated cells in the
BFA-free medium for 60 min resulted in nearly complete restoration of
the normal localization of both the ZnT7-Myc fusion protein and GM130
(Fig. 5b, compare sections G and
J to E and H). These BFA-induced Golgi
disruptions and the recovery of these disruptions by incubation of the
cells with a fresh medium are characteristic features for the proteins
that associate with the Golgi apparatus. These results demonstrate that
ZnT7-Myc resides in the Golgi apparatus of the ZnT7-Myc-expressing CHO
cells.
To examine the function of ZnT7-Myc in the translocation of cytoplasmic
zinc into the Golgi apparatus of the ZnT7-Myc-expressing CHO cells, we
used Zinquin, a zinc-specific fluorescent dye, to detect the
accumulation of free zinc in the Golgi apparatus. When the
ZnT7-Myc-expressing CHO cells were grown in the medium containing 75 µM zinc for 3 h and then exposed to Zinquin, a
bright blue, perinuclear fluorescence were observed (Fig.
6D), whereas much fainter
fluorescence was observed in the vector-transfected CHO cells (Fig.
6C). The Zinquin fluorescence seen in the control cells may
result from the function of the endogenous Golgi ZnT protein(s) in
response to the increased cytoplasmic zinc concentration. When the
vector-transfected CHO cells were grown in a zinc-deficient medium for
3 h before Zinquin treatment, there was no fluorescence detected
(Fig. 6A). However, a few signs of fluorescence
scattered around the nucleus was observed in the ZnT7-Myc-expressing
CHO cells grown in the zinc-deficient medium for 3 h before Zinquin treatment, indicating that the level of zinc accumulation is dependent on the expression of ZnT7-Myc and the zinc concentration in the culture
medium (Fig. 6, B and D). The zinc-bound Zinquin
appears to be in the Golgi apparatus of the ZnT7-Myc-expressing CHO
cells, because the perinuclear blue fluorescence diffused into the
cytoplasmic region of the cells upon BFA treatment (Fig.
6E), and the perinuclear blue fluorescence recuperated with
the removal of BFA (Fig. 6F). Taken together, these results
suggest that the Myc-tagged ZnT7 protein is functional in CHO cells and
that ZnT7 is a zinc transporter that facilitates the rapid zinc
accumulation in the Golgi apparatus in these cells.
To date, genetic and genomic studies have identified more than a
dozen molecules involved in zinc homeostasis in mammalian cells (4-9,
23-28). In this study, we describe the discovery of a gene,
Znt7, which encodes a novel member of the ZnT family. ZnT7
contains many properties described for the members of the ZnT family,
including six predicted transmembrane domains and a histidine-rich loop
between transmembrane domains IV and V. The histidine-rich loop has
been proposed to bind zinc, because histidine is a common ligand for
zinc. We show that the Znt7 gene is expressed in many mouse
tissues including liver, kidney, spleen, heart, brain, small intestine,
and lung, with abundant expression in small intestine and liver and
less expression in heart. We also demonstrate that the expression of
the ZnT7 protein is restricted to the tissues of lung and small
intestine with abundant expression in the proximal segment (duodenum
and part of jejunum) of small intestine. The ZnT proteins have been
shown to play roles in decreasing the cytoplasmic zinc by either
transporting zinc out of cells or sequestrating zinc into intracellular
compartments. We show that ZnT7 leads to zinc accumulation in the Golgi
apparatus when overexpressed in CHO cells. This finding, combined with
the similarity in the amino acid sequence and predicted secondary
structures to the previously described ZnT proteins, strongly indicate
that ZnT7 is a zinc transporter involved in translocation of the
cytoplasmic zinc into the Golgi apparatus (Figs. 4-6).
The presence of ZnT7 in the vesicular compartment in the hBRIE 380, WI-38, and transiently transfected NRK cells (Fig. 4) suggests that
ZnT7 may also be involved in transporting zinc into a unique vesicular
compartment. The vesicular compartment where ZnT7 localizes appears
different from that of ZnT2, ZnT3, ZnT4, ZnT5 or ZnT6 (5, 6, 8, 9, 12,
13), suggesting that those ZnT proteins are dedicated zinc transporters
for different vesicular compartments. The localization of ZnT7 in the
Golgi and vesicular compartments combined with the abundant expression of the ZnT7 protein in the duodenum of small intestine indicate that
ZnT7 may play an important role in zinc absorption in the gut.
Functional characterization of gene homologues in model organisms is an
invaluable reference for investigating human or mouse genes with
unknown functions. In this regard, yeast mutant strains are
particularly informative, because the mutant strains for zinc metabolism are available and have been proven to be useful for characterizing the function of ZnT4 and ZnT6 in zinc homeostasis (7,
9). Several zinc transporters of Saccharomyces cerevisiae have been reported (29-32). The ZRT1 (zinc-regulated transporter) gene
encodes a high affinity zinc uptake protein (29). The zrt1 mutant is defective in growth in zinc-limited medium (29). The ZRT3 and
ZRC1 (zinc resistance conferring) genes encode zinc transporters responsible for the release of the stored zinc from vacuoles and the
sequestration of zinc into vacuoles, respectively (30, 31). Deletion of
the zrt3 gene results in zinc deficiency when cells are
grown in a zinc-limited medium (30). Deletion of the zrc1 gene leads to zinc hypersensitivity when cells are grown in zinc-rich medium (31). Interestingly, however, our attempts to either augment the
zinc-deficient phenotypes of the zrt1 and zrt3
mutants or complement for the function of the zrc1 mutant
with mouse ZnT7 were unsuccessful. Failure to exhibit the function in
yeast mutants may suggest that ZnT7 requires a partner(s) to be
functional and that this protein(s) is not available in yeast cells.
This hypothesis is supported by our results in
zinc-dependent Zinquin fluorescence study in the
ZnT7-Myc-expressing CHO cells (Fig. 6). ZnT7 was able to transport the
cytoplasmic zinc into the Golgi apparatus when the cells were grown in
the medium containing a higher level of zinc. The requirement of an
additional component(s) for function has been previously proposed for
ZnT1 (4). In that study, expression of a truncated ZnT1 protein, in
which the first transmembrane domain of ZnT1 was deleted, has a
dominant negative effect on zinc efflux in wild-type cells, leading to
zinc hypersensitivity. Thus, it is important to investigate the
functional component(s) of ZnT7 in the delivery of zinc into the Golgi
apparatus as well as cytoplasmic vesicles by yeast and/or mammalian
two-hybridization analysis and by truncation/mutagenesis analysis.
To date, seven ZnT genes (including ZnT7 reported here), each encoding
a closely related protein, have been identified and characterized
(4-9). In addition, our preliminary study indicated that there is
another novel member of the ZnT family, ZnT8, which is only expressed
in brain and liver (data not shown). An intriguing question is why
mammalian cells need so many functionally redundant ZnT proteins. A
possible scenario is that the ZnT genes are selected for enhanced
fidelity of a zinc efflux/sequestration process in cells. To maintain
this fidelity of appropriate translocation of the cytoplasmic zinc into
a variety of subcellular compartments, coordinated regulations of the
ZnT protein levels may occur in particular cell types and particular
subcellular compartments.
It has been demonstrated that the mRNA of the ZnT genes except
Znt1 exhibit tissue- and cell type-specific expression
profiles, indicting that these ZnT genes are regulated at the
transcriptional level in different tissues (6-9). The regulatory
elements that can confer tissue- and cell type-specific expression in
the promoter regions of these ZnT genes are currently unknown. The
abundance and subcellular localization of the ZnT proteins may be
further regulated at translational and post-translational levels. For example, the mRNAs of Znt4, Znt6, and
Znt7 were detected in liver by Northern blot analysis,
whereas the proteins are barely detectable on the Western blots (Fig.
3, and data not shown) (7, 9). That is also the case for
Znt3, whose mRNA is highly expressed in testis, yet its
protein is not detectable on the Western blot (6). This translational
regulatory mechanism may play an important role in the restriction of
ZnT protein expression in tissues. Post-translational modifications
have been demonstrated for ZnT2, ZnT4, ZnT6, and ZnT7 (Fig. 3, and data
not shown) (9). These modifications, which may include phosphorylation
and glycosylation, may modulate the stability and/or transport activity
of the ZnT proteins. Furthermore, zinc-induced Golgi-to-vesicle
trafficking has been observed for ZnT4 and ZnT6 in the cultured NRK
cells (9). Although we have not detected that this zinc-induced
Golgi-vesicle trafficking occurs to ZnT7 (data not shown), this
regulation may be critical for some ZnT proteins such as ZnT4 and ZnT6,
leading to a rapid increase of local protein concentrations to
effectively remove excess zinc from cells.
In conclusion, there is evidence that each member of the ZnT family is
dedicated to effluxing or sequestrating the cytoplasmic zinc into a
variety of intracellular compartments in different cells. However,
functional redundancy exists in many tissues, which is evidenced by the
lack of obvious phenotypes in the brain of the Znt3
knock-out mice and in the tissues (except for mammary glands and
otolith) of the lethal milk mutant (14, 15). It is
reasonable to hypothesize that, due to universal importance of zinc in
cellular processes, cells have to retain the genes whose functions are
partially overlapped to ensure survival in case one gene is inactivated.
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DISCUSSION
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cells (8). A
unique feature of ZnT5 is that it has a long amino-terminal portion
(410 amino acids), which is not homologous to any other members of the
ZnT family. The function of this unique sequence is unclear. Recently,
it has been demonstrated that ZnT5 plays important roles in the
maturation of osteoblasts and in the maintenance of normal function of
heart (17). The Znt5 knock-out mice display bone
developmental abnormalities and sudden death due to heart block and
sinus bradycardia (17). We recently characterized a gene encoding ZnT6.
ZnT6 facilitates the translocation of the cytoplasmic zinc into the
trans-Golgi network (TGN) and the vesicular compartment (9).
The intracellular location of ZnT6 was found to be regulated by zinc in
the cultured normal rat kidney (NRK) cells. High extracellular zinc in
the culture medium induced the trafficking of ZnT6 from the TGN
compartment to the vesicular compartment. This zinc-induced protein
trafficking was also seen with ZnT4 in NRK cells, although the
trafficking was less sensitive to the extracellular zinc
concentration (9).
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80 °C overnight.
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Fig. 1.
Comparison of the predicted ZnT7 amino acid
sequence. a, alignment of the amino acid sequences of
rodent ZnT1-7. The amino acid residues that are identical in at least
four sequences are indicated by black boxes. The
conserved amino acid residues are indicated by gray
boxes. The six transmembrane domains are
underlined and numbered. b, dendrogram
illustration of the relationships among the mammalian ZnT family
members. The tree was created using the Genetics Computer Group program
PILEUP.
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Fig. 2.
Expression of mouse Znt7
mRNA. Znt7 mRNA was purified from liver,
kidney, spleen, heart, brain, small intestine, and lung tissues of
C57BL/6J. The Northern blot containing 2 µg of poly(A)+
RNA isolated from indicated tissues was probed with the ORF region
of the murine Znt7 cDNA. The RNA size markers are
indicated on the right.
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Fig. 3.
Expression of the ZnT7 protein in mouse
tissues. Western blots containing 50-µg protein extracts from
mouse tissues (C57BL/6J), including brain, lung, liver, kidney, heart,
and small intestine (proximal, middle, and distal segments), were
probed with a rabbit anti-ZnT7, anti-ZnT2, anti-ZnT4, or anti-ZnT6
antibody followed by a peroxidase-conjugated secondary antibody. ZnT7,
ZnT2, ZnT4, and ZnT6 were visualized using an ECL kit. The protein
markers are shown.
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Fig. 4.
Subcellular localization of ZnT7.
The endogenous ZnT7 protein in hBRIE 380 and WI-38 cells, which were
cultured in the DMEM containing 10% FBS, was detected by an
affinity-purified anti-ZnT7 antibody (1:25 dilution). The ZnT7-Myc
fusion protein in the NRK cells that were transiently transfected with
a plasmid expressing ZnT7-Myc was detected by a monoclonal anti-Myc
antibody (1:100 dilution) (Stressgen). The NRK cells transiently
transfected with the vector alone was used for the control of the
specificity of the anti-Myc antibody. The endogenous ZnT4 and ZnT6
proteins in NRK cells were detected using the anti-ZnT4 and anti-ZnT6
antibodies, respectively. The endogenous GM130 and TGN38 in NRK cells
were detected by the monoclonal anti-GM130 and anti-TGN38 antibodies,
respectively. Alexa 488-conjugated goat anti-rabbit or anti-mouse
antibody (green fluorescent images) or Alexa 594 conjugated
goat anti-mouse antibody (red fluorescent image) were
used as the secondary antibodies for the photomicrographs.
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Fig. 5.
The effects of BFA on the subcellular
localization of ZnT7-Myc in CHO cells. a,
immunolocalization of ZnT7-Myc in the CHO cells that were stably
transfected with a vector or a plasmid expressing ZnT7-Myc. ZnT7-Myc
was detected either by the anti-ZnT7 (section B)
or anti-Myc antibody (section C). The
green and red florescent images were merged to
indicate the area of overlap, which is shown in yellow
(section D). The florescent staining of the
vector control cells is shown (section A).
b, subcellular localization of ZnT7-Myc in the BFA-treated
CHO cells. The ZnT7-Myc-expressing CHO cells were grown for 48 h
and incubated with 5 µg/ml BFA for 0 min (sections E and
H) or at 37 °C for 30 min (sections F and
I) and then recovered by further incubating cells with fresh
culture medium at 37 °C for 60 min (sections G and
J). The ZnT7-Myc protein and the endogenous GM130 were
detected by monoclonal anti-Myc and anti-GM130 antibodies,
respectively. Both perinuclear ZnT7-Myc (section
E) and GM130 staining (section H) disperse in
punctate structures (sections F and I) after 30 min of BFA treatment. The effects were reversed after removal of BFA
and incubation of the cells in the fresh medium for 60 min
(sections G and J).
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Fig. 6.
Zinc-dependent Zinquin
fluorescent staining in ZnT7-Myc-expressing CHO cells.
a, subcellular localization of ZnT7-Myc. The
ZnT7-Myc-expressing CHO cells were cultured in the regular medium
(DMEM/F12 with 10% FBS) for 48 h before zinc treatment. Cells
were then cultured in the zinc-deficient medium, DMEM/F12, containing
10% chelex-treated FBS (A and B) or in the
zinc-deficient medium containing 75 µM ZnSO4
(C-F) for 3 h. Cells were washed and
further incubated in the DMEM/F12 medium containing 10% chelex-treated
FBS and 5 µM Zinquin for 2 h
(A-F). The Zinquin-treated cells were further
incubated with Brefeldin A (5 µg/ml) at 37 °C for 30 min
(E) and recovered from BFA treatment at 37 °C for 60 min
(F) in the presence of Zinquin. The photomicrographs of the
blue fluorescence were obtained by a Nikon Eclipse 800 microscope with a C-9051 filter.
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ACKNOWLEDGEMENTS |
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We especially thank Dr. Lei Zhang, Dr. Jane Gitschier, Dr. Bing Zhou, Dr. Bo Lonnerdal, Dr. Shannon Kelleher, and Reine Yu for technical assistance and advice. We thank the scientists and staff in the Western Human Nutrition Research Center for support in this research. We thank Dr. Lei Zhang for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by United States Department of Agriculture Grant CRIS-5306-53000-008-00D.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF529196 and AF529197.
¶ To whom correspondence should be addressed: Rowe Program in Genetics, University of California Davis, One Shields Ave., Davis, CA 95616. Tel.: 530-754-5756; Fax: 530-754-6015; E-mail: lhuang@whnrc.usda.gov.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M207644200
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ABBREVIATIONS |
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The abbreviations used are: ZnT, zinc transporter; TGN, trans-Golgi network; ORF, open reading frame; EST, expressed sequence tag; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; BFA, Brefeldin A. FBS, fetal bovine serum; CHO, Chinese hamster ovary; NRK, normal rat kidney; FRT, Flp recombinase target.
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