ZnT7, a Novel Mammalian Zinc Transporter, Accumulates Zinc in the Golgi Apparatus*

Catherine P. KirschkeDagger and Liping HuangDagger §

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta  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).

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 -80 °C overnight.

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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).


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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.

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.


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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.

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.


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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.

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.


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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).

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.


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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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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