Two different zinc transport complexes of cation diffusion facilitator proteins localized in the secretory pathway operate to activate alkaline phosphatases in vertebrate cells.

Zinc is an essential component for the catalytic activity of numerous zinc-requiring enzymes. However, until recently little has been known about the molecules involved in the pathways required for supplying zinc to these enzymes. We showed recently (Suzuki, T., Ishihara, K., Migaki, H., Matsuura, W., Kohda, A., Okumura, K., Nagao, M., Yamaguchi-Iwai, Y., and Kambe, T. (2005) J. Biol. Chem. 280, 637-643) that zinc transporters, ZnT5 and ZnT7, are required for the activation of zinc-requiring enzymes, alkaline phosphatases (ALPs), by transporting zinc into the lumens of the Golgi apparatus and the vesicular compartments where ALPs locate and converting apoALPs to holoALPs. ZnT6 is also located in the vesicular compartments like ZnT5 and ZnT7. However, the functions of ZnT6 and relationships among these three transporters have not been characterized yet. Here, we characterized the cellular function of ZnT6 together with ZnT5 and ZnT7 by gene-targeting studies using DT40 cells. ZnT6-deficient DT40 cells showed low ALP activity, suggesting that ZnT6 is required for the activation of zinc-requiring enzymes like ZnT5 and ZnT7. Combined disruptions of three transporter genes and re-expressions of transgenes revealed that ZnT5 and ZnT6 work in the same pathway, whereas ZnT7 acts alone. Furthermore, co-immunoprecipitation studies revealed that ZnT5 and ZnT6 formed hetero-oligomers, whereas ZnT7 formed homo-oligomers. Interestingly, the Ser-rich loop in ZnT6, a potential zinc-binding site, was dispensable for the zinc-supplying function of ZnT5/ZnT6 hetero-oligomers, suggesting that the His-rich loop in ZnT5 may be important for zinc binding and that the loop in ZnT6 may acquire another function in the hetero-oligomer formation. These results suggest that two different zinc transport complexes operate to activate ALPs.

The cation diffusion facilitator (CDF) 1 family is a family of metal transport proteins found in diverse organisms from prokaryotes to eukaryotes (1)(2)(3)(4)(5)(6)(7). To date, eight CDF proteins designated ZnT1-8 (for zinc transporters 1-8) have been characterized in mammals (8 -15). Members of this protein family have the same predicted membrane topology of six membranespanning domains and the cytoplasmic His-rich loop between membrane-spanning domains IV and V (2,3,6), with some exceptions such as ZnT5, which has a long N-terminal portion with extra membrane-spanning domains, and ZnT6, which lacks most of the histidine residues in a potential metal binding His-rich loop while retaining a Ser-rich loop (12,13). Most CDF proteins have been characterized as zinc transporters that facilitate zinc efflux from the cytosol and mobilize the cytosolic zinc into intracellular organelles (1)(2)(3)(4)(5)(6)(7). In most cases the mobilization of zinc into the organelles is shown to contribute to zinc storage and/or detoxification during zinc excesses; in such cases the CDF proteins are localized to vacuoles and endosome/ lysosome compartments (9, 11, 16 -18).
There are a couple of CDF proteins localized in the secretary pathway, where zinc is important for the activity of many proteins. The Saccharomyces cerevisiae Msc2 protein, which is the major route of zinc entry into the endoplasmic reticulum, is required to maintain the proper function of the endoplasmic reticulum (19). A number of secretory, membrane-bound, or organelle-resident enzymes biosynthesized and become functional in the secretory pathway require zinc as an essential component for their catalytic activity; e.g. matrix metalloproteinases (20), angiotensin-converting enzyme (21), and alkaline phosphatases (ALPs) (22). We recently showed that vertebrate ZnT5 and ZnT7 proteins are required for zinc incorporation into ALPs in the Golgi apparatus and the vesicular compartments to convert apoALPs to holoALPs (23).
ZnT6 is also localized to the Golgi apparatus and is thought to be implicated in the delivery of zinc into the Golgi lumen in addition to ZnT5 and ZnT7 (13). However, the direct evidence of ZnT6 function in the secretory pathway has not, to date, been reported. In this study, we examined the cellular function of ZnT6 along with that of ZnT5 and ZnT7 by means of gene disruption studies in vertebrate cells and found that ZnT6 plays a role in the activation of tissue-nonspecific ALP (TNAP) and needs the co-expression of ZnT5 for its function, whereas neither ZnT5 nor ZnT6 needs to be co-expressed for ZnT7 to activate ALP in vertebrate cells. Furthermore, our biochemical analysis identified the formation of hetero-oligomeric complexes of ZnT5 and ZnT6 and the homo-oligomeric complexes of ZnT7. * This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y. Y.-I. and T. K.) and by the Sasakawa Scientific Research Grant from The Japan Science Society (to T. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The

MATERIALS AND METHODS
Plasmid Construction-Two targeting constructs designed to disrupt the chicken ZnT6 (cZnT6) gene were constructed. The ϳ13-kb cZnT6 gene was amplified with a pair of gene-specific primers using DT40 genomic DNA as a template by LA-Taq (TaKaRa). Each amplified DNA fragment was used to map the restriction sites. The long or short arm was PCR-amplified and subcloned downstream or upstream of the drug selection marker cassettes including drug-resistant genes (Bsr R or His R ) flanked by mutant loxP sites (24). Plasmids that express human ZnT5 (hZnT5), chicken ZnT6 (cZnT6), human ZnT6 (hZnT6), or human ZnT7 (hZnT7) were constructed by inserting each cDNA into a pA-Puro vector and a pA-Zeocin vector (23). To construct pA-HA-hZnT6, we fused the DNA fragment encoding a hemagglutinin (HA) epitope (YPY-DVPDYA) plus the start Met to the first ATG codon of hZnT6 cDNA (HA-hZnT6) and inserted it into pA-Puro and pA-Zeocin. Similarly, we constructed pA-FLAG-hZnT5 by inserting FLAG-hZnT5 cDNA (12) into pA-Puro and pA-Zeocin. To construct pA-hZnT7-FLAG or pA-hZnT7-HA, we fused the DNA fragment encoding the FLAG epitope or the HA epitope in-frame with the 3Ј-end of hZnT7 cDNA and inserted it into a pA-Puro and pA-Zeocin. The pANMerCreMer plasmid encoding the tamoxifen-regulated chimeric Cre recombinase was a kind gift from Dr. Michael Reth (25). All plasmids were linearized with appropriate restriction enzymes prior to electroporation.
Generation of Mutant Cells-Our experimental strategy and the targeting constructs we used are shown in the supplemental data available in the on-line version of this article. ZnT7 Ϫ/Ϫ cells, ZnT5 Ϫ cells, and ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells were established as described previously (23). To obtain ZnT6 Ϫ/Ϫ cells, we transfected the wild-type DT40 cells sequentially with ZnT6-Bsr R and ZnT6-His R targeting constructs. ZnT6 Ϫ/Ϫ cells were transfected with ZnT5-Neo R targeting construct to produce ZnT5 Ϫ ZnT6 Ϫ/Ϫ cells. To generate ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells or ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells we excised the drug selection marker cassettes in ZnT7 Ϫ/Ϫ cells or ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells according to methods described elsewhere (24). Briefly, these cells stably harboring the plasmid containing pANMerCreMer were cultured for 2 days in the presence of 200 nM 4-hydroxytamoxifen (Sigma), which translocates the MerCreMer protein into the nucleus and, thereby, the MerCreMer protein recombines DNA at mutant loxP sites. Excision of the drug selection marker cassettes was confirmed by Southern blotting or genomic PCR after limiting dilution. The cells for which the excision was confirmed were transfected sequentially with ZnT6-Bsr R and ZnT6-His R targeting constructs to obtain ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells or ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells.
Southern Blotting-Twenty micrograms of genomic DNA prepared from DT40 cells were digested with appropriate restriction enzymes for 24 h. After digestion, DNA was electrophoresed on agarose gel and transferred to a nitrocellulose membrane filter in 0.4 N NaOH. The membrane was hybridized to the radiolabeled DNA probes and exposed to an imaging plate (Fuji). Radiographic images were obtained using a BAS 2500 bioimaging analyzer (Fuji).
RNA Preparation and Northern Blotting-Total RNA was extracted from DT40 cells using Sepasol I (Nacalai Tesque). The RNA (20 g) was electrophoresed on agarose gel and transferred to nitrocellulose membrane filter in 20ϫ SSC. The membrane was hybridized to the appropriately radiolabeled cDNA probes. Radiographic images were obtained as described above.
Measurement of TNAP Activity-The total cellular protein prepared from the cells was lysed in ALP lysis buffer. Five micrograms of protein was used for measuring the TNAP activity as described previously (23). One hundred microliters of substrate solution (2 mg/ml p-nitrophenyl phosphate in 1 M diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl 2 ) was added, and p-nitrophenol released by TNAP was measured by the absorbance at 405 nm. Shrimp ALP (Roche Applied Science) was used as a standard.
Inductively Coupled Plasma Mass Spectrometry Analysis-DT40 cells (1 ϫ 10 8 ) were washed twice with phosphate-buffered saline and lyophilized. The lyophilized cells (ϳ20 mg) were mineralized after the addition of concentrated nitric acid (2.5 ml) by using a microwave digestion system with temperature control (Multiwave, PerkinElmer Life Sciences) in a polytetrafluoroethylene tube. Digested samples were quantitatively diluted, 64 Zn was measured by inductively coupled plasma mass spectrometry (ELAN DRC II, PerkinElmer Life Sciences), and dynamic reaction cell technology was employed to minimize matrix effects. The reference material was durum wheat flour (NIST-RM 8436, United States Department of Commerce), which was analyzed with the samples for quality control of the zinc analysis. Each sample was analyzed in duplicate for zinc concentration.
Immunofluorescence Staining-Immunofluorescence analysis of FLAG-hZnT5 or HA-hZnT6 was performed as described previously (23). Briefly, DT40 cells were harvested, washed with phosphate-buffered saline, and incubated on coverslips coated with poly-L-lysine for 30 min following fixation with 4% paraformaldehyde at room temperature for 30 min and permeabilization with 0.1% TritonX-100 at room temperature for 10 min. The cells were immunostained with anti-FLAG antibody M5 (Sigma; 1: 400 dilution), followed by donkey anti-mouse IgG conjugated with Alexa 594 (Molecular Probes) as the secondary antibody, and with anti-HA antibody 3F10 (1: 250 dilution), followed by goat anti-rat IgG and rabbit anti-goat IgG conjugated with Alexa 488 (Molecular Probes) as the secondary and third antibodies. The stained cells were observed under a confocal laser-scanning microscope (Fluoview, Olympus).
Preparation of the Membrane Fraction-The membrane fraction was prepared as described previously with minor revisions (23). Briefly, DT40 cells (ϳ2 ϫ 10 7 ) were resuspended in 1 ml of cold homogenizing buffer and homogenized with 60 strokes of a 7-ml Dounce homogenizer. To remove the nucleus, we centrifuged the homogenate at 400 ϫ g for 10 min at 4°C. The post-nuclear supernatant was centrifuged at 20,400 ϫ g for 60 min at 4°C. The pellet was lysed in ALP lysis buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl 2 , and 0.1% TritonX-100) and stocked at Ϫ70°C until use.
Immunoprecipitation Analysis-DT40 cells (2 ϫ 10 7 ) were collected and washed with phosphate-buffered saline. The cells were then lysed with 1 ml of Nonidet P-40 lysis buffer (50 mM HEPES-HCl, pH 7.4, 100 mM NaCl, 1.5 mM MgCl 2 , and 1% (v/v) Nonidet P-40) containing proteinase inhibitors (Nacalai Tesque), followed by rotating for 2 h at 4°C. The whole cell lysates were adjusted to 1 g/l with Nonidet P-40 lysis buffer. An aliquot of the lysates was precipitated with three volumes of acetone for 2 h at Ϫ20°C and centrifuged at 20,400 ϫ g for 15 min at 4°C. The pellet was lysed with 5ϫ Ling's solubilizing buffer (150 mM sucrose, 50 mM Tris-HCl, pH 8.0, 20 mM dithiothreitol, 10% SDS, and 5 FIG. 1. Low TNAP activity in ZnT6-deficient DT40 cells. TNAP activity of the total cellular protein was assayed and is shown as milliunits (mU) per milligram of cellular protein. The total cellular protein was prepared from wild-type (WT) cells, hZnT6-expressed WT cells, ZnT6 Ϫ/Ϫ cells, and cZnT6-or hZnT6-expressed ZnT6 Ϫ/Ϫ cells. Expression of TNAP and ␤-actin mRNAs was examined by Northern blot analysis (lower section). Each value is the mean Ϯ S.D. of triplicate experiments, and the section for Northern blotting shows representative results.
mM EDTA) and used as the input fraction of the immunoprecipitation. Other aliquots of the lysates were immunoprecipitated with monoclonal antibodies, anti-FLAG M2 (Sigma; 1:200 dilution), or anti-HA 3F10 (Roche Applied Science; 1:200 dilution) in the presence of 2% bovine serum albumin. After rotating for 1 h, 25 l of protein G-Sepharose beads (Amersham Biosciences) was added and rotated for 2 h at 4°C. After centrifugation at 400 ϫ g for 5min, the pelleted beads containing the immunoprecipitates were washed three times with Nonidet P-40 lysis buffer and lysed in the 5ϫ Ling's solubilizing buffer. An equal volume of 2ϫ urea buffer (8 M urea, 30 mM sucrose, 10 mM Tris-HCl, pH 8.0, 4 mM dithiothreitol, 2% SDS, and 1 mM EDTA) was added and incubated at 37°C for 30 min before electrophoresis.
Immunoblot Analysis-Proteins were separated with electrophoresis through 8% SDS-polyacrylamide gels. After the transfer of proteins to nitrocellulose membrane (Hybond-ECL, Amersham), the blot was blocked with blocking solution (5% skim milk and 0.1% Tween 20 in phosphate-buffered saline) and then incubated with anti-hZnT5 (

RESULTS
Low TNAP Activity in ZnT6-deficient DT40 Cells-To investigate the cellular function of ZnT6, we generated ZnT6-deficient DT40 cells (ZnT6 Ϫ/Ϫ cells) as described under "Materials and Methods." Because ZnT6 has been shown to be localized to the subcellular compartments that are involved in the secretory pathway such as the Golgi apparatus, it may be employed in supplying zinc to zinc-requiring enzymes biosynthesized and matured through the secretory pathway, a role similar to that of ZnT5 and ZnT7 (23). To examine this possibility, we measured the TNAP activity in the ZnT6 Ϫ/Ϫ cells. We previously established the assay by using the TNAP activity as a marker FIG. 2. ZnT5, ZnT6, and ZnT7 all contribute to the activation of TNAP. A, ZnT5, ZnT6, and ZnT7 all contribute to the activation of TNAP. TNAP activity was measured using total cellular protein prepared from wild-type (WT), ZnT5 Ϫ , ZnT6 Ϫ/Ϫ , ZnT7 Ϫ/Ϫ , ZnT5 Ϫ ZnT6 Ϫ/Ϫ , ZnT5 Ϫ ZnT7 Ϫ/Ϫ , ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ , and ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells (upper section). Expression of cZnT5, cZnT6, cZnT7, TNAP, and ␤-actin mRNAs is also shown (lower section). Each value is the mean Ϯ S.D. of triplicate experiments, and the sections for Northern blotting show representative results. B, the amount of total cellular zinc is similar among wild-type (WT) cells and all the mutant cells. The total cellular zinc content was measured using the lyophilized cells by inductively coupled plasma mass spectrometry (see "Materials and Methods") and is shown as micrograms of zinc per gram of lyophilized cells. Each value is the average of two independent experiments. to measure the zinc supply to zinc-requiring enzymes in the secretory pathway (23). The TNAP activity in ZnT6 Ϫ/Ϫ cells was reduced to ϳ20% of that in wild-type cells, suggesting that ZnT6 is also required for the activation of TNAP. This reduced activity was restored by expressing the exogenous ZnT6 cDNA from either chicken (see GenBank™ accession number AY986776) or human (ϳ50 or 70% activity, respectively, compared with that in wild-type cells) (Fig. 1, upper section). No significant difference was detected in the expression of TNAP mRNA among these cells (Fig. 1, lower section). The TNAP activity in ZnT6 Ϫ/Ϫ cells expressing cZnT6 or hZnT6 disappeared when cultured in the zinc-free medium containing Chelex-treated fetal calf serum, and the addition of zinc to the medium restored the activity in a zinc-dependent manner (data not shown). These results indicate that ZnT6 contributes to the conversion of apoTNAP to holoTNAP as in the cases of ZnT5 and ZnT7.
ZnT5 and ZnT6 Work in the Same Pathway, but ZnT7 Works Alone-Three CDF proteins, ZnT5, ZnT6, and ZnT7, seem to work on the activation of TNAP. We therefore investigated the contribution and relationship among these transporters to the expression of TNAP activity by means of the multiple gene disruptions of these transporters. First, we measured the TNAP activity using the total cellular protein prepared from single gene disruptant cell lines, namely ZnT5 Ϫ cells, ZnT6 Ϫ/Ϫ cells, and ZnT7 Ϫ/Ϫ cells. The TNAP activity in ZnT7 Ϫ/Ϫ cells was reduced only by 30%, whereas the activity in ZnT5 Ϫ cells was reduced by 70%. The TNAP activity in ZnT6 Ϫ/Ϫ cells was reduced by 80%, which was somewhat similar to the case of ZnT5 Ϫ cells ( Fig. 2A, upper section). Then, we assayed double gene disruptant cell lines, specifically ZnT5 Ϫ ZnT6 Ϫ/Ϫ cells, ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells, and ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells. The TNAP activity in ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells was severely diminished to Ͻ1% of that in wild-type cells, as was the case in ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells ( Fig. 2A, upper section) (also see Ref. 23), whereas the activity in ZnT5 Ϫ ZnT6 Ϫ/Ϫ cells maintained ϳ30% of the TNAP activity in wild-type cells ( Fig. 2A, upper section), which is not lower than that in the single gene disruptants, ZnT5 Ϫ cells and ZnT6 Ϫ/Ϫ cells, suggesting that ZnT5 and ZnT6 work in the same pathway supplying zinc to TNAP. In addition, the TNAP activity in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells was severely reduced (Ͻ1% of that in wild-type cells; Fig. 2A, upper section), implying that ZnT7 works in different pathway from those of ZnT5 and ZnT6 to supply zinc to TNAP. In all cell lines, the expression levels of TNAP mRNA were unaffected ( Fig. 2A,  lower section).
These mutant cells showed normal microscopic morphology and showed sensitivity to a high zinc concentration similar to that of the wild-type cells. Total cellular zinc in the mutant cells was comparable with that in wild-type cells in the range of 60 -75 g/g of lyophilized cells (Fig. 2B), which confirms that the cellular functions of ZnT5, ZnT6, and ZnT7 are for the zinc supply into the lumens of the secretory compartments and not for storage and/or detoxification of zinc in an acidic endosomal/ lysosomal compartment, as has been proposed for ZnT2 (9,26).
Consistently, when we expressed ZnT5 and ZnT6 transgenes in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells (human ZnT5 N-terminally tagged with FLAG epitope (FLAG-hZnT5) and human ZnT6 N-terminally tagged with HA epitope (HA-hZnT6) were used in this setting), TNAP activity was restored only in the cells expressing both genes but not in the cells expressing either one of the two transporters (Fig. 3A). On the contrary, when we expressed the ZnT7 transgene in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells (human ZnT7 C-terminally tagged with FLAG epitope (hZnT7- FIG. 3. ZnT5 and ZnT6 work in the same pathway, but ZnT7 works alone. A, co-expression of ZnT5 and ZnT6 is required for the restoration of the reduced TNAP activity in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells. TNAP activity was measured using total cellular protein prepared from wild-type (WT) cells, ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells, and ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing FLAG-hZnT5 and HA-hZnT6 alone or together (upper section). The expression of FLAG-hZnT5 and HA-hZnT6 in these cells was confirmed by immunoblot analysis using membrane protein prepared from the indicated cells. Calnexin (CNX) is shown as a control (lower section). B, the expression of ZnT7 alone is functional for the activation of TNAP in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells. TNAP activity was measured using total cellular protein prepared from wildtype (WT), ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells, and ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing hZnT7-FLAG (upper section). Expression of hZnT7-FLAG in these cells was confirmed by immunoblot analysis using membrane protein prepared from the in- FLAG) in this setting), ZnT7 could restore TNAP activity and required neither ZnT5 nor ZnT6 (Fig. 3B). These results further support the fact that the function of ZnT5 is dependent on ZnT6, but the function of ZnT7 is not in terms of supplying zinc to TNAP.
ZnT5 and ZnT6 Interact Each Other to Form Hetero-oligomers, and ZnT7 Forms Homo-oligomers-As described above, ZnT5 and ZnT6 are involved in the same pathway supplying zinc to the vesicular compartments. We next addressed the question of whether these two gene products worked in sequential order or simultaneously. To get a clue to answer this, we examined co-staining of ZnT5 and ZnT6, even though their reported subcellular localizations were similar to each other. ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing FLAG-hZnT5 and HA-hZnT6 were subjected to indirect immunofluorescence staining and showed co-localization of ZnT5 and ZnT6 (Fig.  4A). This result may imply that ZnT5 and ZnT6 work together.
Then, we examined the physiological interaction between ZnT5 and ZnT6 by co-immunoprecipitation experiments using whole cell lysates prepared from these cells. Immunoprecipitation using the anti-FLAG antibody followed by blotting with the anti-HA antibody detected a protein of the size expected for HA-hZnT6 in the cells expressing both FLAG-hZnT5 and HA-hZnT6 (Fig. 4B, upper section). Similarly, the anti-HA antibody could co-precipitate a protein of the size expected for FLAG-hZnT5 only when the cells expressed both transporters (Fig.  4B, middle section). These results indicate that ZnT5 and ZnT6 form hetero-oligomeric complexes. We next examined whether or not ZnT7 shows an oligomer formation, because not only ZnT5 and ZnT6 (as we described above) but also other CDF proteins were recently shown to form oligomeric complexes (18,27). As shown in Fig. 5, the co-immunoprecipitation experiments revealed that hZnT7-FLAG and hZnT7 C-terminally tagged with the HA epitope (hZnT7-HA) physically interacted with each other when we expressed hZnT7-FLAG and hZnT7-HA in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells. This result indicates that ZnT7 forms homooligomeric complexes. Unlike ZnT7, neither ZnT5 nor ZnT6 formed homo-oligomeric complexes in similar experimental settings (data not shown).
The Ser-rich Loop in ZnT6 Is Not Essential for ZnT5/ZnT6 Hetero-oligomeric Complexes to Activate TNAP-As mentioned above, most of the ZnT transporters characteristically possess the cytoplasmic His-rich loop (2,3,6), which is proposed to be a potential zinc-binding site (16,28), although its precise role has not yet been elucidated. ZnT6 lacks most of the histidine residues within this loop. Thus, we examined whether the loops in ZnT5 and ZnT6 were essential for their functions based on our findings described above (the hetero-oligomer formation of ZnT5 and ZnT6). For this purpose, we made mutant proteins lacking these loops that we designated as hZnT5⌬His and hZnT6⌬Ser mutant proteins; the amino acids from 542 to 578 in the His-rich loop in hZnT5 were deleted in hZnT5⌬His, those from 164 to 189 in the Ser-rich loop in hZnT6 were deleted in hZnT6⌬Ser (Fig. 6A), and the TNAP activity in the cells expressing these mutants was assayed. The TNAP activity was not restored by the co-expression of hZnT5⌬His with HA-hZnT6 in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells (Fig. 6B left). The com- Whole cell lysates were prepared from the indicated cells. FLAG-hZnT5 and HA-hZnT6 were immunoprecipitated with antibodies against the FLAG and HA epitopes, respectively. The immunoprecipitates (IP) were subjected to immunoblot analysis using antibodies against the FLAG or HA epitopes. For an estimation of the amount of FLAG-hZnT5 and HA-hZnT6 in whole cell lysates, 50% of the aliquot of lysates was subjected to immunoblot analysis in parallel after acetone precipitation (Input, lower section).

FIG. 5. ZnT7 forms homo-oligomers.
Whole cell lysates prepared from ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing hZnT7-FLAG and hZnT7-HA alone or together were subjected to immunoprecipitation (IP) as described for Fig. 4B. plex formation was not impaired because hZnT5⌬His was co-immunoprecipitated with HA-hZnT6 (Fig. 6B right), suggesting that the mutation introduced in the ZnT5 gene did not affect the conformation of ZnT5; if any, the mutation affected the zinc-supplying function of ZnT5. In contrast, the co-expression of HA-hZnT6⌬Ser and FLAG-hZnT5 partly restored TNAP FIG. 6. The Ser-rich loop in ZnT6 is not essential for ZnT5/ZnT6 hetero-oligomeric complexes to activate TNAP. A, diagram of hZnT5⌬His and hZnT6⌬Ser mutants. Human ZnT5 has the His-rich loop between membrane-spanning domains XIII and XIV (black loop, upper left), and hZnT6 has the Ser-rich loop between membrane-spanning domains IV and V (black loop, lower left). The amino acids from 542 to 578 in the His-rich loop were deleted in hZnT5⌬His (upper right), and the amino acids from 164 to 189 in the Ser-rich loop were deleted in hZnT6⌬Ser (lower right). B, the His-rich loop in ZnT5 is essential for ZnT5/ZnT6 hetero-oligomeric complexes to activate TNAP. The TNAP activity was measured using total cellular protein prepared from wild-type (WT) cells, ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing both hZnT5 and HA-hZnT6, and ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing both hZnT5⌬His and HA-hZnT6 (left section). Whole cell lysates prepared from the indicated cells were subjected to immunoprecipitation (IP) followed by immunoblot analysis as described for Fig. 4B (right). C, the Ser-rich loop in ZnT6 is not essential for ZnT5/ZnT6 hetero-oligomeric complexes to activate TNAP. The TNAP activity was measured using total cellular protein prepared from wild-type (WT) cells, ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing both FLAG-hZnT5 and HA-hZnT6, and ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells expressing both FLAG-hZnT5 and HA-hZnT6⌬Ser (left section). Whole cell lysates prepared from the indicated cells were subjected to immunoprecipitation (IP) as described for Fig. 4B (right section). D, hZnT5⌬His shows a dominant negative effect in DT40 cells. The TNAP activity was measured using total cellular protein prepared from wild-type (WT), ZnT5 Ϫ , ZnT7 Ϫ/Ϫ , and ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells expressing or not expressing hZnT5⌬His. Expression of hZnT5⌬His or calnexin (CNX) as a control is shown in the lower section. activity in ZnT5 Ϫ ZnT6 Ϫ/Ϫ ZnT7 Ϫ/Ϫ cells (up to 40% of that in HA-hZnT6-expressing cells) (Fig. 6C left). Again, the mutation introduced in the ZnT6 gene had little effect on the conformational changes of ZnT6, as HA-hZnT6⌬Ser was co-immunoprecipitated with FLAG-hZnT5 (Fig. 6C right). These results indicate that the His-rich loop in ZnT5 is essential for activating TNAP, whereas the Ser-rich loop in ZnT6 seems to have more moderate roles so that ZnT6 seems to lose zinc-transporting activity and transport zinc only by the formation of hetero-oligomeric complexes. In addition, we observed that hZnT5⌬His protein showed dominant negative effects in the activation of TNAP when expressed in either wild-type cells, ZnT5 Ϫ , ZnT7 Ϫ/Ϫ or ZnT5 Ϫ ZnT7 Ϫ/Ϫ cells (Fig. 6D), which implied that the introduced mutant proteins may interfere with the endogenous hetero-oligomer formation of ZnT5 and ZnT6 in DT40 cells. DISCUSSION In vertebrate cells, three zinc transporters, ZnT5, ZnT6, and ZnT7, have been shown to be localized to the Golgi apparatus and the vesicular compartments and thought to be implicated in the entry of zinc into their lumens, thereby supplying zinc to numerous enzymes biosynthesized and matured in the secretory pathway (12)(13)(14). Of these three, we recently reported that ZnT5 and ZnT7 are required for the activation of TNAP (23). In the case of ZnT6, however, there was no direct evidence showing that ZnT6 supplies zinc to zinc-requiring enzymes in the secretory pathway. Here, we showed that ZnT6 is involved in the expression of TNAP activity by the use of gene disruption studies in DT40 cells. Then why are the three transporters localized to the same compartments? The three might have different specificities to target proteins. However, the actual number of functional zinc transporters in the secretory pathway known to date is two and not three, because we have shown direct evidence that ZnT5 and ZnT6 form hetero-oligomeric complexes, which was also suggested in a most recent report by Ellis et al. (29).
The formation of ZnT5/ZnT6 hetero-oligomeric complexes has been considered to be essential for their functions, because both genes need to be expressed to activate TNAP. There are many transporters or channels that form hetero-oligomers to manifest their own biological activity, and some of them form hetero-oligomers not only for their activity but also for their subcellular translocation. For example, the ATP-binding cassette transporters ABCG5 and ABCG8 are not trafficked to plasma membrane for excretion of their substrate out of the cells until the heterodimers are formed; each of them is retained in the endoplasmic reticulum if expressed alone (30). This is not the case for ZnT5 and ZnT6. The subcellular localization of ZnT5 and ZnT6 was the same when they were expressed alone or together (data not shown and Fig. 4A). Rather, they require each other for the zinc-supplying function.
Our mutation studies revealed a fascinating aspect of ZnT6. That is, during evolution ZnT6 seems to have lost most of the histidine residues from the loop between membrane-spanning domains IV and V (13) and appears to exert transport function by forming complexes with ZnT5. Moreover, ZnT6 lacks two essential histidine residues in membrane-spanning domains II and V (6) but has many potential protein kinase C and CK2 phosphorylation sites (13). ZnT6 may function as a component modulating the activity of ZnT5 in the ZnT5/ZnT6 heterooligomeric complexes and elicit fine regulation of zinc transport activity, which might be related to the characteristic of ZnT6 of redistributing toward the periphery of the cells with the addition of zinc (13).
Increasing numbers of zinc transport systems have been identified in the subcellular organelles or plasma membrane for zinc delivery to zinc-requiring proteins, enzymes (19,23), and transcription factors (31). Further studies are required to extend our understanding of how the zinc transporters exert their zinc-transporting activities and cooperate to maintain zinc homeostasis.