Zn2+-stimulated Endocytosis of the mZIP4 Zinc Transporter Regulates Its Location at the Plasma Membrane
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m310799200
ISSN1083-351X
AutoresByung‐Eun Kim, Fudi Wang, Jodi Dufner‐Beattie, Glen K. Andrews, David Eide, Michael J. Petris,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoZinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis. Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis. Zinc is an essential nutrient for all organisms because it is required by a variety of enzymes that are involved in critical areas of metabolism. However, zinc is also potentially toxic when allowed to accumulate beyond cellular needs. Thus, homeostatic mechanisms have evolved to precisely regulate intracellular levels of this nutrient. Genetic studies in bakers' yeast, Saccharomyces cerevisiae, identified the first eukaryotic zinc import protein, ZRT1 (1Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (458) Google Scholar, 2Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (641) Google Scholar). The ZRT1 protein shows significant sequence homology to the IRT1 protein from Arabidopsis thaliana, which is primarily an iron transporter that can also transport zinc (3Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1115) Google Scholar). Both ZRT1 and IRT1 were the founding members of the ZIP (ZRT1-IRT1-like protein) super-family of metal transporters that exist in all eukaryotes (for review, see Ref. 4Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (439) Google Scholar). The hallmark features of ZIP proteins include eight transmembrane domains, the fourth of which contains fully conserved histidyl and glycyl residues in a putative amphipathic α-helix. In humans, 14 ZIP proteins have been identified by data base sequence comparisons (5Eide D.J. Pfluegers Arch. Eur. T. Physiol. 2004; (in press)Google Scholar), and at least two of these proteins, hZIP1 and hZIP2, function as zinc importers when expressed in K562 cells (6Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 7Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Recently, another ZIP family member, hZIP4 (SLC39A4), was shown to be mutated in the inherited disorder of zinc deficiency, acrodermatitis enteropathica (AE) 1The abbreviations used are: AEacrodermatitis enteropathicaHAhemagglutinin antigenHEKhuman embryonic kidneyMCDmethyl-β cyclodextrinPBSphosphate-buffered salineTPENN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. (8Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 9Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (437) Google Scholar). acrodermatitis enteropathica hemagglutinin antigen human embryonic kidney methyl-β cyclodextrin phosphate-buffered saline N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. AE is characterized by symptoms of zinc deficiency (10Lorincz A.L. Arch. Dermatol. 1967; 96: 736-737Crossref PubMed Scopus (4) Google Scholar), such as dermatological lesions, changes in the small bowel mucosa, reduced weight gain, and immune and reproductive dysfunction (11Baudon J.J. Fontaine J.L. Larregue M. Feldmann G. Laplane R. Arch. Fr. Pediatr. 1978; 35: 63-73PubMed Google Scholar, 12Bohane T.D. Cutz E. Hamilton J.R. Gall D.G. Gastroenterology. 1977; 73: 587-592Abstract Full Text PDF PubMed Google Scholar, 13Braun O.H. Heilmann K. Pauli W. Rossner J.A. Bergmann K.E. Eur. J. Pediatr. 1976; 121: 247-261Crossref PubMed Scopus (21) Google Scholar, 14Chesters J.K. J. Inherit. Metab. Dis. 1983; 6: 34-38Crossref PubMed Scopus (13) Google Scholar). The primary basis of AE is hypothesized to be the reduced uptake of dietary zinc by intestinal cells (15Atherton D.J. Muller D.P. Aggett P.J. Harries J.T. Clin. Sci. (Lond.). 1979; 56: 505-507Crossref PubMed Scopus (57) Google Scholar, 16Lombeck T. Schnippering H.G. Ritzl F. Feinendegen L.E. Bremer H.J. Lancet. 1975; 1: 855Abstract PubMed Scopus (79) Google Scholar) because patients respond positively to dietary zinc supplements (11Baudon J.J. Fontaine J.L. Larregue M. Feldmann G. Laplane R. Arch. Fr. Pediatr. 1978; 35: 63-73PubMed Google Scholar, 12Bohane T.D. Cutz E. Hamilton J.R. Gall D.G. Gastroenterology. 1977; 73: 587-592Abstract Full Text PDF PubMed Google Scholar, 17Ohlsson A. Acta Paediatr. Scand. 1981; 70: 269-273Crossref PubMed Scopus (18) Google Scholar, 18Krieger I. Evans G.W. Zelkowitz P.S. Pediatrics. 1982; 69: 773-777PubMed Google Scholar). Consistent with this hypothesis was the finding that the affected protein in AE, hZIP4, and the murine homolog, mZIP4, are most abundantly expressed in the small intestine (8Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), and the mZIP4 protein is located at the apical membrane of intestinal enterocytes in zinc-deficient mice (8Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 19Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Moreover, mZIP4 has been shown to stimulate zinc uptake when expressed in the human embryonic kidney cell line, HEK293 (19Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Patients with AE provide in vivo evidence of the importance of hZIP4 in dietary zinc uptake; however, skin fibroblast cell lines derived from AE patients also display zinc deficiency phenotypes and have reduced zinc uptake (20Vazquez F. Grider A. Biol. Trace Elem. Res. 1995; 50: 109-117Crossref PubMed Scopus (18) Google Scholar, 21Grider A. Lin Y.F. Muga S.J. Biol. Trace Elem. Res. 1998; 61: 1-8Crossref PubMed Scopus (12) Google Scholar). These findings suggest that hZIP4 is likely to function in the uptake of zinc in a variety of cell types. The importance of mammalian ZIP4 in dietary and cellular zinc uptake has prompted recent studies investigating whether the expression of the mZIP4 gene is regulated by zinc availability. Studies using mice have established that mZIP4 mRNA levels in the small intestine and embryonic visceral yolk sac are induced under conditions of dietary zinc deficiency, suggesting that the transcription or stability of mZIP4 mRNA is regulated by zinc availability (19Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In this study, we investigated whether the mZIP4 protein is regulated by zinc availability. Using transfected HEK293 cells, a functional hemagglutinin antigen (HA)-tagged form of the mZIP4 protein was localized in cytoplasmic vesicles within the perinuclear region of the cell. There was considerable overlap between mZIP4-HA and the transferrin receptor in recycling endosomes, and mZIP4-HA was shown to cycle via the plasma membrane. Significantly, zinc deficiency conditions resulted in the accumulation of the transporter at the plasma membrane, and this was associated with increased zinc uptake activity. The addition of low micromolar zinc concentrations stimulated the rapid endocytosis of mZIP4-HA. The endocytosis of mZIP4-HA was also stimulated by manganese, cobalt, and cadmium, although this required higher concentrations relative to zinc. Notably, a zinc-responsive localization was also found for the hZIP4 protein. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis. Reagents, Cell Lines, and Antibodies—HEK293 cells expressing either the HA-tagged mZIP4 protein or pcDNA3.1-puromycin vector, were isolated after transfection using the LipofectAMINE 2000 reagent (Invitrogen). The hZIP4 cDNA cloned into the pcDNA3.1-puromycin vector was kindly provided by Jane Gitschier (UCSF) and tagged at the carboxyl terminus with the HA epitope using PCR and standard molecular biology protocols. Puromycin-resistant cells were established by the selection of transfected cells with 2 μg/ml puromycin in the growth medium (Sigma). mZIP4-HA expression was analyzed by Western blotting and immunofluorescence microscopy using the polyclonal rabbit anti-HA antibody (Sigma). All cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 units/ml penicillin and streptomycin in a 5% CO2,37 °C incubator. This medium contains ∼2 μm Zn2+. Anti-mouse and anti-rabbit antibodies conjugated to horseradish peroxidase were purchased from Roche Applied Science. Alexa-488 and -588 antibodies were purchased from Molecular Probes. Transferrin receptor antibodies were from Santa Cruz Biotechnology. Anti-tubulin antibodies, methyl-β cyclodextrin (MCD), Chelex 100, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) were from Sigma. Zinc depletion of 10% fetal bovine serum in Dulbecco's modified Eagle's medium was performed using Chelex 100 prepared as described previously with minor modifications (22Messer H.H. Murray E.J. Goebel N.K. J. Nutr. 1982; 112: 652-657Crossref PubMed Scopus (17) Google Scholar). Briefly, a solution of 2% (w/v) Chelex 100 resin in 10% fetal bovine serum in Dulbecco's modified Eagle's medium was incubated overnight with constant stirring followed by filtration through a 0.2-μm filter. Immunoblot Analysis of mZIP4-HA Protein—Cells cultured in 25-cm2 flasks were scraped into ice-cold phosphate-buffered saline (PBS) and pelleted by centrifugation. After several washes in ice-cold PBS, the cells were lysed by sonication in lysis buffer containing 62 mm Tris-Cl, pH 6.8, 2% SDS, 100 mm dithiothreitol, and protease inhibitor mix (Roche Applied Science). Samples were centrifuged for 10 min at 16,000 × g, and 20-μg protein lysates were separated using 4–20% SDS-PAGE, transferred to nitrocellulose membranes, and detected by chemiluminescence using anti-HA antibodies (1:1,000) followed by horseradish peroxidase-conjugated secondary antibodies (1:5,000). Immunofluorescence Microscopy to Assess Surface and Intracellular Pools of mZIP4-HA Protein—Cells were grown in 24-well trays for 24 h on sterile glass coverslips. In some experiments, TPEN and/or zinc (as ZnCl2) was added to the medium at indicated concentrations and times. To detect the total pool of mZIP4-HA protein, cells were washed twice with 1 ml of ice-cold PBS and fixed for 10 min at 25 °C using 4% paraformaldehyde. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked for 1 h with 1% bovine serum albumin and 3% skim milk in PBS, and then probed with the anti-HA antibodies (1:1,000) followed by Alexa-488 anti-mouse antibodies (1:1,000). Samples were viewed with a 60× objective using a Leica DMRE microscope fitted with a Retiga Ex digital camera. To label only the surface pool of mZIP4-HA, the Triton X-100 permeabilization step was omitted, and fixed cells were blocked and probed with the anti-HA and Alexa-488 antibodies as described above. Immunofluorescence analysis of mZIP4-HA endocytosis was assessed by detecting the uptake of anti-HA antibodies from basal medium, medium containing 10 μm TPEN, or medium containing both 10 μm TPEN and 10 μm ZnCl2. Briefly, cells were pregrown in basal medium, chilled on ice to inhibit protein trafficking, and then incubated for the indicated times in the above medium at 37 °C in the presence of 2 μg/ml anti-HA antibodies. Cells were then transferred to ice to prevent further trafficking of mZIP4-HA, washed twice with ice-cold PBS, and the surface-bound antibodies were removed by three incubations for 2 min in 2 ml of acidic buffer on ice (100 mm glycine, 20 mm magnesium acetate, 50 mm potassium chloride, pH 2.2). After a further two washes with 2 ml of PBS on ice, the cells were fixed, permeabilized, and processed for immunofluorescence as described above. Detection of mZIP4-HA Protein Levels at the Plasma Membrane—The pool of mZIP4-HA at the plasma membrane was assessed by measuring the levels of anti-HA antibodies bound to the surface of HEK/mZIP4-HA cells. HEK/mZIP4-HA cells were cultured for 24 h in 6-well trays, washed twice with PBS on ice, and fixed for 10 min in 4% paraformaldehyde without subsequent permeabilization steps. Cells were then blocked using 3% skim milk in PBS and incubated with 5 μg/ml anti-HA antibody for 30 min at room temperature. Cells were washed five times in PBS to remove unbound antibodies and then lysed by sonication in SDS lysis buffer described above. Cell lysates containing the solubilized anti-HA antibodies that were bound to the mZIP4-HA protein at the plasma membrane were separated using 4–20% SDS-PAGE, transferred to nitrocellulose membranes, and the anti-HA antibodies were then detected using horseradish peroxidase-conjugated antibodies (1: 5,000) by chemiluminescence (Roche Applied Science). Tubulin protein levels were detected on parallel immunoblots using anti-tubulin antibodies (1:40,000; Sigma). Assay of Zinc-stimulated mZIP4-HA Endocytosis—The endocytosis of mZIP4-HA was determined by measuring the uptake of anti-HA antibodies added to the cultured medium of HEK/mZIP4-HA cells. Cells were pregrown in 6-well trays for 24 h in basal medium and then incubated for the indicated times at 37 °C in basal medium, Chelextreated medium, or TPEN-containing medium containing 5 μg/ml anti-HA antibodies and the indicated amounts of zinc or other metals. Cells were washed twice with 2 ml of PBS on ice, and surface-bound antibodies were removed by three washes in 2 ml of ice-cold acidic buffer (above). Cells were harvested by scraping into 1 ml of ice-cold PBS and pelleted by centrifugation at 1,000 × g. The cell pellets were solubilized in SDS buffer (above), and 20 μg of lysates containing internalized anti-HA antibodies were separated using 4–20% SDS-PAGE, transferred to nitrocellulose membranes, and detected by chemiluminescence using horseradish peroxidase-conjugated secondary antibodies, as described above. 65Zn Uptake Assays—HEK/mZIP4-HA and HEK/vector cells were seeded in 24-well poly-l-lysine-coated plates 48 h prior to zinc uptake assays. Cells were preincubated for 1 h in basal medium or medium supplemented with 10 μm ZnCl2, 10 μm TPEN, or 10 μm ZnCl2 plus 10 μm TPEN. Cells were then washed with ice-cold uptake buffer (15 mm HEPES, 100 mm glucose, and 150 mm KCl, pH 7.0) and then incubated in prewarmed uptake buffer containing 5 μm65ZnCl2 (PerkinElmer Life Sciences) in a shaking 37 °C water bath for 5 min. Assays were stopped by adding an equal volume of ice-cold uptake buffer supplemented with 1 mm EDTA (stop buffer). Cells were collected on nitrocellulose filters (Millipore; 0.45-μm pore size) and washed three times with 10 ml of stop buffer. Parallel experiments were conducted at 0 °C for cell surface 65Zn binding, which was subtracted from the values at 37 °C to obtain net zinc uptake values. Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650 counter. Zinc uptake was calculated using a standard curve and normalized to protein concentrations of cell lysates. We have demonstrated recently that expression of the mouse ZIP4 cDNA stimulates zinc uptake in transiently transfected HEK293 cells (19Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). The initial focus of the present study was to identify the subcellular distribution of the mZIP protein. A HA epitope tag was fused to the carboxyl terminus of mZIP4 to allow detection of the mZIP4-HA protein using anti-HA antibodies (Fig. 1A). The HEK293 cell line was transfected with the mZIP4-HA plasmid construct, and several independent populations of cells expressing mZIP4-HA were isolated by selection with puromycin to generate HEK/mZIP4-HA cells. In each population, ∼80% of the cells stably expressed the mZIP4-HA protein as determined by immunofluorescence microscopy using anti-HA antibodies (data not shown). Western blot analysis using anti-HA antibodies revealed a specific protein in HEK/mZIP4-HA cells which was absent in HEK293 cells transfected with the pcDNA3.1 vector alone (Fig. 1B). The apparent molecular mass of 90 kDa was larger than the expected size of 70 kDa for mZIP4-HA. This discrepancy was likely the result of glycosylation of mZIP4-HA because the expected size was observed when HEK/mZIP4-HA cells were treated with the glycosylation inhibitor, tunicamycin (data not shown). We then investigated whether altered zinc availability could affect the abundance or apparent molecular mass of the mZIP4-HA protein. HEK/mZIP4-HA cells were cultured for 4 h under conditions made zinc-deficient by supplementing the medium with the membrane-permeable zinc chelator, TPEN. However, this treatment did not alter the abundance or electrophoretic mobility of the mZIP4-HA protein (Fig. 1B). The effect of excess zinc (100 μm) in the medium was also tested, and again no apparent changes in abundance or apparent molecular mass of the mZIP4-HA protein were observed (Fig. 1B). We then explored the possibility that the intracellular distribution of mZIP4-HA is regulated by zinc. In HEK/mZIP4-HA cells cultured in basal medium, immunofluorescence microscopy using anti-HA antibodies revealed that mZIP4-HA was distributed in cytoplasmic vesicles that were concentrated in the perinuclear region (Fig. 1C). There was no signal detected in HEK293 cells transfected with the empty vector (data not shown). A striking result was obtained when TPEN was added to the medium for 1 h to generate a zinc-deficient condition. The distribution of mZIP4-HA was shifted toward the periphery of cells, and labeling of the protein at the plasma membrane was clearly apparent (Fig. 1D). This TPEN-induced relocalization of mZIP4-HA was suppressed when equal amounts of zinc and TPEN were added together to the medium (Fig. 1E), suggesting that the redistribution of mZIP4-HA using TPEN alone was likely because of zinc limitation. The redistribution of mZIP4-HA was also observed when HEK/mZIP4-HA cells were cultured in medium from which metals had been previously extracted using the metal-chelating resin, Chelex 100, and this effect was suppressed by replacing zinc (data not shown). These effects of TPEN and Chelex treatments were specific for mZIP4-HA because these treatments did not affect the locations of several proteins known to cycle or reside within endocytic compartments, including the transferrin receptor, the copper transporter, hCtr1, the early endosome marker EEA1, and the ATP7A copper exporter (data not shown). These data suggest that the location of mZIP4-HA is zinc-responsive and that zinc limitation increases its abundance at the plasma membrane. The observation that under basal medium conditions, the mZIP4-HA protein was concentrated in perinuclear vesicles, together with the redistribution to the plasma membrane upon zinc limitation, suggested that a significant fraction of the protein exists in the recycling endosomal compartment. Thus, we investigated whether mZIP4-HA colocalized with the transferrin receptor, a marker of recycling endosomes. In HEK/mZIP4-HA cells cultured in basal medium, the transferrin receptor was located in a perinuclear vesicular compartment (Fig. 2B, green), and there was considerable overlap with the location of mZIP4-HA protein (Fig. 2A, red), as indicated in the merged images (Fig. 2C, yellow). These findings suggested that mZIP4-HA is abundant in recycling endosomes and may cycle between this compartment and the plasma membrane. To investigate further whether mZIP4-HA cycles via the plasma membrane, we tested whether treating cells with an endocytic inhibitor would lead to an increase in the levels of mZIP4-HA at the plasma membrane. HEK/mZIP4-HA cells were incubated in medium containing the general endocytosis inhibitor, MCD, which inhibits both clathrin- and caveolae-mediated endocytic pathways (23Rodal S.K. Skretting G. Garred O. Vilhardt F. van Deurs B. Sandvig K. Mol. Biol. Cell. 1999; 10: 961-974Crossref PubMed Scopus (840) Google Scholar). MCD treatment resulted in the accumulation of mZIP4-HA at the cell surface (Fig. 2F). This finding supported the hypothesis that mZIP4-HA cycles between endosomal compartments and the plasma membrane. Our studies then focused on whether zinc limitation resulted in increased levels of mZIP4-HA at the plasma membrane. The extracellular location of the HA epitope at the carboxyl terminus of mZIP4 enabled us to label only the surface fraction of mZIP4-HA protein with anti-HA antibodies. This was achieved by using anti-HA antibodies to probe intact HEK/mZIP4-HA cells that were fixed, but not permeabilized. Weak punctate staining on the surface of HEK/mZIP4-HA cells was observed when these cells were cultured in basal medium (Fig. 3A). The punctate nature of this staining suggested that mZIP4-HA may localize within distinct microdomains of the plasma membrane, as found for other surface proteins (24Nabi I.R. Le P.U. J. Cell Biol. 2003; 161: 673-677Crossref PubMed Scopus (618) Google Scholar). Notably, in TPEN-treated HEK/mZIP4-HA cells there was a marked increase in the levels of surface-bound anti-HA antibodies (Fig. 3A). This surface staining was specific for mZIP4-HA because there was no labeling of HEK293 cells transfected with the vector alone (Fig. 3A). Together with earlier experiments with permeabilized cells, these data suggested that zinc limitation increases the level of mZIP4-HA at plasma membrane. To demonstrate more quantitatively the increased surface expression of mZIP4-HA in zinc-limited cells, we used an immunoblotting method developed previously to measure changes in surface levels of the hCtr1 copper transporter under conditions of varying copper availability (25Petris M.J. Smith K. Lee J. Thiele D.J. J. Biol. Chem. 2003; 278: 9639-9646Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). The strategy involved using immunoblots to measure the levels of anti-HA antibodies that were recovered from the surface of intact (i.e. nonpermeabilized) HEK/mZIP4-HA after these cells were probed with anti-HA antibodies. HEK/mZIP4-HA cells were pretreated with cycloheximide to inhibit new protein synthesis. Cells were then were exposed to basal or TPEN-containing medium, fixed with paraformaldehyde, and the intact cells were then probed with the anti-HA antibodies to label mZIP4-HA protein at the plasma membrane. After extensive washing of cells to remove unbound antibodies, the cells were lysed, and the solubilized anti-HA antibodies were detected by Western blotting with horseradish peroxidase-conjugated secondary antibodies. As a control, the anti-HA antibody was run on the same gel and detected as a single 100 kDa band (Fig. 3B, lane 10). There was a low abundance of mZIP4-HA at the plasma membrane of HEK/mZIP4-HA cells grown in basal medium, as evident from the low levels of surface-bound anti-HA antibodies (Fig. 3B, lane 2). However, the treatment of HEK/mZIP4-HA cells with TPEN resulted in a rapid increase in the levels of anti-HA antibodies bound to the cell surface, indicating increased levels of mZIP4-HA at the plasma membrane (Fig. 3B, lanes 3–6). This increased surface binding of anti-HA antibodies was saturated within 30 min of the addition of TPEN (data not shown). There were no detectable anti-HA antibodies bound to the surface of untreated or TPEN-treated HEK293 cells stably transfected with the empty pcDNA3.1 vector (Fig. 3B, lane 1, and data not shown). These findings confirmed independently the immunofluorescence microscopy results and suggested that zinc limitation increased the level of mZIP4-HA protein at the plasma membrane. Moreover, the rapidity of this response, together with its occurrence in the absence of new protein synthesis, indicated that it involved a post-translational mechanism. We then investigated whether this TPEN-induced accumulation of mZIP4-HA at the plasma membrane was reversible upon the addition of zinc. Starting with HEK/mZIP4-HA cells treated with for 1 h with 10 μm TPEN (Fig. 3B, lane 6), the levels of mZIP4-HA protein at the cell surface were assessed over time following an incubation in medium containing 10 μm zinc. A reduction in surface levels of mZIP4-HA was observed within 2 min of zinc supplementation. This finding suggests that zinc stimulates the rapid removal of mZIP4-HA from the plasma membrane of zinc-limited cells. One explanation for the increased levels of mZIP4-HA at the plasma membrane under zinc-limiting conditions is that this is a homeostatic response, which promotes increased zinc uptake by these cells. To test this hypothesis, HEK/mZIP4-HA cells were pregrown in either basal or zinc-deficient medium, and then zinc uptake assays were performed over 5 min in buffer containing 65Zn, as described under "Experimental Procedures." There was no difference in 65Zn uptake in HEK/mZIP4-HA cells pretreated with either basal medium or elevated zinc (Fig. 4). However, a pretreatment of the HEK/mZIP4-HA cells with TPEN to increase the level of mZIP4 at the plasma membrane resulted in a significantly elevated 65Zn uptake activity. The addition of zinc to the TPEN-containing medium suppressed this increased uptake activity in the HEK/mZIP4-HA cells, suggesting that the increased zinc uptake after TPEN treatment was the result of zinc deficiency. Notably, zinc uptake differed little, if any, in the HEK/vector control cells preexposed to basal medium, TPEN, or TPEN plus zinc. This suggested that the TPEN-induced increase in zinc uptake activity in HEK/mZIP4-HA cells was dependent on mZIP4-HA expression. In control experiments performed in parallel, an increase in the level of mZIP4-HA at the plasma membrane similar to that shown in Fig. 3 was observed (data not shown). These data support the hypothesis that the increased levels of mZIP4-HA protein at the plasma membrane in zinc-deficient conditions are likely a homeostatic mechanism to stimulate increased zinc uptake. In Fig. 3, the speed with which the addition of zinc to zinc-deficient cells reduced the levels of mZIP4-HA levels at the plasma membrane prompted us to test whether this was accomplished through increased endocytosis of the protein. The endocytosis of mZIP4-HA was assessed using a strategy similar to that used previously to measure the endocytosis of the copper transporter, hCtr1 (25Petris M.J. Smith K. Lee J. Thiele D.J. J. Biol. Chem. 2003; 278: 9639-9646Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). We surmised that if mZIP4-HA undergoes endocytosis from the plasma membrane, anti-HA antibodies added to the growth medium would bind to surface mZIP4-HA and be internalized. HEK/mZIP4-HA cells were exposed for 5 min to medium containing anti-HA antibodies, rapidly cooled on ice, and the surface-bound anti-HA antibodies were removed by washing cells with ice-cold acidic buffer (data not shown). The internalized anti-HA antibodies were then detected by immunofluorescence microscopy. When HEK/mZIP4-HA cel
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