Human Molecular Genetics Advance Access originally published online on January 6, 2004
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Human Molecular Genetics, 2004, Vol. 13, No. 5 563-571
DOI: 10.1093/hmg/ddh049
Acrodermatitis enteropathica mutations affect transport activity, localization and zinc-responsive trafficking of the mouse ZIP4 zinc transporter
1Departments of Biochemistry and Nutritional Sciences, University of Missouri, Columbia, MO 65211, USA and 2Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160-7421, USA
Received November 11, 2003; Accepted December 17, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The Zip4 protein is involved in dietary zinc uptake from the intestinal lumen. The human ZIP4 gene (SLC39A4) was identified because of its association with acrodermatitis enteropathica (AE), a genetic disorder of zinc absorption. To date, several SLC39A4 mutations have been identified in AE patients. To investigate the effects of these mutations on function of the Zip4 transporter, we introduced six AE-associated missense mutations into the orthologous mouse ZIP4 gene for functional expression in cultured cells. All mutations decreased 65Zn uptake activity of mZip4, thereby providing a causal link to AE. The mutants fell into two groups based on their phenotypic effects. Several alleles (G340D, L382P, G384R, G643R) failed to localize on the cell surface at high levels. These defects were attributable to misfolding and/or mislocalization in the secretory pathway. Two other alleles (P200L and G539R) accumulated to high levels in the plasma membrane and had wild-type apparent Km values for 65Zn uptake. However, these mutations decreased the Vmax of uptake to
30% of wild-type. We showed previously that wild-type mZip4 is regulated post-translationally in response to zinc status. In zinc-replete cells, mZip4 is found largely in intracellular compartments. In zinc-limited cells, surface levels increase markedly because the rate of endocytosis decreases. Surprisingly, endocytosis of both P200L and G539R is no longer zinc responsive; these proteins are endocytosed at a slow rate regardless of zinc status. These effects suggest a zinc sensing mechanism for regulating Zip4 trafficking in response to zinc. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Zinc is an essential nutrient because of the many roles it plays. Its importance is especially clear when considering the symptoms of dietary zinc deficiency. These include immune system dysfunction, growth retardation and mental disorders. Similar symptoms are also found in patients with acrodermatitis enteropathica (AE), a recessive inherited disease of humans (MIM 201100). Patients with AE have impaired absorption of dietary zinc and these symptoms can be ameliorated by increased dietary zinc levels (1–4). Moreover, fibroblasts isolated from AE patients have decreased zinc uptake activity and zinc content (5,6). These results suggested that AE is caused by mutations affecting a protein responsible for zinc transport into intestinal enterocytes and other cells.
Support for this hypothesis was obtained recently with the identification of the SLC39A4 gene. SLC39A4 was identified as the likely AE gene by homozygosity mapping and subsequent analysis of candidate genes in the mapped region (7). Most AE patients studied had mutations in SLC39A4 not found in normal individuals (8–10). SLC39A4 encodes the hZip4 protein. Consistent with its proposed role in zinc transport, hZip4 is a member of the ZIP family of metal ion transporters (11). Studies of other ZIP transporters have indicated a conserved role of these proteins in zinc uptake in bacteria, fungi, plants and mammals (12–15). SLC39A4 mRNA was detected throughout the small intestine and colon, i.e. the sites of dietary zinc absorption (8). We have recently cloned and characterized the closely related mouse mZIP4 gene and determined it to be an ortholog of hZip4 (16). These proteins share 78% amino acid sequence similarity. The mZIP4 gene is abundantly expressed in the same tissues as the human gene and mZip4 protein accumulates at the apical surface of intestinal enterocytes.
Zinc uptake in the intestine is responsive to zinc status. Under deficient conditions, this process is up-regulated to enhance the acquisition of zinc (17). This regulation may occur through both transcriptional and post-translational control. First, mZIP4 mRNA levels are increased in zinc-deficient mice, suggesting that transcription of this gene may be responsive to zinc status (16). Second, in vitro studies have indicated that the subcellular location of both hZip4 and mZip4 change in response to zinc status (18). In zinc-deficient cells, Zip4 is abundant on the plasma membrane. In zinc-replete cells, the protein is found in intracellular compartments. This post-translational regulation occurs, at least in part, through the control of the endocytosis rate of the Zip4 protein. Zinc deficiency was shown to slow endocytosis of the Zip4 protein specifically leading to increased surface levels. Studies in vivo have suggested that this post-translational regulation may also occur in the intact animal (16).
Many of the mutations found in SLC39A4 of AE patients were predicted to impair function of this protein (8–10). Several of these alleles were frameshifts and deletions mutations. Many others were missense mutations that altered the identity of only a single amino acid. These alleles provide a valuable resource to probe the function and regulation of the Zip4 transporter. In this report, we show that AE mutations disrupt the transport activity, subcellular localization and/or zinc-responsive protein trafficking of the mZip4 protein.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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AE mutations cause defects in zinc uptake activity
Recent reports identified several missense mutations in SLC39A4 that were associated with AE. For several of these alleles, the amino acids altered were conserved in the orthologous mouse ZIP4 gene. Therefore, we introduced these mutations into mZIP4 to probe their effects using various assays previously developed for studying the function of this protein (16,18). The mutations and their locations are summarized in Fig. 1. P200L is located in the extracellular amino-terminal domain whereas G340D, L382P, G384R, G539R and G643R (corresponding to hZip4 G330D, L372P, G374R, G526R and G630R, respectively) are all located in predicted transmembrane domains. These alleles were expressed in transfected cells from the cytomegalovirus (CMV) promoter and included a carboxy-terminal hemagglutinin antigen (HA) epitope tag. This tag is located on the extracellular surface of the plasma membrane (18) (see below).
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To test the effects of these mutations on zinc transport, plasmids expressing these mutant alleles were transiently transfected into HEK293 cells and then assayed for 65Zn uptake activity. HEK293 cells are particularly useful for this purpose because they have a lower level of endogenous zinc uptake activity than do many other cell lines tested (16) (data not shown). In these experiments, 65Zn was supplied at 2 µM, the apparent Km of wild-type mZip4-HA (16), so this assay was sensitive to potential changes in apparent Km and/or Vmax. Transfection of HEK293 cells with the wild-type gene resulted in a 3-fold increase in 65Zn uptake activity when compared to cells transfected with the vector alone (Fig. 2A). In contrast, all six mutant alleles showed reduced rates of zinc uptake activity relative to cells expressing wild-type mZip4-HA (P<0.01). Varying degrees of impairment were observed with the G643R mutant being the most severely affected and P200L showing a more modest defect relative to wild-type.
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Despite equal transfection efficiencies (data not shown), the different proteins accumulated to varying levels and in various forms (Fig. 2B). Immunoblotting showed that cells expressing wild type mZip4-HA had a prominent band at
80 kDa and a less intense band at 76 kDa. The predicted molecular mass of mZip4-HA (72 kDa) is significantly smaller than the observed size; at least part of this discrepancy is explained by N-linked glycosylation of the protein (see below). Many of the mutations resulted in substantially reduced protein levels (e.g. L382P, G384R and G643R) and increased accumulation of smaller molecular mass forms. P200L and G340D accumulated to near wild-type levels but also had increased amounts of a smaller form. Only G539R showed an abundance and immunoblot pattern similar to wild-type.
Many AE mutations cause defects in N-glycosylation of the mZip4 protein
To further explore the effects of these mutations on mZip4 function, we established stably transfected populations of CHO cells expressing these different alleles. While these stable CHO transfectants expressed 70% less mZip4-HA protein than transient transfectants, relative protein levels and apparent molecular masses were similar to those observed in transiently transfected HEK293 cells (data not shown). To investigate the potential role of N-glycosylation in (a) the discrepancy between observed and predicted apparent molecular mass of the wild-type protein and (b) the effect of the mutations on mZip4-HA electrophoretic mobility, cells were treated with the N-glycosylation inhibitor tunicamycin for 16 h prior to analysis of the proteins by immunoblotting. Inhibiting N-glycosylation of the wild-type protein resulted in a shift in apparent molecular mass from 80 (major band) and 76 (minor band) kDa to a single band at 72 kDa, i.e. the predicted size of mZip4-HA (Fig. 2C). Analysis of the mutant proteins yielded a similar result with all higher molecular mass bands shifting to a single band at 72 kDa. Similar results were obtained when glycosyl groups were removed enzymatically with PNGaseF (data not shown). These data indicate that the different forms of mZip4-HA observed in wild-type and mutant-expressing cells are the result of variations in the extent of N-glycosylation.
Many AE mutant proteins are mislocalized
The altered N-glycosylation states apparent for many of the mutants suggested that these proteins have defects in localization and/or protein folding. To assess effects of AE mutations on protein localization, we first examined the level of mZip4-HA protein found on the cell surface in stably transfected cells. These experiments were possible because the HA epitope tag on the carboxy-terminus of the protein is located on the extracellular surface (Fig. 1) (18). Surface localization was assessed first by indirect immunofluorescence microscopy of cells that were fixed but not permeabilized prior to antibody treatment. Surface labeling with anti-HA antibody was strong for wild-type mZip4-HA, and appeared even stronger for P200L and G539R (Fig. 3A). Not surprising given their lower total accumulation, surface staining for L382P and G384R was substantially weaker and undetectable for G643R. G340D surface levels were also low despite its abundant total accumulation.
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This immunofluorescence microscopy analysis of surface mZip4-HA levels was complemented with a more quantitative immunoblot assay of surface-bound antibody (18). In this experiment, fixed non-permeabilized cells were incubated with anti-HA antibody, washed free of unbound antibody, and protein extracts were then prepared. The amount of antibody bound to the cell surface was then assessed by immunoblot detection of the anti-HA antibody recovered in the protein extracts using a secondary antibody. As shown in Figure 3B, no surface-bound antibody was detected in vector-only transfectants. In cells expressing mZip4-HA, levels of surface bound antibody closely correlated with the surface levels of mZip4-HA protein observed by immunofluorescence microscopy. No differences in tubulin levels were observed indicating equal loading of the protein in each lane.
To assess the effects of these mutations on subcellular protein localization, we performed immunofluorescence confocal microscopy on permeabilized cells stably expressing wild-type and mutant mZip4-HA proteins. Cells expressing wild-type mZip4-HA showed abundant vesicular staining that was dispersed throughout the cytoplasm (Fig. 4). Clearly, most of the mZip4-HA protein in these cells is intracellular as we have observed previously (18). We recognize that high-level expression of wild type mZip4-HA from the CMV promoter may alter its subcellular distribution relative to the native protein expressed from its own promoter. Nonetheless, this approach serves as a useful assay of effects of AE mutations on the distribution of mZip4-HA. Cells expressing P200L also showed a pattern of distribution similar to wild-type. In contrast, all other mutants showed clear staining of the nuclear envelope and a reticulated pattern of fluorescence extending from the nucleus into the cytoplasm suggestive of ER retention. These results, coupled with the detected defects in glycosylation, indicate mislocalization of all AE mutants but P200L.
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Effects of P200L and G539R mutations on 65Zn uptake
The processing defects, mislocalization and/or low abundance of G340D, L382P, G384R and G643R provide a clear explanation for their decreased uptake activity. The link between P200L and G539R and AE was less apparent given that these two alleles are abundantly expressed at the plasma membrane. To more closely investigate the effects of these mutations on zinc uptake, we determined the apparent Km and Vmax values for wild-type mZip4-HA and these two alleles in transiently transfected HEK293 cells. Stable lines were not used for these assays because the lower protein expression led to uptake rates only slightly higher than background levels (data not shown). Again, HEK293 cells were used because of their lower endogenous zinc uptake activity. We determined the initial rate of 65Zn uptake over a range of concentrations (Fig. 5A). As was observed in Figure 2, there was reduced zinc uptake activity in the mutants. Michaelis–Menten kinetic constants derived from these data indicated no change in apparent Km but Vmax values were substantially reduced in the mutants. Apparent Kms were 1.7±0.1, 1.9±0.2 and 2.1±0.3 µM and Vmax values (adjusted to remove the contribution of the endogenous system observed in the vector control cells) were 8.9±0.2, 3.6±0.3 and 2.4±0.3 pmol/min/mg protein for wild-type, P200L and G539R, respectively. The decreased Vmax in the mutants occurred despite there being equivalent levels of mZip4-HA protein detectable on the surface of these transiently transfected cells (Fig. 5B). Thus, these mutant transporters have reduced transport capacity relative to the wild-type protein. The higher level of protein expression obtained in transient transfectants can apparently override the differences in plasma membrane levels observed for these proteins in stable transfectants.
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P200L and G539R have defects in zinc-responsive protein trafficking
We recently discovered that both mZip4 and hZip4 are regulated at a post-translational level by zinc (18). Wild-type Zip4 protein constitutively cycles between the cell surface and intracellular vesicular compartments. Zinc deficiency causes wild-type Zip4 to be endocytosed at a reduced rate and this results in increased surface levels of the protein. As a consequence, the zinc uptake capacity of the cell increases because of the greater number of transporters on the cell surface. Thus, we hypothesize that zinc-stimulated Zip4 endocytosis is a mechanism of zinc homeostasis serving in vivo to control transporter activity in response to zinc status.
The slightly higher surface levels of P200L and G539R observed in stably transfected cells grown in basal medium (Fig. 3) suggested that zinc-regulated trafficking may be altered for these mutant proteins. To test this hypothesis, we first determined the effects of zinc treatment and deficiency on surface protein levels as judged by immunofluorescence microscopy of non-permeabilized cells. These experiments were performed with cells stably expressing mZip4 alleles because the higher level of expression in transiently transfected cells leads to constitutively high surface levels of mZip4. TPEN [N,N,N',N'-tetrakis(2-pyridyl-methyl)ethylenediamine], a membrane-permeable zinc chelator, was used to induce zinc deficiency. TPEN treatment for 1 h caused surface levels of wild type mZip4-HA to increase relative to cells grown in basal medium (Fig. 6A). Thus, the concentration of zinc in basal medium is sufficiently high to stimulate endocytosis of wild type mZip4. Similar results were obtained when cells were grown in media made zinc deficient by prior extraction of the metal with Chelex-100 resin (18), indicating that this is not a pharmacological effect of TPEN.
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In contrast, surface levels of P200L and G539R were high regardless of zinc status. Even treating cells with 100 µM zinc failed to reduce surface levels of these proteins. The failure of P200L and G539R to respond to zinc status was also apparent when surface levels of the protein were assayed using immunoblotting (Fig. 6B). TPEN treatment of cells expressing wild type mZip4-HA resulted in an
6-fold increase in surface levels. TPEN or zinc treatment had little if any effect on surface levels of P200L and G539R. These results suggested that, unlike the wild-type protein, the endocytosis rate of P200L and G539R was unresponsive to zinc. To test this conclusion, we used a direct assay of mZip4-HA endocytosis (18). Endocytosis of mZip4-HA can be assayed by examining the internalization of surface bound anti-HA antibody added to living cells in the growth medium. In these experiments, cells were pre-grown in Chelex-treated media to induce zinc deficiency, chilled to stop endocytosis and surface mZip4-HA protein was labeled with anti-HA antibody. The cells were then warmed to 37°C, allowed to internalize the surface-bound antibody for 5 min, and then quickly chilled again to stop further endocytosis. Antibodies remaining on the cell surface were then removed by washing with acidic buffer. This treatment effectively removes all surface-bound antibodies and allows specific detection of internalized antibodies (18). The cells were then lysed and internalized antibody levels assayed by immunoblotting. As shown in Figure 7, zinc concentrations as low as 0.5 µM were sufficient to stimulate endocytosis of the mZip4-HA protein. In contrast, zinc concentrations as high as 20 µM had little effect on the internalization of P200L or G539R. No antibody internalization was observed in vector-only transfectants confirming that these effects are mZip4-HA dependent. The failure of zinc to stimulate endocytosis of P200L and G539R explains, at least in part, the high surface levels of these mutant proteins in basal and zinc-treated cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In this report, we describe the first functional analysis of alleles of acrodermatitis enteropathica. This analysis has led to several insights regarding the function and regulation of the mammalian Zip4 transporter. Consistent with their role in AE, all six of the mutations we characterized caused defects in the function of mZip4 detectable using the various in vitro assays we have developed. Because of the close overall similarity between mZip4 and hZip4 (16) and the conservation of the affected residues in both proteins, we predict that these alleles will cause similar defects in the human protein.
Results from this study indicated that wild type mZip4 is N-glycosylated. Three potential glycosylation sites (N192, N219 and N272) are found in the amino-terminal domain before the first predicted transmembrane domain. These are the only potential sites within the protein indicating that the amino terminus is on the extracellular surface of the membrane (Fig. 1). This topology is found among other ZIP transporters. At this time, it is unclear if all three potential N-glycosylation sites in mZip4 are modified or if only a subset are used.
The AE mutations studied here fell into two general classes; mZip4 alleles that fail to accumulate on the cell surface at high levels (G340D, L382P, G384R and G643R) and those that do (P200L and G539R). All of the mutant proteins that failed to accumulate in the plasma membrane showed apparent defects in N-glycosylation. None of these mutations change the sequence of the predicted N-glycosylation sites. Therefore, incomplete glycosylation is probably caused indirectly by misfolding of the protein such that it is no longer a good substrate for glycosylation and/or by mislocalization within the secretory pathway. Analysis of the total accumulation and intracellular distribution of these proteins further supported the model that they are aberrant. All of the mutations in this class are altered in conserved residues within transmembrane domains. These mutations change nonpolar residues to charged (D, R) or helix-disrupting (P) amino acids that are likely to disrupt protein folding. The lower total accumulation of some mutant proteins, e.g. L382P, suggests that they may be degraded by quality control mechanisms within the ER. The level, distribution, or activity of these mutant proteins could not be improved by growing cells at lower temperature prior to assay (data not shown). Reduced growth temperatures have corrected apparent folding defects of mutants in other transporters (19,20).
P200L and G539R differed from the other alleles; they accumulated to high levels in the plasma membrane and yet had decreased uptake activity. A close examination of their transport properties in transiently transfected cells with equal surface protein levels indicated that these two mutants had wild type affinities for Zn2+ (as judged from their apparent Kms). However, the Vmax values of the mutant proteins were only 30% of wild-type (Fig. 5). G539R replaces a non-polar residue at the beginning of transmembrane domain V with a positively charged residue. Given that the substrate, Zn2+, is also positively charged, this may interfere with transport due to electrostatic repulsion.
P200L is located in the domain amino terminal to transmembrane domain I. The effects of this mutation on mZip4 function indicate the importance of this unusual domain in transport. The amino-termini of both mouse and human Zip4 contain several potential zinc ligands (16). These residues may participate in zinc transport perhaps by binding zinc in the intestinal lumen and increasing the local concentration of substrate available for the transporter. A similar role has been proposed for potential copper-binding sites in the extracellular amino-terminus of yeast and human Ctr1 (21). Whatever the role of the amino-terminal domain in Zip4, the identification of P200L as an AE-causing allele indicates the importance of this domain in Zip4 function. As further support of this hypothesis, several additional AE mutations affecting the amino-terminus of human Zip4 have recently been identified (P84L, R95C, N106K, R251W, Q303H and C309Y) (8–10). Future studies will define the role of this domain in Zip4 function.
We propose that the defects in uptake activity observed for P200L and G539R are their causal link with AE. However, these mutations had an unexpected effect that may also shed light on the mechanism of zinc-responsive mZip4 protein trafficking. Previous studies indicated that mZip4 accumulates on the cell surface under zinc-limiting conditions and is removed from the cell surface under replete conditions (18). These studies established that surface levels are controlled, at least in part, by the effects of zinc on the rate of mZip4 endocytosis. Zinc-deficient cells endocytose the mZip4 protein more slowly than replete cells while the internalization of other proteins is unaffected. This zinc-responsive trafficking is likely a regulatory mechanism to control the cell surface levels, and hence the uptake activity, of the transporter. In contrast to the wild-type transporter, P200L and G539R failed to respond to zinc and accumulated on the cell surface at high levels regardless of the zinc status.
As shown in Figure 7, endocytosis of wild-type mZip4 increased with zinc treatment while little or no such increase was observed with the mutants. Thus, these mutants are showing defects consistent with an impaired mechanism of zinc sensing to control mZip4 trafficking. This sensing mechanism is as yet unknown. One possible mechanism of zinc sensing is suggested by recent studies of the MNK copper transporting P-type ATPase. MNK traffics between the trans-Golgi network (TGN) and the plasma membrane (22). In cells grown in basal medium, MNK is predominately localized to the TGN where it delivers copper to secreted cuproenzymes. In high copper, MNK traffics to the cell surface where it effluxes excess copper to the extracellular environment. Copper-induced trafficking of MNK was found to be closely linked with its transport activity, suggesting that transport and copper sensing were coupled (23). Our results with mZip4 P200L and G539R mutants are also consistent with a similar link between transport activity and trafficking. Alternatively, P200L and G539R may fail to traffic because their reduced uptake activity results in lower levels of intracellular zinc relative to cells expressing wild-type mZip4-HA. We do not believe this model to be likely because the contribution of mZip4 to total uptake in our stably expressing transfectants (as opposed to transient transfectants) is very small (25% of the total). Thus, we do not expect that cells expressing wild-type or mutant mZip4 will differ greatly in their intracellular zinc levels.
Finally, one of the mutations we studied, G643R (equivalent to G630R in hZip4), resulted in complete loss of detectable zinc uptake activity. Despite the severity of this defect in mZip4 function, this mutation is not lethal in humans and patients with this allele were successfully treated with dietary zinc supplements. This result strongly suggests that additional uptake pathways for zinc exist in the apical surface of the intestine that function under zinc-replete conditions. The identities of those other pathways are still unknown.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Plasmid constructions
Mouse ZIP4 cDNA encoding the long isoform was cloned into an expression vector as described previously (16). Mutations were introduced into the mZIP4-HA clone using the QuikChange site-directed mutagenesis kit (Stratagene). Mutants were sequenced in their entirety to confirm the absence of additional mutations.
Cell culture and transfection methods
Human embryonic kidney cells (HEK293) were cultured in DMEM (Invitrogen) plus 0.45% glucose and Chinese hamster ovary cells (CHO) were cultured in DMEM (Invitrogen) plus 0.1% glucose, 2% proline under 5% CO2. All culture media contained 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 100 µM MEM non-essential amino acids (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). HEK293 (2x105) or CHO (1.0x106) cells were seeded in 25 mm2 flasks and transfected with the plasmid DNAs. Transfections were performed using Lipofectamine 2000 (Invitrogen). Transfection efficiencies were typically 60% for the HEK293 cells used in 65Zn uptake assays. Stable CHO cell lines were selected with 5 µg/ml puromycin (Sigma) 48 h after transfection.
65Zn uptake assays
Transiently transfected HEK293 cells were used for 65Zn uptake assays as described previously because these cells have a low level of endogenous uptake activity (16). In brief, cells (2x105) were seeded in 24-well poly-L-lysine coated plates and transfected with the plasmid DNAs. After 36–48 h post-transfection, the cells were used for 65Zn uptake assays. Cells were washed once in uptake buffer (15 mM HEPES, 100 mM glucose, and 150 mM KCl, pH 7.0) and then added to prewarmed uptake buffer containing the specified concentration of 65ZnCl2 (PerkinElmer Life Sciences Inc.) and incubated in a shaking 37°C water bath for 15 min. Assays were stopped by adding an equal volume of ice-cold uptake buffer supplemented with 1 mM EDTA (stop buffer). Cells were then collected on nitrocellulose filters (Millipore; 0.45 µm pore size) and washed three times in stop buffer (10 ml of total wash volume). Parallel experiments were conducted at 0°C to estimate 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. In a parallel experiment, cells were washed three times with ice-cold uptake buffer and resuspended in 0.1% SDS, 1% Triton X-100, PBS buffer for cell lysis. Protein levels were then determined using the Bio-Rad DC protein assay. Zinc uptake rates were calculated and normalized to protein concentrations of cell lysates. Michaelis–Menten constants were determined by nonlinear interpolation of the data using Prism (version 3.0a for Macintosh, GraphPad Software, San Diego, CA, USA). Analysis of variance (ANOVA) and paired t-tests were used for statistical comparison.
Immunoblotting
Cells were cultured for 48 h post-transfection in six-well trays, washed three times with ice-cold PBS, scraped into PBS, and collected by centrifugation. After three additional washes in ice-cold PBS, the cells were lysed by sonication in buffer containing 62 mM Tris–Cl (pH 6.8), 2% SDS, 5 mM dithiothreitol, 1 mM EDTA and protease inhibitor mixture (Roche Molecular Diagnostics). Loading buffer was added to the protein extracts and then boiled for 5 min. Unless indicated otherwise, 20 µg of protein extracts were separated using 4–20% gradient SDS-PAGE ready gels (Bio-Rad), transferred to nitrocellulose membranes, and probed using an anti-HA polyclonal antibody (1 : 1000, Sigma) followed by an anti-rabbit antibody conjugated to horseradish peroxidase (HRP, Pierce Endogen). As a loading control, the membranes were stripped of antibodies by incubating at 60°C for 30 min in stripping buffer [62.5 mM Tris–HCl, 100 mM 2-mercaptoethanol, and 2% (w/v) SDS], washed in TBST, and reprobed with a 1 : 40 000 dilution of mouse anti-tubulin (Sigma) primary antibody and 1 : 10 000 dilution of HRP-conjugated anti-mouse IgG (Pierce Endogen) as secondary antibody.
For immunoblot analysis of mZip4-HA surface levels, the cells were cultured in six-well trays. In some experiments, ZnCl2 or N,N,N'N'-tetrakis (2-pyridyl-methyl) ethylenediamine (TPEN) was added to the media at the indicated concentrations and times. Cells were washed three times with PBS on ice, fixed in 3.7% paraformaldehyde for 30 min at 4°C, blocked, and incubated for 1 h at room temperature with 1 : 500 primary rabbit anti-HA antibody (Sigma), and then washed five times with PBS to remove unbound antibodies. The cells were then lysed by sonication in buffer containing 62 mM Tris–Cl (pH 6.8), 2% SDS, 5 mM dithiothreitol, and protease inhibitor mixture (Roche Molecular Diagnostics). Lysates containing the solubilized anti-HA polyclonal antibodies that were bound to the mZip4-HA protein at the plasma membrane were separated by SDS–PAGE, transferred to nitrocellulose membranes, and the anti-HA antibodies were then detected using anti-rabbit HRP antibodies (1 : 000) by chemiluminescence (Roche Applied Science).
Endocytosis of mZip4-HA was determined by assaying the uptake of anti-HA antibodies added to the cultured media of cells expressing mZip4-HA and mutant derivatives. Cells were grown on six-well plates in basal medium or in basal medium supplemented for 1 h with 100 µM zinc or 5 µM TPEN. The cells were chilled on ice 10 min and then rewarmed and incubated in basal, zinc- or TPEN-supplemented media containing 5 µg/ml anti-HA antibodies for 5 min. During this time, antibodies bound to the HA epitope are internalized by endocytosis. Cells were transferred to ice to prevent further endocytosis, washed three times with ice-cold PBS and surface-bound antibodies were removed by five washes with ice-cold acidic buffer (100 mM glycine, 20 mM magnesium acetate, 50 mM potassium chloride, pH 2.2). Our previous studies demonstrate that this treatment is sufficient to remove all detectable surface-bound antibodies (18). After two additional washes with ice-cold PBS, the cells were collected, lysed and analyzed for anti-HA antibodies by immunoblotting as described above.
Immunofluorescence microscopy
To assay the surface levels of mZip4-HA, cells were grown in 24-well plates for 48 h on sterile glass coverslips. In some experiments, ZnCl2 or TPEN was added to the media at the indicated concentrations and times. The cells were then washed three times with ice-cold PBS, fixed in 3.7% paraformaldehyde for 30 min at 4°C, and washed three times with PBS. Cells were blocked for 1 h with PBS plus 5% normal goal serum (Jackson ImmunoResearch Laboratories Inc.) and 1% bovine serum albumin (Sigma). Cells were incubated for 1 h at room temperature or overnight at 4°C with a 1 : 500 dilution of primary rabbit anti-HA antibody (Sigma). The cells were then washed five times in PBS followed by incubation for 1 h with the appropriate anti-rabbit or anti-mouse IgG secondary antibody conjugated with Alexa 488 or Alexa 647 (Molecular Probes). The cells were washed again with PBS and then examined with an Olympus 1X-70 microscope fitted with an MRC-600 confocal laser (Bio-Rad Laboratories, Hercules, CA, USA). The intracellular distribution of mZip4 proteins was examined in a similar manner using cells that were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Permeabilized cells were incubated with 2 µg/ml DNAase-free RNase (Roche Molecular Biochemicals) at 37°C for 30 min, and the nuclei was stained with propidium iodide (PI, Sigma) prior to mounting.
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ACKNOWLEDGEMENTS |
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The authors thank Elizabeth Rogers for critical reading of the manuscript. This work was supported by NIH grants DK063975 (to G.A. and D.E.) and GM58265 (D.E.) and grants from the National Natural Science Foundation of China to F.W. (grant nos 30170805 and 39970641).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO 65211, USA. Tel: +1 5738829686; Fax: +1 5738820185; Email: eided{at}missouri.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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