Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells

M. Greenough, L. Pase, I. Voskoboinik, M. J. Petris, A. Wilson O'Brien, and J. Camakaris

Department of Genetics, University of Melbourne, Parkville, Victoria 3010, Australia

Submitted 6 April 2004 ; accepted in final form 13 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH2-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper.

Menkes disease; PDZ; copper; trafficking


MENKES DISEASE is an X-linked recessive disorder that causes severe systemic copper deficiency and is usually fatal before 3 years of age. The systemic copper deficiency results from defective absorption in the gut and reabsorption in the kidney and is associated with copper accumulation in these tissues (8). Menkes disease is caused by mutations in the ATP7A (MNK) gene, which encodes a copper-translocating P-type ATPase (6, 33, 54). Heavy metal P-type ATPases are characterized by their ability to translocate heavy metals across membranes (32). Observations in cultured Menkes disease patient fibroblasts and MNK overexpression studies in Chinese hamster ovary (CHO) cells suggest a role for the MNK protein in copper efflux (1, 5, 28, 42). The steady-state localization of MNK is in the trans-Golgi network (TGN), where it delivers copper to copper-dependent enzymes in the secretory pathway (15, 37, 40, 56). We previously demonstrated (39) that MNK traffics to the plasma membrane (PM) when levels of copper are elevated. This facilitates efflux of excess copper from cells and restores normal copper homeostasis. Evidence was also provided that MNK normally cycles constitutively between the TGN and the PM in basal medium (38). Previous studies have focused on elucidating the molecular signals that control MNK trafficking in nonpolarized cultured cells. For example, NH2-terminal domain metal-binding sites 5 and 6 in the wild-type MNK protein are required for copper-induced translocation to the PM (49), a region containing transmembrane domain 3 provides a Golgi localization signal (15), and a dileucine motif proximal to the COOH-terminal tail of MNK is an endocytic signal required for effective recycling from the PM to the TGN (38). Recent studies have established that mutations in conserved regions that block copper transport activity also prevent copper-responsive trafficking to the PM (41, 52). However, little is known about the localization or copper-responsive trafficking of MNK in polarized cells, where defective copper egress in the gut and kidney leads to the severe systemic copper deficiency observed in Menkes disease (8).

In epithelial cells, transport proteins are specifically targeted to particular membranes to effect vectorial transport of fluid, solutes, and electrolytes (29). Targeting from the TGN and endocytic compartments to either apical (AP) or basolateral (BL) membrane domains is regulated by sorting and/or retention mechanisms (10). Basolateral targeting signals are almost exclusively found in the cytoplasmic domains of transmembrane proteins and usually contain either tyrosine or dihydrophobic motifs (2). Studies have shown these motifs to interact with adaptor proteins AP1 (44) and AP2 (45, 46), which are involved in clathrin assembly at the TGN and the PM, respectively. Furthermore, basolateral sorting determinants often overlap internalization signals (21, 22). Because the hydrophobic dileucine L1487L1488 of wild-type MNK is required for internalization of MNK from the PM to the TGN, we investigated whether it also acts as a basolateral targeting motif.

PDZ domain proteins interact with PDZ target sequences usually located at the carboxyl termini of membrane-associated proteins (20). These interactions have been implicated in stabilization/retention of membrane proteins at specific membranes in polarized cells (34). The COOH terminus of MNK has the amino acid sequence DTAL, which fits the consensus target motif for class 1 PDZ proteins (20).

Defects or alterations in trafficking of membrane proteins have been linked with disease (25, 27, 30). Furthermore, a known mutation in a Menkes disease patient has been shown to inhibit MNK trafficking in cultured fibroblasts (1, 26). In the current study our aims were to characterize the normal localization of MNK in polarized cells under varying copper conditions and to identify signals in the COOH-terminal domain of MNK that regulate its trafficking to discrete membrane locations in polarized Madin-Darby canine kidney (MDCK) cells.


    MATERIALS AND METHODS
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Cell culture, DNA constructs, and transfection. MDCK cells (American Type Culture Collection no. CCL-34) were maintained in Eagle's basal medium with Earle's salts (Thermotrace, Melbourne, Australia) supplemented with (in mM) 2 L-glutamine, 1.2 NaHCO3, and 20 HEPES with 10% fetal calf serum (CSL, Melbourne, Australia). MNK constructs were produced by in vitro mutagenesis as previously described (49). Nomenclature for constructs is as follows: wtMNK, full-length wild-type MNK; L1487A-L1488A, mutation of the dileucine proximal to the COOH terminus to dialanine; {Delta}DTAL, truncation of a putative PDZ binding domain; 116, CXXC-to-SXXS mutations in metal-binding sites 1–6 (49, 53). Dr. D. Strausak and Prof. J. Mercer (Center for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, Australia) provided the 116 cDNA. A summary of the mutant constructs is presented in Fig. 1. Parental MDCK cells were transfected with either wild-type or mutated MNK cDNA with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. A chimeric construct, which has the cytoplasmic tail and transmembrane region of rat nucleotide pyrophosphatase phosphodiesterase (NPP)3 (4, 31) and the extracellular domain of human transferrin receptor (TfR), was transfected as above and used as an apical membrane marker. Prof. J. W. Goding (Dept. of Pathology and Immunology, Monash Medical School, Prahran, Australia) provided the NPP3-TfR construct. Transfection-quality DNA was derived from Escherichia coli with a Nucleobond PC100 DNA midiprep kit (Macherey-Nagel, Duren, Germany) or an Endofree Plasmid Maxi kit (Qiagen, Hilden, Germany). For the stable transfection of wtMNK, colonies were selected in 500 µg/ml G418 (Invitrogen) for ~3 wk before screening for protein expression by indirect immunofluorescence microscopy. For transient transfections (all mutant constructs) cells were seeded onto glass coverslips or Costar Transwell (24-mm diameter, 0.4-µm pore size; Corning, Acton, MA) membranes at 60,000 cells/cm2 2 days before transfection and were fixed 48 h after transfection. A x100 oil objective lens and a Leica Polyvar microscope were used to screen for MNK expression in transfected cell lines.



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Fig. 1. Schematic representation of MNK showing a summary of the mutants produced by site-directed in vitro mutagenesis. The COOH-terminal mutants, L1487A-L1488A and {Delta}DTAL, are shown as a sequence alignment against the wild-type (wt) protein (A). Cysteines in the NH2-terminal metal-binding sites (MBS), CXXC, were substituted by serines, SXXS, to produce a nontrafficking mutant, 116 (B). The sequence location of the metal-binding motifs has been described previously (49).

 
Processing of cells for MNK localization studies. Cells were grown on glass coverslips or Costar Transwells until polarized, as assessed by uniform expression of the tight junction marker ZO-1 (zonula occludens) or the adherin junction marker E-cadherin under indirect immunofluorescence. The antibodies to ZO-1 and E-cadherin (uvomorulin) have been used extensively in other subcellular localization studies (3, 7, 17, 23, 43) and are available commercially (see below). Conditions for copper-induced MNK trafficking, fixation, and indirect immunofluorescence were described previously (39). Copper was added to growth medium as aqueous copper chloride and mixed immediately. Copper chloride does not precipitate in medium at the concentration of 315 µM used in all MNK trafficking experiments.

Antibodies. Polyclonal anti-rabbit MNK antibodies were described previously (5). Antibodies against Na-K-ATPase were a gift from Dr. Judy Callaghan (Dept. of Pathology and Immunology, Monash Medical School, Prahran, Australia). Anti-Golgi marker to protein 58K was purchased from Sigma-Aldrich (St. Louis, MO). The antibody anti-OKT9, a mouse monoclonal antibody used for the detection of the apical marker NPP3/TfR, was a gift from Prof. J. W. Goding. Anti-uvomorulin (E-cadherin) was purchased from Sigma-Aldrich and used as a marker for tight cell-cell contacts in polarized cells. Monoclonal mouse anti-ZO-1, a widely used antibody for detection of tight junctions associated with polarized epithelial cells, was purchased from Zymed Laboratories (South San Francisco, CA). For detection of primary antibodies, the Alexa Fluor range of IgG-fluorophore conjugates (Molecular Probes, Eugene, OR) was used.

Confocal microscopy. Confocal imaging was performed with an Optiscan F900e Personal Confocal System with a krypton-argon laser (Optiscan, Melbourne, Australia). An Olympus BX60 microscope with a x60 PlanApo oil-immersion lens was used for visualization of samples. F900e control software was used for image capture and analysis. At least 20 polarized cells, expressing varying levels of the transfected MNK constructs, were analyzed per sample, and representative images are shown in Figs. 2, 3, 5, and 6. Cell thickness was between 10 and 15 µm, scanned in the X-Y axis in 1-µm increments to create three-dimensional (3D) image stacks. Generally, 20 scans per image were required to bring out detail in membrane structures. Maximum-brightness images were obtained by flattening the 3D stack into a single image. Images in the X-Z axis were obtained from cross sections of the X-Y scans. Images were taken at normal, x1.25, or x1.5 software zoom and saved as RGB TIFFs to maintain acceptable image quality. Images were then opened in Photopaint 10 (Corel) for channel separation and brightness/contrast correction.



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Fig. 2. Localization of wtMNK in relation to several marker proteins and evidence of polarization. MNK (A) is shown to be present at the Golgi apparatus in basal medium as demonstrated by colocalization with the Golgi marker protein 58K (B). The merged image of wtMNK-58K is shown in C, where MNK appears green and 58K is red (colocalization is yellow). Uniform polarized monolayers are evident by the expression pattern of ZO-1 (D) and E-cadherin (E). The merged image of ZO-1-E-cadherin (F) shows that ZO-1 is expressed at the upper lateral junctions whereas E-cadherin is distributed more widely at the lateral membranes (ZO-1 appears green and E-cadherin appears red). The Na-K ion exchange pump Na-K-ATPase is expressed mainly at the lateral surface of kidney cells (G), and significant colocalization of Na-K-ATPase is observed with E-cadherin (H). The merged image of Na-K-ATPase-E-cadherin is shown in I, where Na-K-ATPase appears green and E-cadherin appears red (colocalization is yellow). Smaller images at bottom of each panel show X-Z cross sections of the corresponding X-Y images. Bars = 10 µm.

 


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Fig. 3. Confocal microscopic images demonstrating MNK trafficking and recycling in polarized Madin-Darby canine kidney (MDCK) cells. In cells stably transfected with wt human cDNA, MNK was evident in a location consistent with the Golgi in basal medium (A), relocalized to the basolateral (BL) membrane in elevated-copper medium (D), and recycled back to the Golgi after being incubated in elevated copper for 3 h and then returned to basal medium for 2 h (G). The cells were colabeled with ZO-1 to show tight cell-cell junctions in each case (B, E, and H, respectively), and the corresponding MNK-ZO-1 merged images are shown in C, F, and I, respectively, where MNK appears green and ZO-1 appears red. Smaller images at bottom of each panel show X-Z cross sections of the corresponding X-Y images. Basal medium contained 0.8 µM total copper and elevated-copper medium contained 315 µM total copper, measured by atomic absorption spectroscopic analysis. Bars = 10 µm.

 


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Fig. 5. Mutation of the NH2-terminal domain metal-binding sites inhibits copper-induced MNK trafficking. Localization of 116 mutant MNK corresponds to the Golgi in basal medium (A), elevated-copper medium (B), and basal medium after elevated copper treatment (C). MNK is labeled green, and E-cadherin is labeled red. Smaller images at bottom of each panel show X-Z cross sections of the corresponding X-Y images. Bars = 10 µm.

 


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Fig. 6. Mutational analysis of MNK to identify key COOH-terminal sequences involved in trafficking of MNK in polarized MDCK cells. Confocal microscopic images are shown, which demonstrate the distribution of transiently transfected MNK mutants in polarized MDCK cells. The dileucine mutant MNK, L1487A-L1488A, is mainly localized to a region consistent with the AP membrane in basal medium (A), elevated-copper medium (B), and basal medium after the removal of elevated-copper medium (C). The normal basal Golgi localization of MNK is not affected by truncation of the last 4 amino acids of the COOH-terminal DTAL, a putative PDZ motif (D). However, in elevated-copper medium MNK/{Delta}DTAL mislocalizes to the AP membrane and intracellularly (E). The MNK/{Delta}DTAL still has the ability to recycle back to the Golgi when returned to basal medium for 2 h after exposure to elevated-copper medium for 3 h (F). The chimeric protein NPP3 TM/cyto-HTFR EC localizes predominantly at the AP membrane in MDCK cells (4, 31), making it useful for visualizing the normal distribution of wtMNK in basal growth medium (G), exposed to 300 µM copper for 3 h (H), and when cells are returned to basal medium for 2 h after exposure to 315 µM copper for 3 h (I). MNK protein is labeled green, and E-cadherin and NPP3 TM/cyto-HTFR EC are labeled red. Smaller images at bottom of each panel show X-Z cross sections of the corresponding X-Y images. Bars = 10 µm.

 
Surface biotinylation. MDCK cells were trypsinized, counted, and seeded onto Costar Transwells (24-mm diameter) at 60,000 cells/cm2 5 days before surface biotinylation was performed. Tight junctions were assessed for paracellular barrier formation before biotinylation with a phenol red diffusion spectroscopic assay as previously described (24). Direct surface labeling of MNK with biotin was performed using methods we described recently (36). The concentration of copper used in biotinylation experiments was 315 µM, the same as that used in confocal imaging analysis. To eliminate the possibility that nonspecific binding of basal MNK to streptavidin beads contributes to the pool of surface-labeled protein, a nonbiotinylated control was performed, which demonstrated that nonbiotinylated MNK was not detectable (data not shown). Visualization and quantification of labeled proteins were performed with a Typhoon 9200 PhosphorImager and Imagequant software (Amersham Biotechnology, Piscataway, NJ). All biotin labeling experiments were performed at 4°C. Surface biotinylation could not be performed on cells expressing the mutant constructs, because the number of cells expressing MNK after transient transfection was too low to permit adequate sensitivity of detection via this technique and several attempts to produce stable cell lines expressing the mutant constructs with cationic lipid transfection methods were unsuccessful.


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Characterization of MNK localization in polarized MDCK cells. The study was initiated by isolating MDCK cells stably transfected with the MNK cDNA construct. MDCK cells grow as polarized cell monolayers that possess two distinct PMs, AP and BL membranes, separated by tight junctions. In basal growth medium MNK localization was found to significantly overlap that of the Golgi marker 58K (Fig. 2, A, B, and C), consistent with a TGN localization for MNK observed previously in nonpolarized cells (15, 39). ZO-1, E-cadherin, and Na-K-ATPase are expressed in polarized MDCK cells at specific membrane locations, which makes them useful in protein localization studies (19). Tight junctions formed between polarized MDCK cells mark the AP-BL membrane boundaries, and the tight junction protein ZO-1 has been used extensively as a marker in permeability and loss of barrier function studies (17). In the current study ZO-1 was used as an immunohistochemical marker of polarization and a visual reference for AP-BL membrane boundaries to help characterize MNK localization (Fig. 2, D and F). E-cadherin is localized to the adherin-rich region of the lateral boundaries of polarized MDCK cells (16), located directly below the tight junctions, and was used as a lateral membrane marker (Fig. 2, E–F and H–I). Na-K-ATPase is a protein that normally localizes to the BL membrane of polarized kidney cells (55), and in the current study we confirmed this localization to be the lateral region of MDCK cells (Fig. 2G). Na-K-ATPase could not be used directly as a marker for MNK localization studies because the antibodies used for detection of Na-K-ATPase and MNK were both rabbit polyclonal and hence could not be used together. However, significant colocalization of Na-K-ATPase and E-cadherin was observed at the lateral membranes (Fig. 2, G, H, and I), which made E-cadherin a useful marker in determining MNK localization when Na-K-ATPase could not be used. The colocalization of Na-K-ATPase and E-cadherin has been described in a study that demonstrates that interaction of the {beta}-subunit of Na-K-ATPase and E-cadherin is required for epithelial polarization (43) and supports the use of E-cadherin to demonstrate the polarized state of the MDCK cells used for MNK localization.

MNK undergoes copper-induced relocalization to BL membrane. MDCK cells have a low detectable level of endogenous MNK that makes them ideal for studying the effects of mutations in transfected mutant MNK cDNAs. To investigate the role of MNK trafficking in polarized epithelia, MDCK cells were transfected with wild-type MNK and various MNK cDNA constructs containing mutations in distinct regions of the protein (see Fig. 1). Wild-type MNK localized at the Golgi of cells in basal medium (Fig. 3, A and C) was found to relocalize to the BL membrane after elevated copper treatment (Fig. 3, D and F). The restoration of basal copper levels to these copper-treated cells resulted in the return of MNK to the Golgi (Fig. 3, G and I). Uniform expression of ZO-1 under all copper conditions confirmed that these cells were polarized (Fig. 3, B, E, and H).

Surface biotinylation. To independently confirm the apparent basolateral localization of wild-type MNK we examined the levels of MNK in pools of proteins isolated from BL or AP membranes. AP or BL surface proteins were biotinylated with sulfo-NHS-SS-biotin, precipitated with streptavidin-agarose, separated by SDS-PAGE, and then detected with anti-MNK antibodies (36). In basal medium, the relative abundance of MNK in BL membranes was greater than that in AP membranes (Fig. 4A), suggesting that in the absence of elevated copper MNK is targeted to the BL membrane. When the copper concentration in the AP chamber was increased to 315 µM, there was a 2.3-fold increase in MNK at the BL surface, whereas levels of MNK at the AP surface were not altered by this treatment (Fig. 4B). When media containing 315 µM copper were added to the BL chamber, AP levels of MNK were unchanged but there was a fourfold increase in MNK at the BL surface (Fig. 4C). Adding 315 µM copper to both AP and BL chambers resulted in a 3.8-fold increase in MNK at the BL surface (Fig. 4D). These findings independently confirmed our immunofluorescence analyses and suggested that elevated copper stimulates the relocalization of MNK to the BL membrane.



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Fig. 4. Western blot and corresponding chart demonstrating the localization of biotinylated MNK protein in MDCK cells grown on Transwell semipermeable membranes are shown. The amount of biotinylated MNK on the apical (AP) membrane (lane 1) and BL membrane (lane 2) was detected under basal copper conditions (A). The amount of biotinylated MNK on the AP (lane 3) and BL (lane 4) membranes was detected when elevated-copper medium was added from the AP chamber (B). The amount of biotinylated MNK on the AP (lane 5) and BL (lane 6) membranes was detected when elevated-copper medium was added from the BL chamber (C). The amount of biotinylated MNK on the AP (lane 7) and BL (lane 8) membranes was detected when elevated-copper medium was added from both chambers (D). The Western blot was scanned with a PhosphorImager (see MATERIALS AND METHODS). Data are shown as relative increase over basolateral MNK protein in basal copper medium and are the means of 3 independent experiments using duplicate wells each time.

 
Mutation of the six NH2-terminal metal binding sites inhibits copper-induced MNK trafficking. Strausak et al. (49) previously demonstrated in cultured CHO cells that NH2-terminal domain metal-binding sites 5 and 6 are critical for copper-induced relocalization from the TGN. The mutant 116, which has all six metal-binding sites mutated, does not respond to elevated copper by relocalizing to the PM. Because polarized cells may have mechanisms different from those of CHO cells for protein trafficking, it was important in the present study to see what effect, if any, mutation of all NH2-terminal metal-binding sites had on MNK trafficking. In polarized MDCK cells, 116 was localized at a region consistent with the Golgi in basal medium (Fig. 5A) and failed to relocalize from this location in elevated-copper medium (Fig. 5B) or when basal medium was restored after elevated-copper treatment (Fig. 5C). These results indicate that the NH2-terminal metal-binding domain of MNK is required for copper-induced trafficking to the BL membrane of polarized MDCK cells.

Identification of trafficking signals in COOH-terminal domain of MNK. To further characterize the copper-induced relocalization of MNK to the BL membrane, we used site-directed mutagenesis to identify candidate motifs critical for this targeting process. The dileucine internalization motif L1487-L1488 was of particular interest in this study because it was previously shown to be important for MNK recycling from the PM to the TGN in nonpolar cells (14, 38). In polarized MDCK cells the dileucine mutant L1487A-L1488A was mislocalized in basal medium, with only residual levels apparent at the Golgi (Fig. 6A). Interestingly, in elevated-copper conditions the weak labeling of L1487A-L1488A in the Golgi was no longer observed and there was an increased level of MNK above the level of E-cadherin staining, consistent with an apical or subapical localization (Fig. 6B). The L1487A-L1488A MNK failed to relocalize to the Golgi when cells were returned to basal medium (Fig. 6C). These data suggest that the dileucine motif L1487-L1488 is essential for basolateral targeting of MNK in elevated copper and endocytic retrieval from the PM after a restoration of copper homeostasis. Examination of the regions downstream of Leu1488 revealed a putative PDZ target motif, 1497DTAL1500. We investigated the role of this putative PDZ target motif in basolateral localization of MNK in MDCK cells by truncation of the 1497DTAL1500 sequence. This truncated form of MNK was localized to the Golgi in basal medium (Fig. 6D), dispersed to a region consistent with the AP membrane and intracellularly after elevated-copper treatment (Fig. 6E), and recycled to the Golgi when basal medium copper levels were restored after elevated-copper treatment (Fig. 6F). The localization of MNK at the AP membrane and intracellularly in elevated copper, along with its ability to recycle back to the Golgi in basal medium, suggested that the COOH-terminal PDZ binding domain is important for retention of MNK at the BL membrane when copper levels are elevated. To confirm the apparent AP membrane localization of mutants described above, MDCK cells stably expressing wtMNK were transfected with the chimeric protein NPP3 TM/cyto-HTFR EC, which resides primarily at the AP membrane of MDCK cells (4, 31). When cells were incubated in basal medium, transferred to elevated-copper medium, and returned to basal medium after copper treatment (Fig. 6, G, H, and I, respectively), the AP membrane was very clearly labeled with NPP3 TM/cyto-HTFR EC, similar to the localization of the L1487A-L1488A mutant MNK (as in Fig. 6, A–C) as well as the 1497DTAL1500 truncated MNK in elevated copper (Fig. 6E).


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In polarized MDCK cells cultured under basal conditions, we have demonstrated the localization of MNK to the Golgi complex (Figs. 2A and 3A). This finding is consistent with the TGN localization of MNK in nonpolar human and rodent cell lines, where it functions to deliver copper to copper-dependent enzymes in the secretory pathway (11, 28, 39, 40). Furthermore, systemic copper deficiency in Menkes disease is caused by failure of copper absorption in the gut and reabsorption in the kidney associated with large accumulations of copper in these tissues (8). We therefore hypothesized that MNK would traffic to the BL membrane of polarized epithelial cells. Two independent techniques, confocal immunofluorescence microscopy (Fig. 3D) and surface biotinylation (Fig. 4, B–D), demonstrated a steady-state shift of MNK to the BL membrane of MDCK cells in response to elevated copper (315 µM). Utilizing the sensitivity of surface biotinylation we were able to detect a very low level of transfected wtMNK at both the AP and BL membranes in basal medium (Fig. 4A), indicative of constitutive trafficking. Using confocal microscopy analysis we observed a steady-state shift of MNK from the TGN to the BL membrane at a medium copper concentration of 126 µM, and this appeared to be complete at 315 µM, indicating a dose-dependent response in MDCK cells. Trafficking of MNK to the BL membrane of polarized cells is consistent with its role in copper absorption in the gastrointestinal tract and its reabsorption in the kidney (9, 18, 50). We also showed that the NH2-terminal domain metal-binding domain is required for copper-induced translocation of MNK from the Golgi to the BL membrane (Fig. 5B).

The mechanisms for sorting and targeting proteins to specific membranes in polarized epithelial cells are only partially understood (4). However, several recent studies have elucidated key domains of the cytoplasmic region of many membrane proteins. Basolateral targeting determinants may be complex in that they are often composed of several motifs with overlapping signals. For example, dileucine motifs may be read differently at specific subcellular sites according to their surrounding structural context (48). In the case of MNK it is known that a dileucine motif in the COOH-terminal cytoplasmic domain, L1487-L1488, is required for endocytic retrieval from the PM (14, 37, 38). We have identified an additional role for this particular dileucine motif to include BL membrane targeting in polarized epithelial cells. This dual role for dileucine motifs is similar to the dual endocytic and polarized targeting roles for a dileucine motif in the macrophage IgG receptor FcRII-2B in MDCK cells (21). Other studies have shown dileucine residues to be important BL membrane signaling motifs, but not as endocytic motifs (4, 13), and further studies have shown that dileucine motifs only target proteins to endosomal/lysosomal compartments (47). It is unclear why removal of the BL targeting dileucine motif of MNK results in AP membrane targeting (Fig. 6, A–C). It is possible that the default trafficking pathway of MNK, without BL sorting via the dileucine motif, is to the AP membrane. Alternatively, mutation of the L1487-L1488 motif may have exposed a "cryptic" apical sorting signal as has been suggested in some other systems (35).

PDZ target motifs are mostly localized at the COOH terminus of membrane proteins. The mislocalization of {Delta}DTAL in copper-supplemented medium (Fig. 6E) suggests that the putative PDZ target motif is important in sorting or BL membrane retention of MNK. In light of data from studies on other membrane proteins (34), an interaction of the PDZ target motif with PDZ binding proteins may be important for selective stabilization/retention of MNK at the BL membrane under conditions of elevated copper. This is consistent with our recent observation (36) from surface biotinylation studies of a rapid recycling pool of MNK proximal to the PM at elevated copper levels. A possible role of PDZ interaction in sorting of MNK cannot be excluded, and elucidation will require kinetic analysis (36), which was not possible in the current study because of relatively low transfection frequencies and difficulties in isolating stable cell lines (unpublished data).

Functional diversity of signaling mechanisms of membrane-associated proteins, including dileucine and PDZ motifs, has been highlighted by many recent studies in polarized cells. Furthermore, overlapping signals and the importance of amino acids surrounding previously characterized motifs suggest that secondary and tertiary protein structure is critical in determining intracellular localization via specific protein-protein interactions (4). Given our recent findings suggesting that there is a copper-regulated kinase phosphorylation of MNK (51), it is possible that signaling via phosphorylation mediates the "reading" of sorting signals as observed in some other systems (12). In the current studies, the identification of two signals, a dileucine motif and a PDZ target motif, has provided insight into the mechanisms of MNK trafficking to the BL membrane in polarized epithelial cells, where absorption/reabsorption is vital for maintaining systemic copper homeostasis. The copper-regulated trafficking of MNK to specific membrane domains in polarized cells provides an exciting paradigm for understanding the molecular interactions involved in regulating the function of a transporter through changes in its subcellular localization.


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This work was supported by the National Health and Medical Research Council (Australia), J. N. Peter's Bequest, Wellcome Trust, and Rowden White Trust.


    ACKNOWLEDGMENTS
 
We thank Dr. J. Callaghan, Prof. J. W. Goding, and Prof. J. Mercer and Dr. D. Strausak for supplying materials as described in MATERIALS AND METHODS.

Present addresses: I. Voskoboinik, Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne, Victoria 8006, Australia; M. J. Petris, Depts. of Biochemistry and Nutritional Sciences, University of Missouri, Columbia, MO 65211.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Camakaris, Dept. of Genetics, Univ. of Melbourne, Parkville, VIC 3010, Australia (E-mail: j.camakaris{at}unimelb.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
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