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Human Molecular Genetics Pages 1293-1300  

Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)
Introduction
Results
   Transfection of MNK cDNA construct and selection of stable MNK-expressing CHO clones
   Analysis of human MNK protein levels in CHO clones
   Copper resistance characteristics of MNK-expressing clones
   Subcellular localization and copper-dependent trafficking of the human MNK protein in CHO-K1 cells
Discussion
Materials And Methods
   Generation and cloning of the MNK cDNA construct
   Cell culture
   Stable transfection
   Antibody production
   Western blot analysis
   MTT cytotoxicity assays
   Indirect immunofluorescence
   Electron microscopy
Acknowledgements
References

Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)

Functional analysis and intracellular localization of the human Menkes protein (MNK) stably expressed from a cDNA construct in Chinese hamster ovary cells (CHO-K1)

Sharon La Fontaine1, Stephen D. Firth1, Paul J. Lockhart1, Hilary Brooks2, Robert G. Parton3, James Camakaris2, Julian F. B. Mercer1,*

1The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital and 2Department of Genetics, University of Melbourne, Parkville, Victoria 3052, Australia and 3Centre for Microscopy and Microanalysis, Department of Physiology and Pharmacology and Centre for Molecular and Cellular Biology, University of Queensland, St Lucia, Queensland 4072, Australia

Received March 30, 1998; Revised and Accepted May 22, 1998

The Menkes protein (MNK or ATP7A) is an important component of the mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans-Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.

INTRODUCTION

Menkes disease is an X-linked inherited disorder of copper transport (1) and results from mutations in the ATP7A gene, which was recently isolated by positional cloning techniques (2-4). The primary defect in patients with Menkes disease is copper deficiency, which results from defective copper uptake from the small intestine and inadequate copper delivery to other tissues. The biochemical and clinical manifestations of the disease can be correlated with a deficiency of critical copper-requiring enzymes (1).

Copper homeostasis is an important biological process because it ensures the availability of copper in required amounts to all cells of all organisms for incorporation into vital enzymes, such as lysyl oxidase and cytochrome c oxidase (1). Copper transport in cells can be viewed essentially as three processes: (i) copper uptake (5,6); (ii) intracellular distribution and utilization; and (iii) copper export (7). There are many protein components involved in each of these processes (8). The protein that is defective in Menkes disease, ATP7A or MNK, is one such protein and is emerging as a key component of the copper transport pathway. Based on its amino acid sequence, MNK is an integral membrane protein that belongs to the P-type ATPase family of cation-transporting proteins. Recently, several Chinese hamster ovary (CHO)-K1 cell lines were selected for resistance to copper. In these cell lines, MNK was overexpressed due to amplification of the endogenous ATP7A gene (9), appeared to be primarily located in the trans-Golgi network (TGN) and could be induced to traffick towards the plasma membrane by increased extracellular copper levels (7). The TGN localization of MNK was recently supported by immunofluorescence analysis of CHO-K1 cells, normal human fibroblasts (10) and HeLa cells (11), but copper-induced trafficking of the endogenous MNK protein in these cells, which had not been selected for copper resistance or overexpression of MNK, was not demonstrated. The presence of MNK in the TGN and at the plasma membrane is consistent with a role for this protein in both intracellular copper transport and copper export.

To investigate the mechanism of the copper-induced trafficking of MNK and copper translocation across cellular membranes, a cDNA construct comprising the MNK coding sequence is necessary. A stable cDNA clone that contained the entire MNK coding region was difficult to obtain despite many attempts using several high copy number plasmid vectors and Escherichia coli strains. The low copy number vectors pWSK29 and pWKS30 (12) enabled stable propagation of the MNK cDNA in E.coli and were used to generate low copy number mammalian expression vectors (13). The validity of these vectors for constitutive and inducible expression of the cDNA-derived MNK transcript arising from the cytomegalovirus (CMV) and sheep metallothionein 1a (sMT-1a) promoters, respectively, was demonstrated in a transient expression system using COS-7 cells (13).

However, elucidation of the function of MNK requires the development of stable cell lines that express full-length MNK from a cDNA construct, followed by cell lines that contain in vitro mutated MNK constructs. This study describes the generation of several stable CHO cell lines that express different levels of the cDNA-derived human MNK protein, which conferred on the cells a copper resistance phenotype. In these cell lines and the parental CHO-K1 cell line, immunocytochemical analyses indicated that MNK was located in the trans-Golgi region of the cell and was induced to relocalize to the cytoplasm and the plasma membrane in the presence of increased extracellular copper. This result was confirmed by electron microscopy, which also showed for the first time the association between MNK and vesicular structures within the cell. We also report copper-induced trafficking of MNK in cells (CHO-K1) that have not been selected for copper resistance or MNK overexpression and functional expression in mammalian cells of MNK protein from a cDNA construct. This study represents an important step towards further structure-function studies of MNK, which will provide important insights into its role in cellular copper transport.

RESULTS

Transfection of MNK cDNA construct and selection of stable MNK-expressing CHO clones

The plasmid pCMB50 (13) was transfected into CHO-K1 cells. Transient expression of MNK protein from this plasmid construct but not from the vector control plasmid, pCMB43, was previously demonstrated in COS-7 cells (13). The hypothesis that MNK is a copper-transporting P-type ATPase, together with the observation that overexpression of MNK was associated with copper resistance in the CHO-K1-derived cell lines CUR2 and CUR3 (7,9) provided the rationale for using a brief period of copper selection to enrich for MNK-expressing transfectant clones. Note that this period of copper selection consisted of ~5 weeks, compared with the period of 6-12 months required to isolate the CUR2/CUR3 cell lines. Four clones were isolated and designated 900-5#4, 600-5#3, 600-6#3 and 600-6#7. Untransfected control cells did not survive the G418 and copper selection regimes. Southern blot analysis indicated that the MNK cDNA construct was present in multiple copies in all clones and that there were significant differences in its copy number between the transfected cell lines (data not shown). Northern blot analysis of RNA from the four clones revealed a specific 4.8 kb plasmid-derived transcript, which included 0.2 kb of 5[prime]-untranslated vector-derived sequence in addition to the 4.6 kb MNK coding region plus 3[prime]-non-coding sequence (data not shown).

Analysis of human MNK protein levels in CHO clones

Western blot analysis of whole cell protein extracts from the clones and control cell lines was carried out using antibodies directed against the MNK N-terminal metal binding region. The 178 kDa MNK protein was detected at the expected levels in CHO-K1, CUR2 and CUR3 cell extracts (Fig. 1). The amount of MNK protein expressed in the transfected clones varied, with clone 600-5#3 expressing MNK at a level similar to CUR3, while clones 900-5#4, 600-6#3 and 600-6#7 expressed MNK at approximately CUR2 levels, as determined over several experiments. In addition, the MNK product of the transfected clones was ~5 kDa smaller than the MNK band in CHO-K1, CUR2 and CUR3 extracts. This result was not due to any sequence abnormalities, since the sequence of the 4.6 kb MNK cDNA was determined and found to be completely normal. The protein expression level of the four clones remained stable over 45 passages, confirming stable integration and expression of the MNK cDNA in these clones.


Figure 1. Western blot analysis of transfectant clones. Whole cell protein extracts were prepared from the four clones, CUR3, CUR2 and CHO-K1 cells, fractionated by SDS-PAGE (7% gel) and transferred to nitrocellulose filters. The filters were probed with an affinity-purified preparation of anti-MNK antibodies directed against the MNK N-terminus (7,9), while the secondary antibody consisted of horseradish peroxidase-conjugated sheep anti-rabbit IgG (AMRAD Biotech). Protein detection was carried out using the Chemiluminescent POD substrate (Boehringer Mannheim). The positions of the molecular weight markers (Bio-Rad) are indicated on the left in kilodaltons (kDa). The position of the endogenous MNK protein is also indicated by an arrow.

Copper resistance characteristics of MNK-expressing clones

For all of the clones, cell survival in increasing amounts of copper was assessed and compared with that of CHO-K1, CUR2 and CUR3, using an MTT colorimetric assay (Fig. 2). At copper concentrations of up to 300-400 µM, survival of the transfected clones was approximately equivalent to that of CUR3. Thereafter, the survival of 600-5#3 parallelled CUR3 survival (Fig. 2D), while the copper resistance of 900-5#4, 600-6#3 and 600-6#7 centred around that of CUR2 (Fig. 2A-C).


Figure 2. Copper resistance characteristics of MNK-expressing clones. The percentage of cells surviving in increasing concentrations of copper was assessed using an MTT cytotoxicity assay. The survival of individual clones ([solid diamond]) was compared with that of CHO-K1 ([Delta]), CUR2 ([circle]) and CUR3 ([square]). (A) 900-5#4; (B) 600-6#3; (C) 600-6#7; (D) 600-5#3. Each data point represents the average of duplicate measurements.

Subcellular localization and copper-dependent trafficking of the human MNK protein in CHO-K1 cells

To determine the subcellular location of the plasmid-derived human MNK protein in CHO-K1 cells, immunofluorescence microscopy was carried out on the transfected clones as well as on the parental CHO-K1 cells, all of which were cultured in basal medium for 48 h. All cell lines showed fluorescent staining in the perinuclear region which was consistent with a TGN localization. The fluorescence intensity corresponded with the level of MNK protein expression in each cell line (Fig. 3A-D). Results obtained with clone 600-6#7 are not shown but were similar. There was also punctate staining within the cytoplasm, indicative of vesicle-like structures. Perinuclear staining was clearly evident in the parental CHO-K1 cell line (Fig. 3A) and was diminished in CHO-K1 and CUR2 cells after pre-incubation of the antibody with the His-tagged MNK fusion protein used for immunization and when pre-immune serum was used (data not shown). The TGN localization of the recombinant MNK was further supported by contraction of the fluorescence signal to form a compact, juxtanuclear signal after incubation of cells in the presence of the fungal metabolite brefeldin A followed by staining with antibodies to either MNK or a resident TGN protein, TGN38, as a control (data not shown).


Figure 3. Subcellular localization and effect of copper on the intracellular distribution of MNK. CHO-K1 (A and E), 900-5#4 (B and F), 600-6#3 (C and G) and 600-5#3 (D and H) were cultured for 48 h in basal medium and then incubated in the absence (A-D) or presence of 189 µM copper (CuCl2) (E-H) for 2-5 h at 37°C. The cells were fixed and stained with a sodium sulphate-precipitated preparation of anti-MNK antibodies, followed by FITC-conjugated sheep anti-rabbit IgG antibodies.

To assess the effect of copper on the intracellular location of MNK, cells cultured in basal medium for 48 h were incubated in medium containing 189 µM copper for 2-5 h and then subjected to immunofluorescence analysis. In all cell lines, including the parental CHO-K1 cell line, treatment with copper caused a decrease in the intensity of the perinuclear signal, with a more dispersed, punctate staining pattern within the cytoplasm (Fig. 3A-H). Staining at the periphery of cells was most apparent in the transfected clones (Fig. 3F-H) and indicated the presence of MNK at the plasma membrane. These results demonstrated that both the endogenous and cDNA-derived MNK protein was able to relocalize in response to copper.

The distribution of MNK, in both the absence and presence of added copper, was further examined at the ultrastructural level using anti-MNK antibodies on ultrathin frozen sections of clone 600-5#3 cells. In untreated cells, specific labelling was observed in the perinuclear area of the cell in small tubular and vesicular profiles associated with the Golgi complex, consistent with a TGN localization (Fig. 4A-C). The bulk of the labelling was associated with small (~50-80 nm), uncoated profiles and not with the neighbouring Golgi cisternae. Negligible labelling was associated with other cellular organelles, such as the endoplasmic reticulum (for example, surrounding the nucleus in Fig. 4B). The plasma membrane also showed negligible labelling for the MNK protein: note the lack of surface labelling in the two cells shown in Figure 4A. In contrast with the control cells, after copper treatment for 2 h every cell profile showed specific labelling for the MNK protein on the plasma membrane (Fig. 4). The labelling was distributed over the entire surface, but was particularly concentrated over surface projections in peripheral areas of the cell. There was no particular concentration of labelling observed in surface clathrin-coated pits. Intracellular labelling for the MNK protein was still observed after copper treatment, which was consistent with an incomplete redistribution to the cell surface under these conditions, as observed by immunofluorescence. In addition to the labelling of putative TGN elements, labelling of tubular profiles in the cell periphery was often observed in copper-treated cells and possibly corresponded to endosomal elements.


Figure 4. Ultrastructural localization of the MNK protein in untreated and copper-treated cells of transfectant clone 600-5#3. Ultrathin frozen sections of 600-5#3 cells were labelled with an affinity-purified preparation of anti-MNK antibodies, followed by 15 nm protein A-gold. (A-C) Representative images of cells that were cultured in basal medium without added copper; (D) the surface of a cell treated for 2 h with CuCl2 prior to fixation. (A) Low power overview showing the general distribution of MNK labelling. The plasma membranes of two cells are closely apposed (p, plasma membrane); note the lack of plasma membrane labelling for MNK and labelling of vesicular and tubular profiles in close proximity to the Golgi cisternae (G) of each cell. (B and C) Higher magnification views of the Golgi region. Labelling is mainly associated with uncoated vesicular elements in close proximity to the Golgi cisternae. The endoplasmic reticulum surrounding the nucleus (N) shows negligible labelling. The labelled elements are often in close proximity to putative clathrin-coated buds (arrowheads). The Golgi cisternae show low labelling. (D) Representative image of copper-treated cells immunolabelled under identical conditions. Note that the number of gold particles associated with the plasma membrane (arrowheads) is greatly increased as compared with control cells [compare with (A)]. Labelling is also observed in peripheral tubular elements [e.g. double arrowheads in (D)], which may correspond to endosomal elements. Bars, 100 nm.

DISCUSSION

This study describes the generation of cell lines that stably express a functional human MNK protein from a genomically integrated cDNA construct. Significant difficulties were encountered by our group and other researchers (14) in generating a full-length MNK cDNA construct that could be stably maintained in E.coli, but in our case these difficulties were overcome by using a low copy number plasmid vector (13). The levels of MNK protein expressed by the clones correlated with their degree of copper resistance and preliminary data indicated that the clones also had an increased ability to efflux copper (data not shown). In the CUR cells copper resistance was associated with amplification of the Menkes gene, MNK, which led to overproduction of MNK and an increased capacity of the cells to efflux copper (9). The fact that the transfectant clones were also resistant to copper due to enhanced copper efflux demonstrated that the overproduced MNK product in these cells was functional and that overexpression of MNK, and not some other factor, was the major contributor to the copper resistance phenotype in both the clones and CUR cells. Note that this study demonstrated the validity and utility of a modified cytotoxic assay (using MTT) for assessing the copper resistance status of cells, which will prove valuable for future studies involving MNK and other copper transport proteins, such as the related Wilson disease protein (WND).

The MNK protein expressed from the cDNA construct was ~5 kDa smaller than the CHO-K1-derived MNK (Fig. 2) and~5 kDa larger than the MNK product that was transiently expressed from the same plasmid construct in COS-7 cells (13). We have also demonstrated that endogenously expressed MNK in normal human fibroblasts is smaller than hamster MNK, but of the same size as MNK expressed from the cDNA construct in both CHO cells and in human Menkes fibroblasts (data not shown). It was recently demonstrated that MNK is glycosylated and that deglycosylation reduced the size of the MNK band on a western blot from 178 to 170 kDa (our unpublished data; 11). It is likely that the observed size differences between human MNK (cDNA-derived and endogenous) and hamster MNK are due to a combination of amino acid differences between the two proteins (~10%; M.J. Petris, personal communication) and glycosylation differences, which may result from sequence differences and recognition of different glycosylation signals by different cell types. The size and possible glycosylation differences between the endogenous and cDNA-derived MNK did not appear to affect the function of MNK produced from the cDNA construct in CHO cells.

Immunofluorescence analysis of the transfectant clones supported the TGN localization of MNK. In addition, MNK relocalization was detectable at significantly reduced extracellular copper concentrations ([ge]20 µM; data not shown) and time course experiments showed that the copper-induced movement of MNK occurred with similar kinetics to that of CUR cells (data not shown). Localization of MNK to the TGN in CHO-K1 cells, human fibroblasts and HeLa cells has been confirmed by other groups (10,11). The current study, in addition, demonstrated that copper-induced relocalization of MNK also occurs in the parental CHO-K1 cells. Therefore, the TGN localization and copper-induced trafficking of MNK are likely to represent normal physiological properties of cells and are not confined to copper-resistant cells or those that overexpress the MNK protein. These observations, together with the fact that in both the CUR cells and the transfected clones the appearance and maintenance of MNK at the plasma membrane was dependent on copper, suggested that the presence of MNK at the plasma membrane in high copper concentrations did not represent an artifact of MNK overexpression. However, a more sensitive anti-MNK antibody or assay would be required to conclusively demonstrate the presence of endogenously expressed MNK at the plasma membrane in the presence of excess copper. In the transfected clones and the CUR cell lines, the increase in MNK protein expression led to a corresponding increase in the degree of copper resistance of the cells, which was shown to be due to enhanced copper efflux from and reduced accumulation of copper in these cells (data not shown; 9). This correlation between protein expression and function in cell lines generated by different means provided further evidence that the data obtained were unlikely to represent artifacts associated with these in vitro systems for analysing MNK function. More likely, the copper-dependent redistribution of MNK represents a general and important component of the cellular copper homeostatic mechanism.

The TGN localization of MNK and its redistribution, via vesicles, to the plasma membrane was confirmed for the first time by immunogold electron microscopic analysis of clone 600-5#3. MNK was found in the perinuclear area of the cell, associated with tubular and vesicular elements identified as the TGN, and was not associated with the endoplasmic reticulum or the Golgi stack. This observation suggested that incorporation of copper into copper-dependent enzymes occurs exclusively in the TGN. At basal copper levels there was negligible plasma membrane labelling. Significantly, at elevated copper levels labelling of the plasma membrane was increased, although not in association with clathrin-coated pits. Therefore, the ultrastructural analysis of MNK localization was entirely consistent with the immunofluorescence data and also demonstrated its association with vesicles within the cell. The identification of MNK in association with cellular vesicles is an important step towards elucidating the copper transport pathway, because it lends support to the hypotheses that either MNK transports copper into vesicles en route to the plasma membrane and the copper is then released from the cell on fusion of the vesicles with the membrane or that the vesicles simply serve to carry MNK to the plasma membrane from where copper is effluxed from the cell. These studies will pave the way for more detailed analyses, which are required to fully define the pathway of MNK in the cell.

The localization of MNK to the TGN and the copper-induced movement of this protein are consistent with previous suggestions of a role for MNK in intracellular copper transport (1,8,15) and enable the cell to regulate the cytoplasmic concentrations of this essential, but potentially toxic, cation. The TGN is located at the trans face of the Golgi apparatus and is the compartment in which secretory proteins are sorted into different types of vesicles for transport to different locations within the cell (16). At low to normal intracellular copper levels MNK is primarily located in the TGN, where it is thought to provide copper to secreted, copper-dependent enzymes such as lysyl oxidase. When intracellular copper levels increase due to elevated extracellular copper, there is a net increase in MNK at the plasma membrane, resulting in increased copper efflux. The location of MNK at the plasma membrane, the reduced intracellular copper accumulation by the cell lines that overexpress MNK and the copper accumulation phenotype of cells from Menkes patients and the mouse models of Menkes disease are consistent with a further role for MNK in the export of excess copper from the cell via an active efflux mechanism.

It was proposed that copper-induced relocalization of MNK formed the basis of cellular copper homeostasis (7), but the molecular basis of MNK trafficking and the signals which determine its intracellular localization are still to be established. A striking feature of MNK and WND is the presence of six conserved motifs in the N-terminal region of these molecules that were recently shown to bind copper (17). It is unlikely that MNK and WND require all six copper binding sites for copper transport activity, since their homologues in yeast and bacteria have only one or two such motifs (18,19). We have previously proposed that the metal binding region may constitute a copper-sensing domain (7), i.e. the level of saturation of the metal binding sites with copper may influence the rate of the exocytic and/or endocytic movement of MNK, via alterations in MNK conformation and protein interactions at the TGN and plasma membrane. The system for functional expression of MNK described in this report will enable future studies using in vitro mutagenesis to test this hypothesis. In addition, the identification of other functional motifs involved in the intracellular location, trafficking and copper transport activity of MNK will be possible. This system will also permit an assessment of the effects of Menkes patient mutations on MNK function. Together, these data may eventually contribute to the development of improved therapeutic strategies for patients with Menkes disease.

MATERIALS AND METHODS

Generation and cloning of the MNK cDNA construct

Total RNA was prepared from a normal human fibroblast cell line, S78, as previously described (20). A cDNA fragment encompassing the 4.5 kb MNK coding region was generated initially as three fragments by RT-PCR using AMV reverse transcriptase (Boehringer Mannheim, Mannheim, Germany) and recommended protocols. The primers employed for RT-PCR and subsequent second round PCR amplification were designed from the previously published sequence of the MNK cDNA (3,4). All DNA manipulations were carried out using standard techniques (21).

Fragment 1 comprised 1.8 kb at the 5[prime]-end of the coding region and was generated from cDNA that was produced using the primer Menkes 2B (5[prime]-CATGTGTTGCGCAGATAAGC-3[prime]). This cDNA was used as a template for PCR with primers Menkes 11A (5[prime]-CGCGCATCCCCGGAAATCAAAATGGACCCAAG-3[prime]) and Menkes 13 (5[prime]-CGTCTCCATTGTCTTATTTCTCG-3[prime]). Menkes 11A removed the original BamHI site around the putative ATG start codon and introduced a BamHI site at the 5[prime]-end of the resultant fragment without altering the amino acid sequence of the coding region. The resulting PCR product was cloned into pBluescript KS II (Stratagene, CA), followed by a further round of PCR with the resultant clone as the template and the primers Menkes 11A and Menkes 14 (5[prime]-GTTGCCCCGCGGTGTTTTGTGAGACTAG-3[prime]). The latter primer introduced a KspI site at the 3[prime]-end, so that the PCR product was digested with BamHI/KspI and cloned into pBluescript KS II to create the plasmid pCMB6.

Fragment 2 comprised 1.54 kb of the central region of the MNK coding sequence and was generated from cDNA produced using the primer Menkes 2D (5[prime]-TCAATGGCTACATCTGTGCC-3[prime]). This cDNA was used as a template for PCR with the primers ME-1F (5[prime]-ATTTAAGGCGGGAAGAAGGAA-3[prime]) and MMNK- 6 (5[prime]-CAATGAGGACTTTGTGCTGC-3[prime]), the product was digested with BamHI/KpnI and then cloned into pBluescript KS II digested with the same restriction enzymes. PCR was carried out using the resultant clone as a template together with the Menkes 15 primer (5[prime]-TGGCACCGCGGGATTCTATACTGCTCCG-3[prime]) and the M13/pUC reverse sequencing primer (Boehringer Mannheim, Mannheim, Germany). Menkes 15 was designed to simultaneously remove the second internal BamHI site and introduce a KspI site, while maintaining the correct amino acid sequence. The product was then digested with KspI/KpnI and cloned into pBluescript KS II. This clone was designated pCMB7.

Fragment 3 was generated from cDNA that was produced using the primer Menkes 6B (5[prime]-TGAAAATTAGCCGGGTGTG-3[prime]). The product that resulted from PCR using this cDNA and the primers Menkes 2C (5[prime]-CACTCCAACTGCTGTGATGG-3[prime]) and Menkes 12 (5[prime]-AACGCAACTCAAATAGGATCC-3[prime]) was digested with KpnI/BamHI and cloned into pBluescript KS II to generate plasmid pCMB8. The Menkes 12 primer permitted the inclusion of 120 bp of sequence downstream of the termination codon and introduced a BamHI site at the 3[prime]-end.

A 4.6 kb BamHI fragment comprising fragments 1-3 was generated and cloned into the BamHI site of vector pWSK29 (12) to generate plasmid pCMB19. The sequence of this cDNA fragment, which contained 18 bp upstream of the putative ATG start codon and 120 bp downstream of the stop codon, was confirmed on both DNA strands using the chain termination DNA sequencing method and the Sequenase Version 2.0 DNA Sequencing Kit (US Biochemical, OH). The cDNA was then excised as a SpeI-SalI fragment and cloned into the NheI and SalI sites of expression vector pCMB43 (13) to generate plasmid pCMB50.

Cell culture

Cells were maintained in basal medium (1.5 µM Cu) which consisted of Dulbecco's modified Eagle's medium (Trace Biosciences, NSW, Australia) or Eagle's basal medium (Trace Biosciences, NSW, Australia), supplemented with 10% fetal calf serum (Trace Biosciences, NSW, Australia), L-proline at a final concentration of 20 mM and 0.2% (w/v) bicarbonate.

Stable transfection

Plasmid DNA for transfection was purified by caesium chloride density gradient centrifugation and linearized with XbaI prior to transfection. CHO-K1 cells were trypsinized and counted. Approximately 5 × 106 cells were transfected with 13 µg DNA by electroporation (GenePulser; Bio-Rad, NSW, Australia) at a field strength of 0.75 kV/cm (0.3 kV and 500 µF) using GenePulser cuvettes with a 0.4 cm electrode gap (Bio-Rad). Cells were allowed to recover for 24 h, after which selection in 600 µg/ml G418 (Life Technologies, NY) was initiated and continued for 15 days. From day 10 following the selection of transfectants in G418-containing medium, MNK-expressing clones were enriched from the pool of G418-resistant clones by addition of 250-350 µM copper to the medium over a 4 week period. From this copper-selected pool of transfectants, a proportion of cells were subjected to further enrichment in either 600 or 900 µM copper over a period of 10 days, to ensure complete loss of non-MNK-expressing clones. Twelve clones that survived in medium containing 600 µM copper and 12 that survived 900 µM copper were isolated after allowing cells to grow and form colonies in normal low copper medium. Four of these clones were chosen for further analysis and were clonally purified.

Antibody production

Primary MNK antibodies were prepared by immunizing rabbits with a fusion protein that was generated and purified using the QIAexpress System (Qiagen, Hilden, Germany) and comprised the N-terminal 590 amino acids of MNK fused to a 6×His tag. The MNK IgG fraction was prepared by sodium sulphate precipitation (22) and a proportion of this preparation was subjected to further affinity purification as previously described (22).

Western blot analysis

Preparation of cell extracts, fractionation of 30 µg total cellular protein by SDS-PAGE, transfer to nitrocellulose (Schleicher and Schuell, Northeim, Germany) and protein detection by chemiluminescence were carried out essentially as previously reported (13). The primary antibody consisted of an affinity-purified preparation of anti-MNK antibodies that was previously prepared (7,9). The secondary antibody consisted of horseradish peroxidase-conjugated sheep anti-rabbit IgG (AMRAD Biotech, Victoria, Australia).

MTT cytotoxicity assays

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cytotoxocity assays for assessing cell survival in increasing concentrations of copper were carried out by a modification of a method described by Mosmann (23). Cells were seeded into 96-well trays at a density that would ensure 100% confluency on the day of harvest. After 24 h, copper was added at appropriate concentrations to duplicate wells and incubation was continued for a further 3 days. The copper-containing growth medium was replaced after 2 days with phosphate-buffered saline containing 50 µg/ml MTT. After incubation for 4 h at 37°C, the MTT-containing medium was removed and the cells were solubilized with acidified isopropanol (200 µl/well). Live cells reduce the MTT dye to a purple colour, so that for each well the absorbance at 600 nm (A600) was read using an EIA plate reader (model no. 2550; Bio-Rad). The reference point was taken as the average reading of four wells that contained cells growing in basal medium and corresponded to 100% survival. For all other wells, the A600 taken as a percentage of the reference point represented the percent survival of cells in copper-containing medium.

Indirect immunofluorescence

Immunofluorescence analysis of CHO-K1 cells and derivatives was carried out essentially as described previously (7). In general, cells were cultured on 13 mm glass coverslips for 48 h. Where appropriate, CuCl2 was added to the growth medium to a final concentration of 189 µM for 2-5 h, unless otherwise specified.

The primary antibody consisted of the sodium sulphate-precipitated preparation of anti-MNK antibodies. The secondary antibodies consisted of fluorescein isothiocyanate (FITC)-conjugated sheep antibodies to rabbit IgG (AMRAD Biotech). Antibody stained cells were visualized using a Zeiss IM-35 inverted microscope, HBO50W lamp, filter set 09 plus accessory filter KP560 for FITC and a 100× oil objective. Photographs were taken on Kodak Ektachrome P1600 film with a Nikon F-601M camera at a magnification of 3200. Exposure times were set manually.

Electron microscopy

Cells of clone 600-5#3 (either untreated or treated for 2 h with 200 µM CuCl2) were fixed in 8% paraformaldehyde in 0.1 M phosphate buffer, pH 7.35, for 1 h at room temperature. They were then washed with 0.2 M phosphate buffer, scraped from the culture dish and pelleted at 10 000 r.p.m. in a microfuge. The cells were then resuspended in warm gelatin (10% in phosphate buffer) and again pelleted at maximum speed in the microfuge. After cooling, the gelatin-embedded cells were infiltrated with PVP-sucrose overnight at 4°C and processed for frozen sectioning as described previously (24). Ultrathin frozen sections (60-80 nm) were labelled, stained and viewed (Jeol 1010; Centre for Microscopy and Microanalysis, University of Queensland) according to published techniques (24). The antibody consisted of the affinity-purified preparation of anti-MNK antibodies.

ACKNOWLEDGEMENTS

We wish to thank Jennifer Paynter and Rosario Reyes for their most valuable technical assistance and Andrew Grimes and Michael Petris for helpful scientific discussions. We also would like to thank Rob Gould and Margaret Lindsay (University of Queensland) for assistance with immunolabelling and David James (University of Queensland) for encouragement throughout this study. This work was supported by funding from the National Health and Medical Research Council and the International Copper Association. S.L. is a BHP Postdoctoral Fellow funded by BHP Pty Ltd.

REFERENCES

1. Danks, D.M. (1995) The metabolic and molecular basis of inherited disease. In Scriver, C.R., Beaudet, A.L., Sly, W.M. and Valle, D. (eds), Disorders of Copper Transport. McGraw-Hill, New York, NY, pp. 2211-2235.

2. Chelly, J., Tümer, Z., Tønnesen, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N. and Monaco, A.P. (1993) Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nature Genet., 3, 14-19. MEDLINE Abstract

3. Mercer, J.F.B., Livingston, J., Hall, B.K., Paynter, J.A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemenack, D. and Glover, T.W. (1993) Isolation of a partial candidate gene for Menkes disease by postional cloning. Nature Genet., 3, 20-25. MEDLINE Abstract

4. Vulpe, C., Levinson, B., Whitney, S., Packman, S. and Gitschier, J. (1993) Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Genet., 3, 7-13. MEDLINE Abstract

5. Dancis, A., Yuan, D.S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J. and Klausner, R.D. (1994) Molecular characterization of a copper transport protein in S. cerevisiae: an unexpected role for copper in iron transport. Cell, 76, 393-402. MEDLINE Abstract

6. Zhou, B. and Gitschier, J. (1997) hCTR1: a human gene for copper uptake identified by complementation in yeast. Proc. Natl Acad. Sci. USA, 94, 7481-7486. MEDLINE Abstract

7. Petris, M.J., Mercer, J.F.B., Culvenor, J.G., Lockhart, P., Gleeson, P.A. and Camakaris, J. (1996) Ligand-regulated transport of the Menkes copper P-type ATPase efflux pump from the Golgi apparatus to the plasma membrane: a novel mechanism of regulated trafficking. EMBO J., 15, 6084-6095. MEDLINE Abstract

8. Vulpe, C.D. and Packman, S. (1995) Cellular copper transport. Annu. Rev. Nutr., 15, 293-322. MEDLINE Abstract

9. Camakaris, J., Petris, M.J., Bailey, L., Shen, P., Lockhart, P., Glover, T.W., Barcroft, C.L., Patton, J. and Mercer, J.F.B. (1995) Gene amplification of the Menkes (MNK; ATP7A) P-type ATPase gene of CHO cells is associated with copper resistance and enhanced copper efflux. Hum. Mol. Genet., 4, 2117-2123. MEDLINE Abstract

10. Dierick, H.A., Adam, A.N., Escara-Wilke, J.F. and Glover, T.W. (1997) Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans Golgi network. Hum. Mol. Genet., 6, 409-416. MEDLINE Abstract

11. Yamaguchi, Y., Heiny, M.E., Suzuki, M. and Gitlin, J.D. (1997) Biochemical characterization and intracellular localization of the Menkes disease protein. Proc. Natl Acad. Sci. USA, 93, 14030-14035.

12. Wang, R.F. and Kushner, S.R. (1991) Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene, 100, 195-199. MEDLINE Abstract

13. La Fontaine, S., Firth, S.D., Lockhart, P.J., Paynter, J.A. and Mercer, J.F.B. (1998) Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein. Plasmid, 39, 245-251.

14. Francis, M.J., Jones, E., Levy, E.R., Chelly, J. and Monaco, A.P. (1996) Functional analysis of the Menkes disease gene. Am. J. Hum. Genet., 59 (suppl.), A149.

15. Herd, S.M., Camakaris, J., Christofferson, R., Wookey, P. and Danks, D.M. (1987) Uptake and efflux of copper-64 in Menkes'-disease and normal continuous lymphoid cell lines. Biochem. J., 247, 341-347. MEDLINE Abstract

16. Griffiths, G. and Simons, K. (1986) The trans Golgi network: sorting at the exit site of the Golgi complex. Science, 234, 438-443. MEDLINE Abstract

17. Lutsenko, S., Petrukhin, K., Cooper, M.J., Gilliam, C.T. and Kaplan, J.H. (1997) N-Terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat. J. Biol. Chem., 272, 18939-18944. MEDLINE Abstract

18. Bull, P.C. and Cox, D.W. (1994) Wilson disease and Menkes disease: new handles on heavy-metal transport. Trends Genet., 10, 246-252. MEDLINE Abstract

19. Solioz, M. and Vulpe, C. (1996) CPX-type ATPases: a class of P-type ATPases that pump heavy metals. Trends Biochem. Sci., 21, 237-241. MEDLINE Abstract

20. Paynter, J.A., Camakaris, J. and Mercer, J.F.B. (1990) Analysis of hepatic copper, zinc, metallothionein and metallothionein-Ia in the developing sheep. Eur. J. Biochem., 190, 149-154. MEDLINE Abstract

21. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

22. Johnstone, A. and Thorpe, R. (1987) Immunocytochemistry in Practice. Blackwell Scientific, Oxford, pp. 50-54.

23. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods, 65, 55-63. MEDLINE Abstract

24. Parton, R.G., Way, M., Zorzi, N. and Stang, E. (1997) Caveolin-3 associates with developing T-tubules during muscle differentiation. J. Cell Biol., 136, 137-154. MEDLINE Abstract

*To whom correspondence should be addressed at: Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Rusden Campus, 662 Blackburn Road, Clayton, Victoria 3168, Australia. Tel: +61 3 9244 7413; Fax: +61 3 9244 7290; Email: jmercer@deakin.edu.au

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