Intracellular localization and loss of copper ... - Semantic Scholar

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Sharon La Fontaine1,+, Stephen D. Firth1,+, Paul J. Lockhart1, Hilary Brooks2, James. Camakaris2,§ and ..... Lush post-graduate scholarship. REFERENCES. 1.
© 1999 Oxford University Press

Human Molecular Genetics, 1999, Vol. 8, No. 6 1069–1075

Intracellular localization and loss of copper responsiveness of Mnk, the murine homologue of the Menkes protein, in cells from blotchy (Moblo) and brindled (Mobr) mouse mutants Sharon La Fontaine1,+, Stephen D. Firth1,+, Paul J. Lockhart1, Hilary Brooks2, James Camakaris2,§ and Julian F. B. Mercer1,3,§,¶ 1The

Murdoch Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia, 2Department of Genetics, University of Melbourne, Parkville, Victoria 3052, Australia and 3Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, Victoria 3125, Australia Received January 11, 1999; Revised and Accepted March 15, 1999

Menkes disease is an X-linked copper deficiency disorder that results from mutations in the ATP7A (MNK) gene. A wide range of disease-causing mutations within ATP7A have been described, which lead to a diversity of phenotypes exhibited by Menkes patients. The mottled locus (Mo, Atp7a, Mnk) represents the murine homologue of the ATP7A gene, and the mottled mutants exhibit a diversity of phenotypes similar to that observed among Menkes patients. Therefore, these mutants are valuable models for studying Menkes disease. Two of the mottled mutants are brindled and blotchy and their phenotypes resemble classical Menkes disease and occipital horn syndrome (OHS) in humans, respectively. That is, the brindled mutant and patients with classical Menkes disease are severely copper deficient and have profound neurological problems, while OHS patients and the blotchy mouse have a much milder phenotype with predominantly connective tissue defects. In this study, in an attempt to understand the basis for the brindled and blotchy phenotypes, the copper transport characteristics and intracellular distribution of the Mnk protein were assessed in cultured cells from these mutants. The results demonstrated that the abnormal copper metabolism of brindled and blotchy cells may be related to a number of factors, which include the amount of Mnk protein, the intracellular location of the protein and the ability of Mnk to redistribute in elevated copper. The data also provide evidence for a relationship

between the copper transport function and copperdependent trafficking of Mnk. INTRODUCTION Significant insights into the biology of copper transport have been gained from the identification and isolation of the genes ATP7A (MNK) (1–3) and ATP7B (WND) (4–6), which are defective in the two inherited disorders of copper transport, Menkes and Wilson diseases, respectively, and more recently from analyses of the proteins encoded by these genes in both mammalian (7–14) and yeast systems (15,16). Menkes disease is an X-linked copper deficiency disorder due to reduced intestinal absorption of copper and defective transport of copper within the body, which leads to generalized copper insufficiency. The reduced activity of critical copper-requiring enzymes, such as lysyl oxidase, cytochrome c oxidase and dopamine-β-hydroxylase, among others, accounts for the biochemical and clinical features of this disease (17). Wilson disease is an autosomally inherited copper toxicosis disorder due to defective biliary excretion of copper which leads to hepatic copper accumulation and consequent liver failure (17). The Menkes (MNK) and Wilson (WND) proteins are transmembrane, P-type ATPase proteins that function to transport copper across cellular membranes (18). Both proteins are located in the trans-Golgi network (TGN) (7–10,12–14,16,18), where presumably they transport copper into the TGN lumen for incorporation into secreted cuproenzymes, such as ceruloplasmin in the case of WND, or lysyl oxidase in the case of MNK. In the presence of elevated extracellular copper, MNK is redistributed to the cytoplasm and plasma membrane from where excess copper is extruded from the cell (8,18). WND relocalizes to an intracellular compartment in response to

+Present address: Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University–Burwood Campus, Building L, 221 Burwood Highway, Burwood, Victoria 3125, Australia §These authors contributed equally to this work ¶To whom correspondence should be addressed at: Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University– Burwood Campus, Building L, 221 Burwood Highway, Burwood, Victoria 3125, Australia. Tel: +61 3 9251 7329; Fax: +61 3 9251 7328; Email: [email protected]

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Figure 1. Schematic representation of the nature and location of the brindled and blotchy mutations with respect to the Mnk protein. Highly conserved motifs within the protein are indicated by black or shaded boxes.

elevated copper (16) and recently it was shown that a mutant form of WND lost the ability to relocalize with increased copper levels (19). Animal models of Menkes and Wilson diseases continue to provide useful tools for extending our understanding of these diseases, the mechanisms that operate to maintain copper homeostasis at the level of the whole animal and for the development of more effective therapeutic strategies. The mottled locus (Mo, Atp7a, Mnk) represents the murine homologue of the ATP7A (MNK) locus (20). The Atp7a gene has been isolated and the encoded protein (Mnk) shows a high degree of similarity, based on sequence and predicted structure, to the human MNK protein (21,22). Mutations within the Atp7a gene lead to a similar diversity of phenotypes to that observed in patients with Menkes disease. Up to 23 alleles at the Mo locus have been identified (23) and 13 have been characterized at the molecular level (24–26). Among these are the mutations that give rise to the brindled (Mobr) and blotchy (Moblo) mouse mutants. The phenotype of the brindled mutant most closely resembles that of classical Menkes disease, in that affected male mice are severely copper deficient at birth, have severe neurological defects and die ~15–17 days after birth (20). The size and quantity of Atp7a mRNA in tissues from this mutant appeared to be normal (21,22). The mutation within the Atp7a

gene was found to be a deletion of 6 bp which leads to an inframe deletion of two amino acids (Ala799 and Leu800) from the region between the fourth transmembrane domain and the transduction domain of the protein (25,26). The blotchy mutant has a much milder phenotype consisting predominantly of connective tissue abnormalities, similar to patients with occipital horn syndrome (OHS), which is characteristic of lysyl oxidase deficiency (20). The causative mutations in both the blotchy mouse and a number of patients with OHS have been identified as splice site mutations within the Atp7a and ATP7A genes, respectively (27,28). Northern blot analysis of tissues from the blotchy mouse revealed the presence of a small amount of normal sized Atp7a mRNA transcript and two larger transcripts resulting from aberrant splicing (21,22). Therefore, the splice site mutation may allow the formation of small amounts of normal Atp7a mRNA. Thus, the brindled and blotchy mutants have very different phenotypes arising from mutations within the same gene. This study represents the first step towards elucidating the molecular basis for these different phenotypes. This report demonstrates that cells from the brindled and blotchy mice show abnormal copper accumulation and retention characteristics and that, unlike normal mouse fibroblasts, the Mnk protein detected in these cells does not redistribute in elevated copper levels. Therefore, based on the phenotypic effects of ATP7A

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Figure 3. Western blot analysis of normal, blotchy and brindled fibroblasts. Whole cell protein extracts were prepared from blotchy and brindled fibroblasts and control cell lines, which included the parental mouse cell line (Normal-2), an adult mouse fibroblast cell line (Normal-1) and a mouse epithelial cell line (Earle’s L cells). Approximately 20 µg of total cell protein was fractionated by SDS–PAGE (7.5% gel) and transferred to nitrocellulose filters. The filters were probed with anti-MNK antibodies directed against the MNK N-terminus (9). The secondary antibody consisted of horseradish peroxidaseconjugated sheep anti-rabbit IgG (AMRAD Biotech). Protein detection was carried out using the Chemiluminescent POD substrate (Boehringer Mannheim, Mannheim, Germany). The positions of the molecular weight markers (Bio-Rad, Hercules, CA) are indicated on the left in kDa.

Figure 2. Copper accumulation and retention by blotchy and brindled fibroblasts. (A) Graph showing the amount of copper that accumulated in cells after 2, 4, 8 and 16 h in medium containing trace amounts of 64Cu. Cell lines included are indicated. Values for accumulation represent the means ± standard error of three independent measurements. (B) Table showing the amount of copper retained in cells after incubation for 16 h in 64Cu-containing medium, followed by incubation for a further 8 h in 64Cu-free medium. Values for retention are expressed as a percentage of the amount of copper that accumulated in cells after 16 h and represent the means ± standard deviation of three independent measurements

mutations that cause Menkes disease, both at the level of cells and in whole animals, this study proposes a link between the copper transport function of MNK and its ability to redistribute with increased copper levels, and provides an explanation at a molecular level for the distinct mutant phenotypes. RESULTS Copper accumulation and retention by fibroblasts from brindled and blotchy mutants The causative mutation within Atp7a in the brindled mouse is an in-frame deletion of 6 bp that encode Ala799 and Leu800, which are located in the small cytoplasmic loop between the fourth and fifth transmembrane domains (25,26; Fig. 1). In the blotchy mouse, multiple Atp7a transcripts, in addition to one of normal size, have been detected previously and all were present at reduced levels (28). These abnormal transcripts appear to be caused by splice site mutations that result in

mRNA splicing abnormalities, such as skipping of the 92 bp exon 11, which leads to the creation of a stop codon at amino acid residue 794 (Fig. 1) and the production of a truncated and non-functional protein. To determine the effect of these mutations on the copper transport characteristics of fibroblasts derived from the brindled and blotchy mutants, copper transport studies were carried out on these cells as well as on fibroblasts from the normal parental mouse (Normal-2) from which the blotchy cell line originated. Cells were incubated in 64Cucontaining medium for 2, 4, 8 and 16 h, after which the amount of copper that had accumulated in cells was determined. Compared with normal fibroblasts, the brindled and blotchy cells accumulated significantly more copper over 16 h (Fig. 2A). In addition, the amount of 64Cu that was retained in cells after a further 8 h in 64Cu-free medium was significantly greater for the mutant cell lines compared with the normal fibroblasts (Fig. 2B). There was no significant difference between the brindled and blotchy cells in the amount of copper accumulated or retained. Mnk protein levels and intracellular distribution in brindled and blotchy fibroblasts Using anti-MNK antibodies directed against the N-terminal region of the human MNK protein (9), western blot analysis was carried out with whole cell extracts from the mutant cell lines, the normal parental cell line from which the blotchy line was derived (Normal-2), a normal mouse adult fibroblast line (Normal-1) and a mouse epithelial cell line (Earle’s L cells). The antibody detected a protein of the expected size (~178 kDa) in all cell lines. Mnk protein levels in the brindled cells were comparable with those in the normal control cells. In contrast, blotchy Mnk was present at much lower levels compared with both normal and brindled Mnk (Fig. 3). A truncated Mnk product corresponding to the exon 11-deleted transcript was not detected, possibly due to rapid degradation.

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Figure 4. Subcellular localization and effect of copper on the intracellular distribution of Mnk in brindled and blotchy fibroblasts. The normal parental cell line and brindled and blotchy cells were cultured for 48 h in basal medium and then incubated in the absence (–Cu) or in the presence (+Cu) of 200 µM copper (as CuCl2) for 2–3 h at 37°C. The cells were fixed and MNK was detected with a sodium sulphate-precipitated preparation of anti-MNK antibodies, followed by FITC-conjugated sheep anti-rabbit IgG antibodies.

To determine the intracellular distribution of Mnk in the brindled and blotchy cell lines and its response to increased extracellular copper levels, normal and mutant cells were incubated in basal medium with or without added copper. Immunofluorescence analysis using the anti-MNK antibodies showed that in basal medium without added copper, Mnk in the control and mutant cells was detected in the perinuclear region, consistent with a TGN location (Fig. 4). With elevated extracellular copper levels, Mnk in normal mouse fibroblasts was redistributed to the cytoplasm and plasma membrane, but the protein detected in brindled and blotchy cells remained within the perinuclear region (Fig. 4). Note that in the blotchy cells, the Mnk expression levels varied from low or undetectable to clearly detectable levels. Therefore, the faint band detected in the western blot represented the average amount of Mnk in a population of cells with variable expression levels, whereas the brindled Mnk band detected by western blot indicated substantially more protein and was consistent with the more uniform expression levels of Mnk in the brindled cells. For the brindled Mnk protein, the failure of the mutant protein to relocalize in high copper was confirmed by introducing the six base deletion (nt 2473–2478, numbering based on GenBank submission) that was identified as the causative mutation for the brindled mutant (25,26) into the 4.6 kb cDNA encoding the human MNK protein (9). The mutant cDNA construct was stably transfected into Chinese hamster ovary (CHO) cells and the intracellular location of the mutant protein in the presence and absence of added copper was determined. Both the wildtype protein (MNK-wt) expressed from the normal human cDNA and the mutant protein (MNK-br) had perinuclear locations in the absence of added copper, but the mutant protein failed to relocalize within 2 h of exposure to 200 µM copper, conditions which promote the relocalization of the normal protein (Fig. 5). This failure to relocalize with elevated copper was also apparent in transiently transfected CHO cells in which the levels of MNK-br expression varied from low to high levels due to differences in the copy number of the introduced plasmid construct (data not shown). Further experiments showed that incubation of cells in increasing copper concentrations (0, 200 and 400 µM) and for longer periods (2, 5 and 24 h) had no effect on the localization of the mutant protein (data not

Figure 5. Effect of the brindled mutation on the intracellular distribution of human MNK in CHO cells. CHO cell lines derived from transfection with either the expression vector pCMB77, pCMB77 containing the wild-type human MNK cDNA (MNK-wt) or pCMB77 with the human MNK cDNA containing the brindled mutation (MNK-br) were cultured for 48 h in basal medium and then incubated in the absence (–Cu) or in the presence (+Cu) of 200 µM copper (as CuCl2) for 2–3 h at 37°C. The cells were fixed and MNK was detected with a sodium sulphate-precipitated preparation of anti-MNK antibodies, followed by FITC-conjugated sheep anti-rabbit IgG antibodies. The CHO cell line containing MNK-wt was kindly provided by M.J. Petris.

shown). Incubation of cells that expressed the wild-type and mutant proteins with the fungal metabolite Brefeldin A led to a condensation of the perinuclear signal to form a tight juxtanuclear signal as observed previously in cells that overexpress MNK (8,18) and typical of TGN located proteins, thus confirming that the mutant protein was not mislocalized (data not shown). Since the normal protein completely relocalizes within 1 h in 200 µM copper, the mutation appeared to cause a complete failure of copper-induced relocalization rather than simply reducing the rate of response. DISCUSSION The mottled mutants are proving valuable for studying the phenotypic effects of different mutations within the Atp7a gene. Fibroblasts from the blotchy mutant were shown previously to accumulate copper to five times the normal level (29). Cells derived from the brindled mutant were also shown to accumulate significantly more and efflux significantly less copper than control cell lines (30,31). The data obtained in this study were consistent with previous results showing an abnormality of copper transport by cells from these mutants. The present data show that there was a significant difference in copper accumulation and retention between the parental cell line (Normal-2) and the blotchy cell line, which corresponded to, and may be accounted for by, the marked reduction in Mnk protein expression level in the blotchy cell line. In contrast, since the expression level of the brindled Mnk was within the normal range, it was likely that the difference in accumulation and retention between brindled and Normal-2 was likely to be due to reduced activity of the brindled Mnk resulting from the brindled mutation. The Mnk protein produced by the brindled and blotchy cells was correctly localized to the TGN, but in both cases the protein did not relocalize in elevated copper conditions. The activities of copper-requiring enzymes are reduced in the brindled mouse (29,32–34), but copper supplementation was shown to increase the activities of a number of these enzymes (33–35). The brindled mutation occurs within the

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small cytoplasmic loop of Mnk, which is postulated to be involved in the transmission of conformational changes between the catalytic site and cation-binding sites (25,26). In addition, it was proposed that the mutant protein has severely reduced copper transport activity rather than being completely inactive, since a complete loss of Mnk leads to prenatal lethality, as is evident with the dappled mutant (20,25). This study shows that the amount of Mnk protein detected in brindled cells was within the normal range of Mnk protein expression levels, but that the protein was unable to undergo copperinduced redistribution from the TGN to the plasma membrane. The effect of this mutation on the trafficking of Mnk was confirmed by deletion of the corresponding amino acids from the human MNK using in vitro mutagenesis and expression of the mutant protein in CHO cells. Expression in CHO cells, in which metallothioneins are not expressed, also demonstrated that the lack of response to copper was a property of the mutant Mnk and not a secondary effect resulting from increased metallothionein levels in the brindled fibroblasts. Although in CHO cells the expression level of MNK-br was decreased compared with MNK-wt, we demonstrated previously that the concentration of copper used in this study (200 µM) promoted MNK relocalization in cells in which MNK levels varied from endogenous (CHO levels) to very high (8,9). With low level expression of Mnk, incubation in elevated copper led to complete loss of the TGN signal, but detection of the protein at the plasma membrane would require a more sensitive antibody (8,9). In cells in which MNK is highly expressed (CUR3 and MNK cDNA-transfected cells), in addition to plasma membrane staining, a residual TGN signal is maintained even in elevated copper, and is presumably due to saturation of the exocytic sorting machinery at the TGN (Fig. 5; 8,9). In this study, MNK-br was clearly expressed at levels above that of the endogenous hamster Mnk (8,9), but movement out of the TGN was not detected after prolonged periods (up to 24 h) in elevated copper (up to 400 µM). At the levels at which MNKbr was expressed, if normal copper-induced trafficking was occurring then a loss of the TGN signal would be clearly detectable. A recent study of the functional consequences of the common H1069Q mutation identified in Wilson disease patients demonstrated that disruption of both copper transport and copper-dependent trafficking of WND was associated with this mutation (19). The effects of the brindled mutation on copper transport and copper-induced trafficking of Mnk were consistent with these observations. These data and recent results obtained in our laboratory from studying mutations within structurally and functionally distinct regions of MNK and WND indicate that mutations that disrupt the copper transport function of these proteins also prevent their copper-induced redistribution. These studies include analysis of the intracellular localization and copper-induced trafficking of MNK in fibroblasts from Menkes patients (L. Ambrosini and J.F.B. Mercer, submitted for publication), analysis of the effects of mutating highly conserved residues within the MNK catalytic core region (D. Strausak, C. Lim, J. Camakaris and J.F.B. Mercer, manuscript in preparation) and the effects of the toxic milk mutation on the copper-induced redistribution of the murine WND homologue (Wnd) (M.B. Theophilos, S. La Fontaine, S.D. Firth, R.G. Parton, R. Gould and J.F.B. Mercer, submitted for publication). Therefore, it appears that copper

transport and copper-induced relocalization of Mnk are intimately linked. The underlying mechanism to explain this observation is unknown, but the consequences of the failure of MNK trafficking can be related to the phenotypes of the mutants. The brindled Mnk was unable to redistribute with elevated copper levels, but based on the survival of the brindled mouse during development, it is likely that the mutant protein retains some copper transport activity and is able to deliver some copper across the TGN lumen. This would explain the restoration of lysyl oxidase activity with copper treatment of the brindled mouse (33–35). Despite an overall milder phenotype, the blotchy mouse has a more severe lysyl oxidase deficiency compared with the brindled mouse (29) and this activity is not restored by copper therapy (35). The explanation for the specific effects on lysyl oxidase and, in particular, the failure of copper therapy is not clear, but our results provide some insight. Based on the presence of small amounts of normal sized Atp7a mRNA in tissues of the blotchy mouse (21,22), low amounts of normal Mnk protein were expected in blotchy cells (28). Indeed we found greatly reduced Mnk levels in the blotchy cells but, unexpectedly, the protein failed to relocalize with elevated copper levels. Based on the effects of mutations on copper transport and copper-induced trafficking of Mnk as discussed above, we suggest that the protein detected at the TGN of the blotchy fibroblasts is an inactive form, possibly derived from a mis-spliced Atp7a mRNA. As noted previously, additional, larger Atp7a transcripts have been detected in blotchy cells but have not been characterized (28). An inactive Mnk at the TGN would explain the severe reduction in lysyl oxidase activity and the failure of this enzyme to respond to copper. To explain the milder phenotype of the blotchy mouse, we postulate that a small amount of normal protein is present in blotchy cells, but is located at the plasma membrane in response to the accumulation of copper which occurs in these cells even in low copper medium. However, the amount of Mnk at the plasma membrane is too low to enable detection by current methods. In the blotchy mouse, the presence of a small amount of active Mnk at the plasma membrane will allow some copper absorption from intestinal epithelial cells, as well as the transport of copper to the brain and some copper efflux from other cells that accumulate copper, and thus may explain the milder course of the disease. In conclusion, the abnormal copper metabolism of brindled and blotchy cells, and therefore the phenotype of brindled and blotchy mice, may be explained by a combination of factors which include the levels and intracellular location of Mnk and the absence of copper-dependent trafficking of the mutant protein. It is clear from this and recent studies (19,36) that analysis of the subcellular distribution of mutant Cu-ATPases can provide valuable insights into the molecular basis for disease phenotypes. These studies are also providing an understanding of how different mutations within the same gene can have differential effects on the gene product, which then leads to vastly different phenotypes. Future studies that address in detail the mechanism of copper-dependent trafficking of Mnk and its relationship with the copper transport activity of the protein will provide further understanding of the basis for disease phenotypes and cellular copper metabolism, as well as directions for developing early intervention strategies.

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MATERIALS AND METHODS DNA manipulations All DNA manipulations were carried out using standard procedures (37). For introduction of the brindled mutation (25,26) into the equivalent position within the human MNK cDNA sequence, a 5'-proximal 2.6 kb EcoRV fragment of the human cDNA (9) was cloned into the vector pBluescript KS II (Stratagene, La Jolla, CA). The resultant plasmid was subjected to in vitro mutagenesis using the Transformer SiteDirected Mutagenesis kit (Clontech, Palo Alto, CA), selection (5'-GCGGCCGCACTAGAACTAGTGG-3') and mutagenic (5'-GCCTGTAGTGAAATTAACTTTGCCTCTGATGTTTTGC-3') primers and recommended protocols. The introduction of the brindled mutation and the integrity of the surrounding sequence was verified using the chain termination method of DNA sequencing and the Sequenase v.2.0 DNA Sequencing kit (US Biochemical, Cleveland, OH). A 536 bp HaeII–EcoRV fragment, which spanned the brindled mutation, was isolated from the mutated plasmid and ligated to a 2.1 kb SpeI–HaeII fragment derived from the 5'-end of the full-length cDNA of plasmid pCMB19 (9,38). The ligated product was then ligated to a 2.2 kb EcoRV–BamHI 3'-fragment isolated from pCMB19 to generate a full-length cDNA that was cloned into the low copy number vector pWSK29 (39) to generate plasmid pCMB133. The full-length cDNA was cloned as an SpeI–SalI fragment into the NheI and SalI sites of the expression vector pCMB77 (40) to generate the final plasmid construct pCMB163. Cell culture and transfection experiments The brindled (30), blotchy and normal (Normal-2) cell lines were isolated as spontaneously transformed clonal lines derived from fetal tissue of brindled, blotchy and normal mice, respectively. The normal cell line (Normal-2) is the parental line from which the blotchy line was derived, Earle’s L cells (NCTC clone 929) were derived from mouse epithelium and the normal adult mouse fibroblast line (Normal-1) was kindly provided by Dr Francesca Walker (The Ludwig Institute, Royal Melbourne Hospital, Victoria, Australia). All cell lines were maintained in basal medium (1.5 µM Cu) which consisted of Eagle’s basal medium (BME; Trace Biosciences, Castle Hill, Australia) supplemented with 10% fetal calf serum (FCS; Trace Biosciences), L-proline at a final concentration of 20 mM and 0.2% (w/v) bicarbonate. CHO-K1 cells were stably transfected with either the linearized expression vector (pCMB77) alone or with linearized pCMB163 containing the brindled mutation. Liposome-mediated transfection was carried out using Superfect reagent (Qiagen, Hilden, Germany) and recommended protocols, followed by selection in G418 (Life Technologies, Grand Island, NY) for 2 weeks. G418resistant transfectant clones were pooled and those resulting from transfection with pCMB163 were tested for MNK expression by indirect immunofluorescence. Western blot analysis Western blot analysis was carried out as described previously (38). The primary antibody consisted of a sodium sulphateprecipitated preparation of anti-MNK antibodies directed

against the N-terminus of the human MNK protein (9). The secondary antibody consisted of horseradish peroxidase-conjugated sheep anti-rabbit IgG (AMRAD Biotech, Melbourne, Australia). Indirect immunofluorescence Immunofluorescence analysis of cells was carried out essentially as described previously (8,9). 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 200 µM for 2–3 h. CHO-K1 cells were fixed with cold acetone, while fibroblast cell lines were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min and quenched with 0.1 M ethanolamine. Non-specific binding was blocked by incubation in 1% gelatin, 1% BSA in PBS overnight, followed by incubation with the primary and secondary antibodies. The primary antibodies were the same as those employed for western blot analysis. The secondary antibodies consisted of fluorescein isothiocyanate (FITC)-conjugated sheep antibodies to rabbit IgG (AMRAD Biotech). 64Cu

accumulation and retention studies

Copper accumulation and retention studies on the normal and mutant mouse fibroblast cell lines were carried out essentially as described previously (41). Cells were cultured in basal medium and then transferred to BME supplemented with 2% FCS and trace amounts of 64Cu (as CuCl2; Australian Radioisotopes, Lucas Heights, Australia). For accumulation studies, cells were incubated in 64Cu-containing medium for 2, 4, 8 or 16 h at 37°C, washed twice in cold BME and harvested by dissolution in 0.1% SDS. For retention experiments, cells were treated in the same way except that after 16 h in 64Cu-containing medium, they were incubated at 37°C for a further 8 h period in 64Cu-free BME. Following accumulation and retention experiments, the amount of 64Cu in the cell lysates was estimated using an LKB-Wallac Ultragamma counter. The amount of protein in the cell extracts was estimated using a previously described protein assay (42). ACKNOWLEDGEMENTS We would like to thank Ms Rosario Reyes for her invaluable technical assistance, Mr Mark Greenough for assistance with copper transport assays and Dr Michael Petris for helpful scientific discussions. We are grateful to Dr Francesca Walker (The Ludwig Institute, Royal Melbourne Hospital, Victoria, Australia) for providing the normal adult mouse fibroblast cell line. This work was supported by funding from the National Health and Medical Research Council, the International Copper Association and the Australian Institute of Nuclear Science and Engineering. P.J.L. is the recipient of an NH&MRC/Dora Lush post-graduate scholarship. REFERENCES 1. Chelly, J., Tümer, Z., Tønneson, 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. 2. Mercer, J.F.B., Livingston, J., Hall, B.K., Paynter, J.A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemenack, D.

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