| Human Molecular Genetics | Pages |
©1999 Oxford University Press |
Defective copper-induced trafficking and localization of the Menkes protein in patients with mild and copper-treated classical Menkes disease
Introduction
Results
Mutation detection in the MNK gene
Western blot analysis of Menkes patients
Effect of mutations on the intracellular localization of MNK
Discussion
Materials And Methods
Cell cultures
cDNA synthesis and PCR amplification
Cloning and sequencing of RT-PCR products
Direct sequencing of genomic PCR products
PCR amplification and sequence analysis of control samples
Western blot analysis
Immunofluorescence
Acknowledgements
References
Defective copper-induced trafficking and localization of the Menkes protein in patients with mild and copper-treated classical Menkes disease
Received May 3, 1999; Accepted May 31, 1999
Menkes disease is an X-linked disorder of copper metabolism. An overall copper deficiency reduces the activity of copper-dependent enzymes accounting for the clinical presentation of affected individuals. The Menkes gene product (MNK) is a P-type ATPase and is considered to be the main copper efflux protein in most cells. The protein is located primarily at the trans-Golgi network (TGN), but relocalizes to the plasma membrane in elevated copper conditions to expel the excess copper from the cell. Here we report the first missense mutation which causes mild Menkes disease, a mutation in a successfully copper-treated classical Menkes patient and the effect of each mutation on the localization of MNK within the cell. Using western blot analysis, MNK was detectable in cells from both patients, but appeared to be mislocalized in the treated case. In the mild Menkes patient, the protein appeared to be located in the TGN but failed to redistribute towards the cell periphery in response to copper. This is the first description of a mutation in a Menkes patient which affects the trafficking of MNK, and the loss of this process is consistent with the clinical phenotype.
INTRODUCTION
Copper is an essential trace element required for the normal functioning of a number of important copper-dependent enzymes including cytochrome c oxidase, superoxide dismutase and lysyl oxidase. However, the properties of copper which make it essential, also make it toxic if present in excess (1). Therefore, homeostatic mechanisms exist to carefully control the copper levels in tissues. Disruptions to these processes have a detrimental effect as illustrated in Menkes disease and Wilson disease, two genetic disorders of copper metabolism. Classical Menkes disease is an X-linked copper deficiency disorder resulting from defective intestinal absorption and distribution of dietary copper. Characterized by neurological degeneration, defective keratinization of hair, and arterial and bone abnormalities, the features of Menkes disease are consistent with the reduced activities of critical copper-requiring enzymes. The disease progresses rapidly and causes death in early childhood (1). Mild Menkes disease is an allelic variant of the classical form where cerebellar ataxia and moderate developmental delay predominate (2). Another allelic variant of Menkes disease is occipital horn syndrome (OHS) which is characterized by marked connective tissue defects (1).
The Menkes gene (MNK; ATP7A) has been cloned (3-5) and, based on its amino acid sequence, encodes a protein of the P-type ATPase family. These integral membrane proteins couple the energy derived from the hydrolysis of ATP with the transport of cations (6) which, together with the remarkable similarity to a bacterial copper-exporting ATPase (7), suggested that the Menkes protein (MNK) was involved in the efflux of copper from cells. This was later demonstrated in copper-resistant Chinese hamster ovary (CHO) cells where overexpression of MNK was associated with enhanced copper efflux (8). The mechanism by which cells regulate this copper efflux has recently been shown to involve copper-induced relocalization of MNK from the trans-Golgi network (TGN) to the plasma membrane from where excess copper is extruded from the cell (9, 10). Although this phenomenon was observed in CHO cells overexpressing MNK, movement of the protein towards the periphery of the cell in elevated copper concentrations has also been reported in the mammary carcinoma cell line, PMC42 (11). The efflux of copper is thought to be defective in cultured fibroblasts from Menkes patients, accounting for the copper accumulation observed in these cells (12, 13).
Here we describe two previously unreported mutations, one in a patient with mild Menkes disease, and the other in a patient with classical Menkes disease who has been treated successfully with copper. We report the effect that these mutations have on the intracellular location and trafficking of the Menkes protein which can then be related to the clinical features observed in each patient.
RESULTS
Mutation detection in the MNK gene
The Menkes gene spans a genomic region of ~140 kb and contains 23 exons ranging in size from 77 to 726 bp (14, 15). For both the mild Menkes and the treated classical Menkes patients, direct sequencing of RT-PCR products from the 4.5 kb coding region was performed to determine the disease-causing mutation.
The mild Menkes patient, GT, was the first reported case of a mild form of Menkes disease (2, 16). At 21 months of age, the patient had developmental delay, abnormal hair and a characteristic facial appearance, features which suggested Menkes disease. The diagnosis was confirmed by copper kinetic studies (2). Cultured fibroblasts were found to accumulate 23 ng of copper/106 cells (normal 4 ± 1 ng) following a 24 h incubation in 64Cu and, after a further 24 h incubation without added isotope, the release of 64Cu was zero (normal 70-80%). Currently aged 20 years, he shows no clinical signs of a connective tissue abnormality. Sequence analysis of the entire 4.5 kb coding sequence of the Menkes gene from GT revealed only one predicted amino acid alteration resulting from a C->T base change at position 4230 (Fig. 1A). This change was not detected by sequencing this region in 45 normal chromosomes, dismissing the likelihood of a polymorphism (data not shown). This mutation would convert Ala1362 to valine in the highly conserved seventh transmembrane domain (TM7). Comparison of this region with other copper-transporting ATPases illustrates the invariant nature of this amino acid (Fig. 2), suggesting that any alteration is likely to be detrimental.
Figure 1. DNA sequence from the mild Menkes patient (GT) and the treated classical Menkes patient (GV). Arrowheads indicate the position where a change from the published MNK sequence (3) was observed. (A) In patient GT, cDNA derived from cultured fibroblasts was sequenced and revealed a C->T (*) base change at nucleotide position 4230 in a GCT codon. This mutation converts Ala1362 to a valine residue. (B) Direct sequencing of cDNA derived from cultured amniocytes from GV identified a 32 bp deletion spanning nucleotides 4269-4301 at the 5[prime] boundary of exon 22. (C) Genomic DNA from this patient was then extracted from cultured fibroblasts and the intron-exon boundary was sequenced with a primer derived from intron 21. The AG of the splice acceptor site (shown in bold) was intact and a genomic deletion was not apparent. The only change observed was an A insertion (*) at position 4277 in exon 22.
Figure 2. Comparison of part of transmembrane seven (TM7) in Menkes (MNK) and Wilson (WND) copper-transporting ATPases from various species. The invariant alanine is shown in bold. Numbers above the human MNK sequence denote amino acid position and those in parentheses indicate literature references.
The treated classical Menkes patient, GV, was diagnosed by copper efflux studies on cultured amniocytes following the death of an affected half-brother at 14 months of age (17). GV was induced at 35 weeks, and a liver biopsy taken on day 2 confirmed the diagnosis and allowed early treatment with copper-histidine (18). At 8 years of age, radiographs showed small, but definite, occipital horns. Now aged 11 years, he is very energetic and physically active. His connective tissue defects, however, have not been corrected. Direct sequencing of the Menkes cDNA from GV identified a 32 bp deletion at position 4269, the 5[prime] boundary of exon 22 (Fig. 1B). To clarify whether the alteration was a genomic deletion or splice site mutation, genomic DNA from the appropriate region was amplified and the splice site was sequenced (Fig. 1C). There was no change in the splice acceptor site, or an intronic deletion, but there was an A insertion at position 4277 within exon 22. Analysis of genomic DNA from GV's mother confirmed this observation, and the possibility of a polymorphism was eliminated after sequence analysis of 45 normal chromosomes (data not shown). The newly inserted base presumably disrupts the recognition signal at the splice junction, causing the first AG pair downstream from the insertion point to become a more favourable splice acceptor site (Fig. 3). According to the method of Shapiro and Senapathy (19) which analyses splice junctions, the sequence surrounding this AG pair represents a potential splice site. This new junction corresponds to the end of the 32 bp deletion in the cDNA and creates a frameshift from the C-terminal end of TM7 with the introduction of a termination codon in the same exon. Having established a splicing abnormality, RT-PCR analysis was performed to determine whether normally spliced transcripts were also produced. Primers flanking the 32 bp deletion were designed to amplify the normal 656 bp product and the 624 bp mutant form. As expected, only the 656 bp product was amplified in the positive control; however, in patient GV, the 656 and 624 bp fragments were observed (Fig. 4). This indicated that both normal and aberrant splicing was occurring. However, the normally spliced form contains the A insertion described above (and so was actually a 657 bp PCR product) which alters the reading frame of the gene. This insertion introduces a premature stop codon 31 amino acids from the insertion point, which would lead to the production of a truncated protein product.
Figure 3. Schematic diagram of the mutation identified in GV. Direct sequencing of the cDNA identified a 32 bp deletion at the 5[prime] boundary of exon 22 (Fig. 1B). Sequence analysis across the intron-exon boundary in genomic DNA revealed an unaltered splice acceptor site (lowercase letters). The only change observed was an A insertion (*) within exon 22. The end of the 32 bp deletion in the cDNA corresponds to the first AG (shown in bold) downstream from this A insertion.
Figure 4. RT-PCR analysis of the region spanning the deletion in patient GV. Primers flanking the 32 bp deletion were used in the PCR amplification of cDNA from normal control fibroblasts and from patient GV. The resulting PCR products were visualized after electrophoresis. In the positive control, a band approximately equal in size to the 653 bp marker was observed, which correlated with the expected 656 bp product. This band was also observed in patient GV, in addition to a smaller fragment, which, based on sequence analysis, represented the 32 bp deleted form. As expected, there were no bands evident in the negative control.
Western blot analysis of Menkes patients
Western blot analysis of cells from these two, and seven other, Menkes patients using a primary antibody raised to the N-terminal metal-binding region of MNK (8) led to the detection of a 180 kDa band in all control cell lines and in three of the nine patients (GV, JT and GT) (Fig. 5A). In the six patients where a 180 kDa band was not detected, staining with an anti-vinculin antibody (Fig. 5B) and Ponceau staining of the membrane (data not shown) confirmed the equivalent loading of protein in these lanes. The 180 kDa band is likely to represent intact MNK since it is absent in most Menkes patients analysed, and is close to the previously reported size of 178 kDa in CHO and HeLa cells (8, 20). An additional 140 kDa band was also observed in the fibroblast cell lines and in the control amniocyte line (which is also fibroblast in nature). This smaller form may be a cleavage product specific to this cell type, but further work is necessary to clarify this observation. Interestingly, a 140 kDa form of the structurally similar Wilson disease protein has been identified in HepG2 cells which localizes to the mitochondria (21).
Figure 5. Western blot analysis of Menkes patients. (A) Analysis of nine Menkes patients and controls with an anti-MNK antibody (8). Control A, BB and GV were derived from amniocytes, Control L, JT and JM originate from lymphoblasts, and Control F, GT, AG, MS, AN and PF from fibroblast cell lines. A band of ~180 kDa was observed in all controls and in three patients; GV, JT and GT. A smaller product of 140 kDa was also observed in Control A, Control F and GT. The different appearance of the lymphoblast samples may be due to glycosylation which causes smearing of bands. (B) Following immunodetection with the anti-MNK antibody, the PVDF membrane was stripped and an anti-vinculin antibody was used to confirm the presence of protein in patient samples where MNK was not expressed. Vinculin is not expressed in lymphoblasts, so a band was not detected in samples of this cell type. For JM, protein loading was confirmed by staining the membrane with Ponceau-S (data not shown).
Effect of mutations on the intracellular localization of MNK
Immunocytochemical analysis was carried out to investigate the effect of the mutations in GT and GV on the intracellular location of MNK. Previous work showed that MNK normally is localized to the TGN, but relocates to the plasma membrane in cells overexpressing MNK that are exposed to elevated copper (9, 10, 20). This copper-induced trafficking, proposed to be part of normal cellular copper homeostasis, was investigated in a control fibroblast cell line (Control F). Under basal conditions, fibroblasts from Control F showed a clear perinuclear signal consistent with TGN localization of MNK (Fig. 6A). This observation was confirmed by culturing the cells in the presence of brefeldin A (BFA), which caused the fluorescence to condense to a juxtanuclear signal, an effect typical of BFA on MNK (9) and other TGN proteins (22, 23) (data not shown). Following the addition of 189 µM copper to the media for 3 h, the perinuclear fluorescence signal in Control F dispersed (Fig. 6B), consistent with movement towards the periphery of the cell. This diffuse staining was more intense than that seen in the fibroblasts of patient AN (Fig. 6E and F) who has a large deletion spanning most of the MNK gene (L. Ambrosini, unpublished data). Therefore, any fluorescence staining of the cytoplasm is due to non-specific staining, consistent with the absence of MNK in the western blot, whereas in Control F the cytoplasmic staining is likely to represent MNK. Although previous work has clearly demonstrated the presence of MNK at the plasma membrane in cells exposed to copper (9, 10, 20), a clear delineation of the plasma membrane is not always apparent, especially in cells expressing endogenous levels of MNK (10). In this case, loss of a TGN signal is indicative of copper-induced trafficking. The TGN localization of MNK was also evident in fibroblasts from patient GT cultured in basal media (Fig. 6C). However, the addition of copper to the media failed to cause relocalization of the protein (Fig. 6D). Instead, a similar perinuclear signal was observed. This observation suggested that the alanine to valine amino acid change in TM7 abolished the copper-induced trafficking of MNK.
Figure 6. Effect of copper on the intracellular localization of MNK in fibroblasts. Cultured fibroblasts from a control cell line, Control F (A and B) and two Menkes patients, GT (C and D) and AN (E and F) were analysed. Cells were grown in basal media for 48 h (-Cu) and, where appropriate, were supplemented with 189 mM CuCl2 for 3 h (+Cu) prior to fixing and staining with the primary antibody, anti-MNK (8). Fibroblasts from Control F showed the expected copper-induced relocalization of MNK from the TGN (A) to the cell periphery (B), but this process was defective in the Menkes patient, GT. In basal media, a clear TGN signal was observed in fibroblasts from GT (C) which persisted following the addition of copper (D). Patient AN (E and F) has a large deletion spanning most of the Menkes gene, so the absence of a signal with the anti-MNK antibody was expected and was consistent with the western blot data.
In the absence of added copper, no perinuclear fluorescence was detected in cultured amniocytes from patient GV, in contrast to the clear TGN signal observed in the control amniocyte line, Control A (Fig. 7A). The fluorescence pattern in GV cells was diffuse (Fig. 7C) and similar to the diffuse fluorescence observed in Control A cells in the presence of added copper (Fig. 7B). Since the western blot analysis demonstrated similar levels of MNK in GV cells compared with controls (Fig. 5A), the lack of a clear perinuclear signal suggested that the Menkes protein in GV was mislocalized, possibly being located on the plasma membrane.
Figure 7. Effect of copper on MNK localization in amniocyte cells. Cultured amniocytes from a control cell line, Control A (A and B), and patient GV (C and D) were investigated. Cells were grown in basal media for 48 h (-Cu) and, where appropriate, were supplemented with 189 mM CuCl2 for 3 h (+Cu) prior to fixing and staining with the primary antibody, anti-MNK (8). Control A showed the expected TGN signal in basal media (A) and the predicted response following the addition of copper (B). In patient GV, however, only a diffuse fluorescence staining pattern was observed in both the absence (C) and presence (D) of added copper.
DISCUSSION
The alanine to valine amino acid change in GT is the first reported missense mutation described in a mild Menkes patient. The only other mutation described causing mild Menkes disease is a base change at the +3 splice donor site of intron 21 which results in both normal and mutant transcripts (24). The missense mutation in GT occurs in the highly conserved TM7 domain at the C-terminal end of the Menkes protein. The conservation of this alanine in such evolutionarily distant organisms as humans and yeast suggests that alteration of this amino acid would have a deleterious effect on copper transport. Although alanine and valine are both hydrophobic non-polar residues, the increased size of the branched side chains of valine may have altered the channel structure of the Menkes protein, reducing the transport of copper. It is of interest that the mutation apparently prevented the normal copper-induced translocation of the protein from the TGN to the plasma membrane. This is the first report of a mutation in MNK affecting trafficking in patient cells. This effect was unexpected based on our previous model that the six N-terminal copper-binding sites are directly involved in sensing the copper status of the cell and inducing the vesicular trafficking of MNK (9). Work from our laboratory on the effect of mutations on trafficking (25) and a similar effect of a mutation in the Wilson protein (26) suggests that the copper transport activity of MNK and its trafficking response are linked.
A second point of interest relates to the molecular basis of the mild Menkes phenotype. The mutant MNK in the TGN presumably retains some copper transport activity, otherwise the patient would have classical Menkes disease, and, being located in the TGN, it can provide copper to lysyl oxidase. This would explain the few connective tissue defects in this patient compared with OHS patients. The mild neurological defects in GT compared with classical Menkes patients also indicate that some copper efflux activity and copper trafficking are retained by the mutant molecule. However, the molecular basis of the trafficking defect is not fully understood as protein motifs within MNK that are responsible for copper-induced trafficking have not yet been identified.
In the copper-treated classical Menkes patient GV, the complex genomic alteration which predicted the formation of a truncated protein lacking most of TM7, and all of TM8 and the C-terminus, still produced detectable levels of MNK on a western blot. However, immunofluorescence studies showed a diffuse staining pattern and not the clear perinuclear signal observed in the control cells. This observation suggested that the mutant protein was mislocalized. A similar staining pattern has been reported recently in cells of a patient with OHS, an allelic variant of Menkes disease (27). Co-localization studies on cells from this patient confirmed that the mutant protein, resulting from the skipping of exon 10, was located in the endoplasmic reticulum. Based on the fact that loss of a C-terminal di-leucine, L1487-L1488, from MNK leads to accumulation of the mutant protein on the plasma membrane (28), we propose that the mutant GV protein is located on the plasma membrane. Although the truncated MNK is predicted to lack most of TM7 and all of TM8, the remaining six transmembrane domains may form a partially functional channel. A partially active MNK located on the plasma membrane, but not in the TGN, is consistent with the patient's response to copper treatment where the neurological defects have been ameliorated, but the connective tissue abnormalities still persist (18). The presence of a partially functional mutant protein is consistent with Kaler's suggestion that Menkes patients require some residual MNK activity to respond to copper therapy (29).
The western blot data presented in this report represent the first study on the expression of MNK in a series of Menkes patients. Of the nine patients analysed, only three showed the 180 kDa band indicative of the Menkes protein. This result suggested that most Menkes patients will not have detectable MNK, and is supported by previous mutation analyses which showed that ~15-20% of Menkes patients have MNK gene deletions (3, 5) and ~80-90% have splice site mutations, frameshifts and premature stop codons (30, 31). Based on these results, we suggest that western blot analysis has potential application in prenatal diagnosis of Menkes disease and may provide a simple alternative to the highly specialized radioactive copper methods currently used.
The two patients described in this study, GT and GV, both express MNK. The presence of MNK in GT was not unexpected, considering the nature of the mutation. The marginally reduced MNK observed in Figure 5A was due to reduced protein loading, as evidenced in the control staining of the membrane with an anti-vinculin antibody (Fig. 5B). The detection of MNK in GV suggests that the predicted truncated protein is stable. However, the truncation was predicted to produce a termination codon at amino acid position 1408, resulting in a protein which is ~10 kDa smaller. A difference of this size should have been detected by the western blots. The reason for this anomaly is unclear, but possible explanations are that the mutant protein may be more heavily glycosylated, or that it binds less SDS than the normal protein. Both these effects would result in slower migration through the gel.
In conclusion, we provide the first report of the functional effects of mutations identified in Menkes patients on the localization and trafficking of MNK. These results explain some of the variant phenotypes exhibited in patients with mutations in the Menkes gene. There are at least three key properties of the mutant MNK which influence clinical phenotype: the amount of residual copper transport activity, the intracellular localization of the protein and the ability of the protein to traffic in response to copper. Classical Menkes disease results from a complete absence of MNK activity, whereas, in the mild Menkes patient, some active protein is present at the TGN supplying lysyl oxidase with sufficient copper to prevent marked connective tissue defects. OHS patients produce a small amount of normal protein which is thought to be located at the plasma membrane due to accumulating copper in the cell. Thus, some copper efflux is possible, but lysyl oxidase cannot receive copper, which explains the preponderance of connective tissue defects. A similar situation is found in the treated classical Menkes patient, GV, possibly with a partially active protein at the plasma membrane and not in the TGN. Consequently, GV's connective tissue defects have not been corrected with copper treatment and he has developed occipital horns. Further investigation of protein expression data, and MNK mutations and their functional effects, will greatly increase our understanding of the clinical features of Menkes disease and copper transport in general, ultimately leading to improvements in the treatment of affected individuals.
MATERIALS AND METHODS
Cell cultures
Fibroblasts were cultured from skin biopsies in Eagle's basal medium (BME; Trace Biosciences, Castle Hill, Australia) supplemented with 10% fetal calf serum (Trace Biosciences). For DNA, RNA and protein extractions, cells were grown to 70% confluency.
cDNA synthesis and PCR amplification
RNA was isolated from cell pellets using a guanidinium hydrochloride procedure as described previously (32). For cDNA synthesis, 5 µg of total RNA was reverse transcribed using 500 ng of oligonucleotide primer, 0.3 mM dNTP, 20 U of RNasin (Promega, Madison, WI), 50 mM Tris-HCl pH 8.5, 8 mM MgCl2, 30 mM KCl, 1 mM dithiothreitol (DTT) and 25 U of AMV reverse transcriptase (Boehringer Mannheim, Mannheim, Germany), in a total volume of 20 µl at 41°C for 90 min. From this reaction, 1 µl was used as the template in subsequent PCR amplification with 0.2 mM dNTP, 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 250 ng of each primer and 1.5 U of AmpliTaq DNA polymerase (Perkin Elmer, Foster City, CA). Five primer sets were used to amplify the entire MNK cDNA: segment 1 (F) 5[prime]-ATTTATCGCTGCCACGGTCTG-3[prime] and (R) 5[prime]-CTTGTGTCAGAGGCTGGCTAA-3[prime]; segment 2 (F) 5[prime]-AATAGTGGCTGTATCACCGGG-3[prime] and (R) 5[prime]-CGTCTCCATTGTCTTATTTCTCG-3[prime]; segment 3 (F) 5[prime]-GTCAAGAAGGATCGGTCAGC-3[prime] and (R) 5[prime]-TGCCTCTTCCACAAGTTTGAC-3[prime]; segment 4 (F) 5[prime]-TTGGAGCAGACACAACCCTTT-3[prime] and (R) 5[prime]-CAATGAGGACTTTGTGCTGC-3[prime]; and segment 5 (F) TGCTCTTAATGCTCAGCAGC-3[prime] and (R) 5[prime]-GTTAAACTGGCAGCACTCTCC-3[prime]. PCR conditions consisted of 35 cycles of amplification at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, with an initial denaturation step of 3 min and a final extension of 5 min.
Cloning and sequencing of RT-PCR products
RT-PCR products were subcloned into pBluescript (Stratagene, La Jolla, CA) according to standard cloning methods (33). Recombinant clones were screened using INSTA-PREP Kit (5 Prime-3 Prime, Boulder, CO) and those with the correct sized insert were subjected to sequence analysis using the Sequenase v.2.0 DNA Sequencing Kit (United States Biochemical, Cleveland, OH).
Direct sequencing of genomic PCR products
Genomic DNA was extracted as described previously (34) and 100 ng was used as a template in the PCR amplification outlined above. The primers used to amplify the region spanning the intron-exon boundaries of exon 22 were (F) 5[prime]-AAACATGGACACGGGGAGAC-3[prime] and (R) 5[prime]-CCTACCAAGAATGACTAGATG-3[prime]. PCR conditions were also as described above. The resulting PCR products were gel purified and used as a template in subsequent sequencing reactions using the Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham, Cleveland, OH) and the following conditions for 30 cycles of amplification: 94°C for 30 s, 50°C for 30 s and 72°C for 90 s.
PCR amplification and sequence analysis of control samples
Control DNA samples were obtained from the DNA Diagnostic Laboratory, The Murdoch Institute. For both sets of control data, 100 ng of genomic DNA was used in each PCR, with the following primers: (F) 5[prime]-GGGAAACGGGTAGCAATGGTG-3[prime] and (R) 5[prime]-GCAGCTATGGGAATTCCAACCAG-3[prime] for sequence analysis of the GT mutation, and (F) 5[prime]-AAACATGGACACGGGGAGAC-3[prime] and (R) 5[prime]-GCAGCTATGGGAATTCCAACCAG-3[prime] for sequence analysis of the GV mutation. The PCR amplification and sequencing conditions were as described above.
Western blot analysis
Cell pellets were sonicated in a buffer of 250 mM sucrose and 10 mM HEPES pH 7.5, centrifuged briefly, and the homogenate spun at 38 000 r.p.m. in a TL-100 ultracentrifuge (Beckman, Fullerton, CA) at 4°C for 1 h. The resultant pellet was resuspended in 62.5 mM Tris-HCl pH 6.8, 5% SDS, and the protein concentration estimated using the BCA Protein Assay (Pierce, Rockford, IL). Approximately 50 µg of total cell protein was fractionated in a 7.5% SDS-PAGE gel and transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA). Protein transfer was verified by staining with Ponceau-S (33). Immunodetection was carried out using the Vistra Western Blotting Kit (Amersham) according to the manufacturer's instructions. The primary antibody used was raised to the six N-terminal metal-binding sites of the Menkes protein as described previously (8). Membranes were stripped using the recommended protocol (Amersham) and incubated with an anti-vinculin antibody (Sigma, St Louis, MO).
Immunofluorescence
Fibroblasts were seeded on 13 mm glass coverslips at 104 cells /ml for 48 h at 37°C. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 10 min, followed by incubation in 0.1% Triton X-100 in PBS for 10 min and 0.2 M ethanolamine in PBS for 2 h, with PBS washes between each step. For copper-induced trafficking and BFA (Sigma) studies, cells were incubated in 189 µM CuCl2 and 5 µg/ml BFA, respectively, for 3 h prior to fixing. After overnight incubation in 1% gelatin at 4°C, cells were incubated with the primary antibody described above according to the method of Petris et al. (9). Coverslips were mounted as described previously (9) and cells visualized using a Zeiss IM-35 inverted fluorescence microscope and a 100× oil objective.
ACKNOWLEDGEMENTS
We wish to thank the patients and their families for their cooperation, D. Newgreen for providing the anti-vinculin antibody, and M. Winsor for assistance with the figures. We are grateful to Drs S. Forrest, H.-H. Dahl and S. La Fontaine for critical review of this manuscript. This work was supported in part by a Block grant from the National Health and Medical Research Council of Australia and the International Copper Association.
REFERENCES
*To whom correspondence should be addressed. Tel: +61 3 9251 7329; Fax: +61 3 9251 7328; Email: jmercer{at}deakin.edu.au
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