Michael J Francis and Anthony P Monaco

1. INTRODUCTION

Menkes disease is a severe neurodegenerative disorder arising from a defect in copper transport. The incidence of this disease has been estimated at 1 in 250,000 (1), although in some populations it can be as high as 1 in 40,000 (2). A less severe form of the disease, occipital horn syndrome (OHS) or X-cutis laxis, resulting from an allelic variant, has also been described. The clinical symptoms of both Menkes disease and OHS are caused by a lack of serum copper and copper-requiring enzymes, such as lysyl oxidase and cytochrome-c oxidase. In normal individuals, dietary copper is taken into the body and the majority is absorbed by the stomach and the small intestine. The copper is transported primarily with ceruloplasmin and is complexed to histidine and albumin. The copper is reduced to Cu(I) and taken up into the cell. In patients with Menkes disease, absorption of orally administered copper is much reduced, and the brain, blood, and liver show decreased levels of copper. However, in many other tissues, there is an increase in copper levels when compared to normal individuals, although there is still a defect in the function of copper-requiring enzymes (3). Cultured fibroblasts from patients show both an excess accumulation of copper and efflux abnormalities from these cells (4-7). These results suggest that the protein responsible for Menkes disease has a direct role in the intracellular transport of copper. This chapter discusses the role of motifs within the Menkes disease protein involved in this transport.

2. IDENTIFICATION, STRUCTURE, AND CELLULAR LOCALIZATION OF THE MENKES DISEASE GENE (ATP7A/MNK)

The gene responsible for Menkes disease (ATP7A) was identified in 1993 and is homologous to a bacterial family of P-type ATPases (8-10). These are integral membrane proteins that use an aspartyl phosphate to transport cations across membranes (Fig. 1). Conserved regions include heavy-metal-binding repeat sequences, GMXCXXC, a phosphatase domain, a DKTGT sequence involved in energy transduction, an ATP-binding domain, and eight transmembrane domains. The ATP7A gene has a 4.5 kb open reading frame and translates a protein of approximately 170 kDa. Deletions, mutations and reduced mRNA synthesis are all evidence that ATP7A is responsible for Menkes disease. Interestingly, mutations in the autosomal homolog of ATP7A (ATP7B/WND) are responsible for the phenotype observed in Wilson's disease patients, another disorder of copper transport. The abnormal

Fig. 1. Proposed gene structure of ATP7A/MNK, highlighting transmembrane domains (1-8) and proposed functional domains. The intracellular trafficking signals and motifs discussed in this chapter are shown in italics: (A) copper-sensing domains, (B) trans-Golgi network (TGN) retention signal, and (C) plasma membrane internalization signal.

Fig. 1. Proposed gene structure of ATP7A/MNK, highlighting transmembrane domains (1-8) and proposed functional domains. The intracellular trafficking signals and motifs discussed in this chapter are shown in italics: (A) copper-sensing domains, (B) trans-Golgi network (TGN) retention signal, and (C) plasma membrane internalization signal.

Fig. 2. Construction of the recombinant full-length MNK cDNA. CDNAs (C) isolated during the cloning of ATP7A (8) were ligated to a 3' RT-PCR product (RT). A MYC epitope tag was added in-frame at the 3' end, resulting in the construct MNKMYC. This full-length epitope-tagged cDNA was subsequently cloned into mammalian expression vectors for trafficking studies.

Fig. 2. Construction of the recombinant full-length MNK cDNA. CDNAs (C) isolated during the cloning of ATP7A (8) were ligated to a 3' RT-PCR product (RT). A MYC epitope tag was added in-frame at the 3' end, resulting in the construct MNKMYC. This full-length epitope-tagged cDNA was subsequently cloned into mammalian expression vectors for trafficking studies.

copper accumulation and retention phenotype observed in cell lines from Menkes patients has been corrected by the expression of the recombinant MNK and WND proteins in these cell lines (11). Both the endogenous and the recombinant proteins at steady state are localized predominantly at the trans-Golgi network (TGN) (12-16). On the addition of copper to cultured cells, a shift in equilibrium is observed and the protein is redistributed to the plasma membrane. The protein returns to the TGN on removal of the copper. It is assumed from this that MNK has a role in copper efflux (12,15,17). Although this trafficking does not require new protein synthesis, both the endocytic and exocytic routes are ATP dependent (12). These observations prompted studies into the characterization and identification of the motifs involved in this intracellular trafficking.

3. EXPRESSION STUDIES FROM A FULL-LENGTH MNK CDNA

In order to confirm the role of newly identified motifs within MNK that were involved in intracellular trafficking, it was essential to generate a full-length cDNA that could be expressed and the resulting protein visualized within mammalian systems. The disruption of putative trafficking motifs within this recombinant protein would help to confirm their role in the copper-induced relocalization of MNK.

3.1. Generation of Recombinant Construct cDNAs and reverse transcription-polymerase chain reaction (RT-PCR) products isolated during the cloning of the ATP7A gene were ligated together to generate the full-length cDNA (Fig. 2). To distinguish between the almost ubiquitous expression of the endogenous protein and the recombinant protein, the recombinant protein was tagged with a specific epitope marker. An in-frame fusion of the MYC epitope (recognized by the monoclonal antibody 9E10) was inserted into the stop-codon position of MNK, resulting in the construct MNKMYC.

3.2. Recombinant MNK Protein Studies

MNKMYC was transfected into numerous human cell lines using a cationic lipid transfection reagent (Superfect-Qiagen). Lysates from the transfected cell lines were separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel. The recombinant MNK protein was detected using the 9E10 antibody and a secondary anti-mouse antibody conjugated to HRP. A band of 180 kDa was observed in accordance with the predicted protein size (data not shown).

3.3. Intracellular Localization of Recombinant MNK

To determine whether the recombinant MNK localized to the same region of the cell as the endogenous form, the cellular localization of MNKMYC was detected using indirect immunofluorescence and confocal laser microscopy. Comparisons were made between the expression pattern of MNKMYC and the human TGN resident marker, TGN46. Figure 3 shows the protein expression of MNK in the human fibroblast cell line MRC5/V2. Both the staining patterns of MNKMYC (Fig. 3B) and TGN46 (Fig. 3A) are visible at a juxtanuclear reticulum location. The colocalization between these two proteins is confirmed by the yellow signal observed when the images are superimposed (Fig. 3C). As endogenous MNK and TGN46 are found in the TGN, these data confirm that the recombinant and endogenous MNK proteins reside in a morphologically identical organelle.

4. CHARACTERIZATION AND IDENTIFICATION OF MOTIFS INVOLVED IN INTRACELLULAR TRAFFICKING OF MNK

We focused our efforts on (1) understanding the role of the six N-terminal copper-binding domains in this copper-induced relocalization, (2) identifying motif(s) involved in retention of MNK to the TGN, and (3) identifying signals that mediate the internalization of MNK from the plasma membrane to the TGN.

4.1. Characterization of N-Terminal Copper-Binding Motifs

The N-terminal domains of both the Menkes protein (MNK) and the Wilson protein (WND) contain evolutionary conserved repeat sequences of the type GMXCXXCXXIE that bind copper, as well as zinc, cobalt, and nickel (18-19). The number of these N-terminal repeats vary in the MNK/WND orthologs from one in Enterococcus hirae and two in Schizosaccharomyces pombe, to six in both

Fig. 3. Recombinant MNK localizes to the TGN. Recombinant MNK containing an in-frame fusion MYC epitope tag was detected by indirect immunofluorescence (B). This signal is almost identical to that of the TGN marker TGN46 (A). This similarity is confirmed by the overlap of the two signals (C). (Reprinted from ref. 16 with permission from Oxford University Press.)

Fig. 3. Recombinant MNK localizes to the TGN. Recombinant MNK containing an in-frame fusion MYC epitope tag was detected by indirect immunofluorescence (B). This signal is almost identical to that of the TGN marker TGN46 (A). This similarity is confirmed by the overlap of the two signals (C). (Reprinted from ref. 16 with permission from Oxford University Press.)

mammalian MNK and WND. The metal-binding properties of these repeats were first established by overexpressing the N-terminal domains of both MNK and WND in Escherichia coli. Both of these proteins can bind copper in vitro and in vivo, and five to six copper molecules are bound for every molecule of the N-terminal domain (19).

Using reducing agents, it was also shown that the copper is most likely to be bound in the Cu(I) form. Further studies confirmed that copper binds as Cu(I) with a stoichiometry of one copper per domain (20), and it is most likely to bind in a linear bi-coordinate manner to the two cysteine residues of the repeat (21). One method employed to assay the role of these copper-binding domains on function has been the complementation of the ccc2 yeast mutant. The gene product of ccc2 supplies copper to Fet3p, which is involved in iron import into the cell. A defective ccc2 protein results in the cells unable to import iron and grow on iron-limited medium (22,23). Both ATP7A and ATP7B can rescue the ccc2 mutant phenotype (24,25). Using this method to assay the effect of mutating the N-terminal repeats on function, the results of Payne and Gitlin inferred that the N-terminal motifs of ATP7A are functionally more important than the C-terminal motifs. The third copper-binding domain is the most important for MNK activity, whereas for ATP7B, the converse is true, and deletion of the first five repeats resulted in normal function (26).

To monitor the role of these repeats in the copper-induced trafficking of MNK from the TGN to the plasma membrane, we performed exhaustive mutagenesis studies of these domains and observed the effects of increased cellular copper levels on intracellular trafficking of MNKMYC in a human fibroblast cell line. It had been previously demonstrated that, unlike the wild-type sequence (GMXCXXC), the mutant sequence, GMXSXXS, will not bind copper (21,27). In our studies, both of the cysteine residues in the copper-binding domains (CBD), were mutagenised to serine (GMXSXXS). Figure 4 shows the effect of increasing copper concentrations on relocalization of wild-type MNK (CBDWT). Under normal physiological conditions, MNK has a perinuclear location. As the copper concentration increases, from 100 to 600 |M, a generally more punctate staining, leading to staining at the plasma membrane, is observed. Removal of copper from the media results in the protein returning to its perinuclear position. Mutagenesis of the cysteine residues to serine in all the repeats (CBDA1-6) abolishes movement to the plasma membrane (Fig. 5). To determine which of the copper-binding domains are involved in the trafficking of MNK to the plasma membrane, a series of mutants containing only one functional copper-binding domain were studied. For example, CBD1WT contains a functional copper-binding domain 1 only. All of these mutants behave identically to that of the wild-type protein and relocalize to the plasma membrane on the addition of copper (Fig. 6). These results suggest that in human fibroblasts, only one functional copper-binding domain is required for movement and that there is no observable difference in the trafficking of MNK from the involvement of any of the individual domains (17).

Fig. 4. MNK moves to the plasma membrane with increasing intracellular copper concentration. Copper was added to cells containing MNK. As the intracellular copper concentration increases (100-600 |lM), MNK moves from the TGN to the plasma membrane. The protein returns to the TGN on the removal of copper from the culture media. (Reprinted from ref. 17 with permission from Oxford University Press.)

Fig. 4. MNK moves to the plasma membrane with increasing intracellular copper concentration. Copper was added to cells containing MNK. As the intracellular copper concentration increases (100-600 |lM), MNK moves from the TGN to the plasma membrane. The protein returns to the TGN on the removal of copper from the culture media. (Reprinted from ref. 17 with permission from Oxford University Press.)

Other studies have shown that in Chinese hamster ovary cells when CBD 4-6 of MNK are mutated, trafficking was abolished and the protein remained in the TGN (28), suggesting that a CBD close to the membrane channel of MNK is essential for copper-induced trafficking. It has been proposed that the N-terminal repeats may function as a copper-sensing domain, with their progressive occupation with copper resulting in the relocalization of the protein (12). All of the above data, although conflicting on the relative importance of each domain, suggest that not all of the copper-binding domains are required for the redistribution of the protein in high levels of copper.

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