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Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Melbourne, Australia
2 To whom correspondence should be addressed. E-mail: jmercer{at}deakin.edu.au.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • copper • Cu-ATPases • Menkes disease • Wilson disease • ATP7A • ATP7B
Copper is used as a cofactor by several important enzymes, including cytochrome c oxidase (in the mitochondrial electron transport chain), superoxide dismutase (part of the protection against reactive oxygen species) and lysyl oxidase, which is needed for the cross-linking of collagen and elastin. Copper is vitally important for brain function. In addition to cytochrome c oxidase, which is essential for energy generation in the brain, copper is present in dopamine ß-monoxygenase, an enzyme involved in the catecholamine synthesis pathway, and in peptidyl 
-amidating monooxygenase, which modifies various peptide neurotransmitters [the biology of copper is comprehensively covered by Linder (1)]. Under certain conditions, however, copper ions are efficient generators of free radicals, which accounts for the toxicity of copper when homeostatic mechanisms are disrupted (1). All organisms have evolved various mechanisms to obtain and distribute copper safely and to excrete the excess; the molecular details of these copper homeostatic mechanisms are becoming better understood.
Genes involved in copper transport
The identification of the genes affected in two human disorders of copper transport, Menkes (ATP7A) and Wilson (ATP7B) diseases, provided important new information about the molecules that regulate systemic and cellular copper concentrations (2– 5). Both genes encode copper ATPases that are members of the P-type ATPases family of cation transporters (4, 5). These proteins are copper efflux pumps that regulate the amount of copper leaving the cell. In addition, they supply copper to secreted cuproenzymes in the transGolgi network (TGN)3. The mechanism allowing the proteins to perform this dual function is described below. The two proteins have a very similar structure that includes eight transmembrane domains, all the hallmarks of P-type ATPases such as an invariant aspartic acid that is phosphorylated, an ATP binding site and a phosphatase domain that hydrolyses the acyl phosphate. In addition, ATP7A and 7B have six copper-binding domains [(metal binding sites (MBS)] with a canonical sequence of GMTCXXC in their N-terminal region. The function of these six domains is not fully understood, but they are proposed to accept copper from cytoplasmic carriers and may deliver copper to the channel formed by the eight transmembrane domains for transport across the membrane (6).
Mutations of ATP7A in humans cause a copper deficiency disorder, Menkes disease, and mutations of ATP7B result in a Cu toxicity condition, Wilson disease. The reason for the very different diseases caused by mutations in genes and proteins of similar structure is in part due to their different pattern of cellular trafficking (discussed below) and distinct pattern of tissue expression. The Menkes gene is expressed in most tissues; the Wilson gene is expressed mainly in the liver, where it is responsible for biliary excretion of copper.
In addition to the Cu-ATPases, other copper-transporting molecules have been identified by genetic approaches using yeast. A picture is emerging of intricate pathways that regulate copper supplies in cells. The copper chaperones and membrane transporters identified in yeast have in turn been found to have orthologs in mammals, demonstrating the high degree of conservation of copper homeostatic mechanisms [reviewed by Pena et al (7)].
The genetic copper deficiency disorders in mice and men
Defects in the Menkes gene demonstrate the importance of adequate supplies of copper during development in humans and there appears to be an even greater requirement for copper in the developing mouse. Mutations that inactivate the Menkes gene cause classical Menkes disease in humans, which is fatal during the first 3 y of life. The same type of mutation causes prenatal lethality in mice (8). For example, the mottled 9H mutation, which is an intragenic deletion in the mouse homolog of ATP7A (9), results in mouse embryos with a range of abnormalities. Many of these embryos appear to have ruptured blood vessels or body walls, perhaps indicative of connective tissue weakness, possibly attributable to a lysyl oxidase deficiency. Supply of copper to the developing fetus requires ATP7A in the placenta, an organ that expresses the gene at relatively high levels (10). The mouse mutants accumulate copper in the placenta, leading to copper-deficient fetuses (11). A range of mutant phenotypes is produced in both mice and humans from different mutant alleles of ATP7A. In humans three clinical variants are known, classical Menkes (the most severe), mild Menkes and a connective tissue disorder, occipital horn syndrome (12). A model to explain these different phenotypes based on the amount of residual ATP7A activity and the intracellular location of the mutant protein has been proposed (13). There are a large number of mutant alleles known in mice and the range of mouse mutant phenotypes is similar to that in humans, with an additional prenatal lethal group (e.g., mottled 9H) not seen in humans. Mutations in Atp7a have been identified in many of these different mutants (8, 14).
Genetic copper toxicoses
Wilson disease is an autosomal recessive copper toxicosis, characterized by a massive accumulation of copper in the liver, with subsequent deposition of copper in other tissues, such as the central nervous system. The disease can present as a liver disease or neurological condition (12). The Wilson gene encodes the ATP7B copper ATPase, which is about 70% identical to the Menkes copper ATPase (5). In the liver, ATP7B supplies copper to the secreted cuproenzyme, ceruloplasmin, which has a role in iron metabolism (15). In addition, ATP7B is thought to deliver copper to the bile when copper levels in liver start to rise. Patients with Wilson disease do not have an active ATP7B (16) and as a result hepatic copper cannot be excreted in the bile and the concentrations rise until liver toxicity develops. There are two rodent models of Wilson disease, the LEC rat (17) and the toxic milk mouse (18). Both models have been shown to have mutations in the ATP7B homolog (17, 19).
The physiology of copper
The copper ATPases, ATP7A and ATP7B, play an important role in the physiological regulation of copper. Figure 1 outlines a model of systemic copper homeostasis and indicates the points at which the ATP7A/B are thought to act in regulating copper levels and distribution in the body. Copper is primarily absorbed in the small intestine, and the amount absorbed is regulated to some extent by the dietary content of copper; the absorption increases when copper intake is low (20). Patients with Menkes disease have low uptake of copper across the small intestine and accumulate copper in the enterocytes, suggesting ATP7A effluxes copper from the enterocyte into the portal circulation. The copper balance in the body is primarily regulated by changes in the biliary excretion of copper (1). Indeed, the liver is central to copper homeostasis and most of the newly absorbed copper first enters this organ after absorption from dietary sources in the small intestine. In the liver, copper is supplied to endogenous enzymes, incorporated into ceruloplasmin and secreted into the blood; if in excess, it is secreted in the bile. If dietary intake of copper rises, the rate of biliary copper is correspondingly increased, maintaining total body copper within acceptable levels. As noted previously, biliary copper secretion is greatly reduced in Wilson disease, suggesting that ATP7B is the copper pump regulating the rate of biliary copper excretion (discussed more later).
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Copper-induced trafficking of the Cu-ATPases: a central mechanism in copper homeostasis
ATP7A was initially localized to the TGN of cells and in this location the protein presumably pumps copper to secreted copper-dependent enzymes such as lysyl oxidase (23). When cells were cultured in media containing high concentrations of copper, ATP7A redistributed to small vesicles and the plasma membrane (23). This redistribution allows excess copper to be pumped out of the cell by the Menkes protein, and when the intracellular copper levels are reduced, the protein returns to the TGN (23). We propose that this process of copper-induced trafficking is one of the fundamental mechanisms responsible for cellular, and presumably physiological, copper homeostasis (6). The Wilson protein undergoes a similar copper-induced trafficking, but the protein moves from the TGN to a vesicular compartment (24) and in polarized hepatocytes a further movement to the apical membrane has been observed (25). Thus, the Wilson protein delivers copper into the vesicles and presumably pumps copper directly into the bile across the apical biliary canalicular membrane. As rising copper concentrations in the hepatocyte induce the trafficking of ATP7B out of the TGN to vesicles and the canalicular membrane, this mechanism is presumably responsible for the increase in biliary excretion of copper when the liver copper levels rise due to excessive dietary intake.
Mechanism of copper-induced trafficking
Because of the importance of copper-induced trafficking of the Cu-ATPases in copper homeostasis, there is interest in determining how copper causes the relocalization of the proteins. Attention has focused initially on the six MBS found in the N-terminal region. Strausak et al. (26) found that only one of MBS 5 or 6 was sufficient for ATP7A to traffic to the plasma membrane in response to copper. A molecule with the first three MBS intact but 4–6 mutated remained in the TGN in the presence of copper, suggesting that the MBS closest to the membrane are important in the trafficking response.
Forbes and Cox (27) have reported functional differences between the MBS of ATP7B. The two MBS closest to the channel were found to be essential for copper transport and the first three N-terminal MBS were not sufficient for Cu transport and could not substitute for MBS 5 and 6. Preliminary data from our laboratory suggests that the trafficking of ATP7B, like ATP7A, requires one of MBS 5 or 6 (Michael Cater, unpublished data).
The trafficking response, however, involves more than the MBS in the N-terminal region, as mutations in diverse parts of the molecule block the trafficking response. For example, a missense mutation in transmembrane domain 7 of ATP7A in a patient with mild Menkes disease results in a molecule that remains in the TGN in high copper (28). Similar effects of mutations blocking trafficking have been reported with ATP7B (29) and the toxic milk missense mutation in transmembrane domain 8 prevents the copper-induced relocalization of the mouse ATP7B (30). It appears likely that the effect of mutations on trafficking may be caused by disruption of the copper transport cycle. During copper transport, the P-type ATPases undergo marked conformational changes, and one of these conformations may expose a trafficking signal. Mutations that prevent the protein acquiring this conformation would prevent trafficking.
The region between MBS 5 and the first transmembrane domain may contain a targeting signal that directs the protein to either the vesicular compartment (ATP7B) or the plasma membrane (ATP7A). Evidence for this is the trafficking behavior of the chimeric molecule in which the corresponding region of ATP7B replaced the N-terminus of ATP7A. This chimera trafficked to the vesicular compartment, like ATP7B (31). Further experiments showed that a chimera in which MBS 1–5 were deleted leaving a molecule with only MBS 6 from ATP7B linked to ATP7A at transmembrane domain 1 still moved to the vesicles in response to copper. A molecule in which all the MBS were deleted did not traffic (31).
In conclusion we propose that the physiological regulation of copper is mainly achieved via the activity of the copper ATPases, ATP7A/B. The regulation is achieved by means of copper-induced trafficking of these proteins, moving them from a biosynthetic role in the TGN to a secretory role at the plasma membrane or vesicles. The differences in targeting of ATP7A and 7B are fundamental to their particular functions in the body and this difference is mediated by sequences around the 5th and 6th MBS. The copper ATPases play an important role in the delivery of copper to the fetus and newborn, and thus are vital for normal development of mammals.
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FOOTNOTES |
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3 Abbreviations used: MBS, metal binding site; TGN, transGolgi network.
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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1. Linder, M. C. (1991) Biochemistry of Copper, Plenum Press, New York.
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. & Glover, T. W. (1993) Isolation of a partial candidate gene for Menkes disease by positional cloning. Nat. Genet. 3: 20–25.[Medline]
3. Chelly, J., Tumer, Z., Tonnerson, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N. & Monaco, A. P. (1993) Isolation of a candidate gene for Menkes disease that encodes a potential heavy metal binding protein. Nat. Genet. 3: 14–19.[Medline]
4. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J. (1993) Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nat. Genet. 3: 7–13.[Medline]
5. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R. & Cox, D. C. (1993) The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nat. Genet. 5: 327–337.[Medline]
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8. Cecchi, C., Biasotto, M., Tosi, M. & Avner, P. (1997) The mottled mouse as a model for human Menkes disease: identification of mutations in the Atp7a gene. Hum. Mol. Genet. 6: 425–433.
9. Mercer, J. F. B., Grimes, A., Ambrosini, L., Lockhart, P., Paynter, J. A., Dierick, H. & Glover, T. W. (1994) Mutations in the murine homologue of the Menkes disease gene in dappled and blotchy mice. Nat. Genet. 6: 374–378.[Medline]
10. Paynter, J. A., Grimes, A., Lockhart, P. & Mercer, J. F. B. (1994) Expression of the Menkes gene homologue in mouse tissues: lack of effect of copper on the mRNA levels. FEBS Lett. 351: 186–190.[Medline]
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16. Thomas, G. R., Forbes, J. R., Roberts, E. A., Walshe, J. M. & Cox, D. W. (1995) The Wilson disease gene: spectrum of mutations and their consequences. Nat. Genet. 9: 210–216.[Medline]
17. Wu, J., Forbes, J. R., Chen, H. S. & Cox, D. W. (1994) The LEC rat has a deletion in the copper transporting ATPase homologous to the Wilson disease gene. Nat. Genet. 7: 541–545.[Medline]
18. Rauch, H. (1983) Toxic milk, a new mutation affecting copper metabolism in the mouse. J. Hered. 74: 141–144.
19. Theophilos, M., Cox, D. W. & Mercer, J. F. B. (1996) The toxic milk mouse is a murine model of Wilson disease. Hum. Mol. Genet. 5: 1619–1624.
20. Turnland, J. R. (1998) Human whole-body copper metabolism. Am. J. Clin. Nutr. 67: 960S–964S.[Abstract]
21. Kodama, H. (1993) Recent developments in Menkes disease. J. Inherit. Metab. Dis. 16: 791–799.[Medline]
22. Ackland, L. M., Cornish, J., Paynter, J. A., Grimes, A., Michalczyk, A. & Mercer, J. F. B. (1997) Expression of Menkes (ATP7a) and Wilson (ATP7b) disease genes in mammary carcinoma cells. Biochem. J. 328: 237–243.
23. Petris, M. J., Mercer, J. F. B., Culvenor, J. G., Lockhart, P., Gleeson, P. A. & 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]
24. Schaefer, M., Hopkins, R. G., Failla, M. L. & Gitlin, J. D. (1999) Hepatocyte-specific localization and copper-dependent trafficking of the Wilson's disease protein in the liver. Am. J. Physiol. 276: G639–G646.
25. Roelofsen, H., Wolters, H., Van Luyn, M. J. A., Miura, N., Kuipers, F. & Vonk, R. J. (2000) Copper-induced apical trafficking of ATP7B in polarized hepatoma cells provides a mechanism for biliary copper excretion. Gastroenterology 119: 782–793.[Medline]
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27. Forbes, J. R., Hsi, G. & Cox, D. W. (1999) Role of the copper-binding domain in the copper transport function of ATP7B, the P-type ATPase defective in Wilson disease. J. Biol. Chem. 274: 12408–12413.
28. Ambrosini, L. & Mercer, J. F. B. (1999) Defective copper-induced trafficking of the Menkes protein in patients with mild and copper-treated classical Menkes disease. Hum. Mol. Genet. 8: 1547–1555.
29. Forbes, J. R. & Cox, D. W. (2000) Copper-dependent trafficking of Wilson disease mutant ATP7B proteins. Hum. Mol. Genet. 9: 1927–1935.
30. La Fontaine, S., Theophilos, M. B., Firth, S. D., Gould, R., Parton, R. G. & Mercer, J. F. B. (2001) Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copper ATPase. Hum. Mol. Genet. 10: 361–370.
31. Mercer, J. F. B., Barnes, N., Stevenson, J., Strausak, D. & Llanos, R. M. (2002) Copper-induced trafficking of the Cu-ATPases: a key mechanism for copper homeostasis. Biometals 16: 175–184.
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