The Cell Biology Of The Cuatpases

Copper homeostasis mediated by both ATP7A/B is dependent on both copper-transport activity and the localization of these proteins to the correct cellular compartment. Both proteins have two major roles in copper homeostasis: delivery of copper to secreted cuproenzymes and efflux of copper from cells. One of the key questions to be answered once the genes were isolated was how a single protein could carry out these both of these functions. The answer to this question is that the dual role is achieved by the alteration of the location of the protein within the cell. The biosynthetic role is carried out when the proteins are in the TGN, and TGN localization has been demonstrated for both ATP7A (32,137,138) and ATP7B (33,42). The efflux role occurs when the proteins are either located on the plasma membrane (ATP7A) (32) or on an intracellular vesicular compartment (ATP7B) (33,42). When copper concentrations in the cell are in the normal physiological range, the proteins are found on the TGN, but exposure of cells to high concentrations of copper results in a relocalization of ATP7A within 30 min to the plasma membrane (32) or a compartment that may be the late endo-some/subapical endosome in the case of ATP7B (41,83). The relocalization of ATP7A was shown to be rapidly reversible when excess copper was removed and not to require new protein synthesis (32). In this study, it was found that the concentration of ATP7A mRNA and protein did not change when cells were exposed to copper, suggesting that the ATP7A gene was not copper responsive. This aspect of copper homeostasis depended solely on a posttranslational mechanism: copper-induced trafficking.

The initial studies on the trafficking of ATP7A/B were carried out the nonpolarized Chinese hamster ovary cells, but in the physiological situation, these proteins will often function in polarized cells and this raised the question as to which membrane, apical or basolateral, the proteins are targeted. It is probable that ATP7A will traffic to the basolateral membrane; however, it has proven difficult to express ATP7A in polarized cells to prove this point (Petris, personal communication), so the membrane target of ATP7A remains to be established. For ATP7B, the situation is clearer; Roelofsen et al. (41) have provided convincing data that, in polarized cultured hepatocytes, this protein traffics in four stages:

(1) at low copper concentrations ATP7B is in the TGN; (2) within 30 min of exposure of the cells to copper, the protein is present in randomly distributed vesicular structures; (3) the vesicles appear to fuse and form clusters that accumulate at the apical pole of the cells; and (4) within 2-3 h, ATP7B is detected on the apical membrane. It is likely that stages 1-3 occur when ATP7B is expressed in nonpolarized Chinese hamster ovary cells, as ultrastructural studies have shown the presence of ATP7B on large vesicular structures that resemble late endosomes (83). These large vesicles may well be equivalent to the subapical vesicles noted by Roelofsen et al. (41). These conclusions, based on studies in cultured cells, are supported by the distribution of ATP7B in rat liver. Schaefer et al. found that in copper-deficient rats, ATP7B was localized in the TGN of hepatocytes, but when animals were copper loaded, the protein was distributed in multiple vesicular structures (42), but these authors did not find any ATP7B on the canalicular membrane. Further evidence that trafficking of the Cu-ATPases is indeed a physiologically relevant process was provided by the finding that both ATP7A and ATP7B are located in the TGN of nonlactating breast tissue, but are redistributed to vesicular structures in the lactating tissue (85,139). Although the physiological stimulus for the change in intracellular location of the proteins in the lactating gland has not been determined, these studies suggest that the trafficking of ATP7A/B is an important part of the delivery of copper into breast milk. The role of ATP7B in this process was suspected from the finding that milk produced by the toxic milk mouse mutant was copper deficient (81) and the Atp7B knockout mouse accumulated copper in the mammary gland (84). Interestingly, the mutant Atp7B in the tx mouse does not relocalize in response to lactation (85), a behavior that is reminiscent of the lack of Cu-induced relocalization observed in a mutant forms of ATP7A found in cells from a patient with mild Menkes disease (52). Indeed, a number of diverse mutations appear to prevent copper-induced trafficking of both ATP7A and ATP7B, but the reason for this is unclear (140,141). The differential effects of mutations on copper transport and trafficking has the potential to generate diverse disease phenotypes, as will be discussed in Section 7.

The importance of Cu-induced trafficking in copper homeostasis has encouraged a number of studies to investigate the way in which copper may stimulate the movement of the protein. Attention has focused on the six metal-binding sites. These studies have used site-directed mutagenesis to alter the MBSs to a non-Cu-binding form (usually changing CXXC to SXXS) or by deletion of one or more of the metal-binding sites. With ATP7A Strausak et al. found that mutation of the first three MBSs did not have any appreciable effect on copper-induced trafficking, but that a molecule in which MBS1-3 were intact and MBS4-6 were mutated was incapable of relocalizing in high copper (134). This work also showed that the first four MBSs could be deleted and the truncated molecule could still traffic in response to Cu. Work by Goodyer et al. showed that an ATP7A molecule with any one of the six MBSs intact could still undergo Cu-induced trafficking (142), a result in apparent contradiction to the finding of Strausak et al. that the mutant with MBS1-3 intact could not traffic. The reason for this discrepancy is at present unclear. Both studies agree, however, that at least one intact MBS is necessary for the trafficking to occur. Although similar studies with ATP7B have yet to be reported, Roelofsen et al. have suggested that the binding of copper to one or more of the copper-binding sites may induce a change in conformation in the protein that exposes an apical targetting signal (41). The same argument could be applied to ATP7A, but in this case, the signal may be a basolateral targeting signal. Discovery of such targeting signals, if they exist, will be an important advance in the analysis of the molecular basis of copper homeostasis.

The Cu-ATPases contain other protein motifs that are important for determining the cellular location of the proteins. Petris et al. and Francis et al. have shown that a dileucine motif in the C-terminal region of ATP7A is required for correct TGN localization (143,144). Mutation of the dileucine to dialanine resulted in a molecule that was constitutively located on the plasma membrane. This effect was shown to be the result of the requirement of the dileucine for retrieval of ATP7A from the plasma membrane to the TGN (i.e., that the dileucine is an endocytic targeting motif). A trileucine motif is found in a corresponding region of ATP7B that may also be responsible for retrieval of this molecule, but this has yet to be demonstrated. A 38-amino-acid sequence in transmembrane 3 of ATP7A was identified as a Golgi localization signal, presumably necessary for the retention of the protein in the TGN (100). Use of ATP7A labeled with a c-myc epitope tag showed that the molecule is undergoing constitutive recycling from the TGN to the plasma membrane, even in basal copper (145). Importantly in the presence of copper, the endocytosis of the protein was not inhibited, suggesting that the mechanism of copper-induced trafficking was not the result of the inhibition of endocytosis, but stimulation of exocytosis. Full characterization of Cu-induced trafficking, the interplay of the various targeting motifs, and the importance of conformational changes in these processes promise to be a fascinating area of investigation for numbers of years to come. Indeed, further characterization of the processes responsible for docking, trafficking, and protein retrieval from the plasma membrane and vesicles and the effect of mutations on these processes will be crucial to the understanding of the regulation of copper homeostasis and the clinical effects of mutations in these molecules.

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