Cellular multitasking: the dual role of human Cu-ATPases in cofactor delivery and intracellular copper balance

Abstract

The human copper-transporting ATPases (Cu-ATPases) are essential for dietary copper uptake, normal development and function of the CNS, and regulation of copper homeostasis in the body. In a cell, Cu-ATPases maintain the intracellular concentration of copper by transporting copper into intracellular exocytic vesicles. In addition, these P-type ATPases mediate delivery of copper to copper-dependent enzymes in the secretory pathway and in specialized cell compartments such as secretory granules or melanosomes. The multiple functions of human Cu-ATPase necessitate complex regulation of these transporters that is mediated through the presence of regulatory domains in their structure, posttranslational modification and intracellular trafficking, as well as interactions with the copper chaperone Atox1 and other regulatory molecules. In this review, we summarize the current information on the function and regulatory mechanisms acting on human Cu-ATPases ATP7A and ATP7B. Brief comparison with the Cu-ATPase orthologs from other species is included.

Figure 1. The major functional domains of human Cu-ATPases

The transmembrane portion of Cu-ATPases is composed of 8 transmembrane segments (dark blue) that form intramembrane copper-binding site(s); the CPC, YN and MxxS motifs contribute to these sites. The ATP-binding domain is composed of the P-domain (light blue) and the N-domain (green) and together with the A-domain (turquoise) is responsible for enzymatic cycle (ATP binding, hydrolysis, phosphorylation, and dephosphorylation). The N-terminal domain has six metal (Cu)-binding subdomains (MBD1-6, red) and the very N-etrminal 63 residues, that are involved in targeting of ATP7B in polarized hepatocytes. The LL letters indicate the di/tri-leucine motif.

Figure 2. Trafficking of human Cu-ATPases in polarized cells

The localization of Cu-ATPases in low (indicated by a thin arrow) and high (indicated by a thick arrow) copper has been described in polarized epithelial cells and tissues. Caco-2 cell serve as a model for the ATP7A trafficking in enterocytes; WIF cells and HepG2 cells are used to study the localization of ATP7B in polarized hepatocytes. The trafficking from the TGN to a vesicular compartment is observed for both ATP7A and ATP7B, however their destination differ. ATP7A traffics towards the basolateral membrane to facilitate copper export into the blood; in hepatocytes ATP7B traffics towards the apical membrane to export copper into the bile (for copper removal).

Figure 3. The involvement of various domains in the transport activity and trafficking of Cu-ATPases

(A). In the absence of copper, the N-terminal domain of Cu-ATPase interacts with the ATP-binding domain and down-regulates the enzyme activity [33]. (B) Atox1 transfers copper to the metal-binding sites 2; this leads to structural changes and allows further transfer of copper to the metal-binding site 4, which may in turn transfer copper to sites 5 and 6 [16, 77]. (C) Recent data suggest that Atox1 may also transfer copper directly to the transmembrane sites, at least in CopA [125]. (D) The binding and hydrolysis of ATP causes the rearrangement of the domains and copper release into the lumen (as suggested in [36]). (E) In elevated copper, the N-terminal dissociates from the ATP-binding domain. This structural change is thought to increase the rate of copper transport into the lumen and expose sites for interaction with cellular trafficking machinery (the example of ATP7B is shown). (F1) The first 63 amino acids may regulate apical trafficking of ATP7B by interaction with specific targeting proteins [53]. (F2) Copper binding also regulates the phosphorylation of Cu-ATPases by kinases [38]. In yeast Cu-ATPase Ccc2, PKA phosphorylation appears to regulate activity [126], the role of the kinase-mediated phosphorylation in either activity or intracellular targeting of human Cu-ATPases is still unknown. The number of copper atoms need to be bound the N-terminal to regulate catalytic activity, kinase-mediated phosphorylation or trafficking remains to be determined