Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites

Abstract

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

“…ATP7B with non-functional MBDs (which cannot bind copper) does not traffic in response to copper elevation (32). Residues S340/341 are located within the long loop connecting MBD3 and MBD4 and are not expected to have any effect on copper binding capacity of N-ATP7B, although this remains to be experimentally verified.…”

Copper-dependent Interaction of Dynactin Subunit p62 with the N Terminus of ATP7B but Not ATP7A

“…Generation of Plasmid Constructs-To generate a bait construct that encoded the entire N terminus of ATP7B, a 1971-bp fragment that encoded amino acids 1-657 was amplified by PCR using pCMB278 (19) as a template, the forward primer 5Ј-ccccatatgATGCCTGAGCAG-GAGAGACAG-3Ј (hWND30) and the reverse primer 5Ј-gggcagctgC-GACGTGTCCTTTCTGAAGAAGGTGAC-3Ј (hWND8) (NdeI and SalI restriction sites in lowercase boldface type). The resulting PCR product was cloned into the NdeI/SalI restriction sites of the GAL4 DNA binding domain (BD) vector pAS2-1 (Clontech) to create plasmid pAS2-1/ATP7B-N (pSLB10).…”

“…To create plasmid constructs that encoded mutated or deleted ATP7B N termini as baits, PCR was carried out using the forward primer hWND30, the reverse primer hWND8, and the following plasmids as templates (19): pCMB398, which encoded ATP7B with both cysteines within the CXXC motif of all six of the metal binding domains mutated to serine (i.e. SXXS); pCMB404, which encoded ATP7B with MBS 1-5 deleted leaving only MBS 6; and pCMB405, which encoded ATP7B with MBS 4 -6 deleted leaving MBS 1-3 intact.…”

“…Several studies have revealed that a large portion of the N-terminal domain is not critical for the overall transport activity and the intracellular redistribution of the human copper-transporting ATPases (10, 14 -18). For example, the N-terminal region of ATP7B encompassing MBS 1-5 (amino acids 63-540) is not essential for copper-induced trafficking, with only one MBS close to the membrane channel necessary and sufficient to support trafficking (19). Hence the large N-terminal region of the ATPases that contain the six MBS may have additional roles and potentially contains other signals or motifs that could be important for the regulated trafficking and targeting of ATP7A and ATP7B (17, 20 -22).…”

Structure of human Wilson protein domains 5 and 6 and their interplay with domain 4 and the copper chaperone HAH1 in copper uptake

“…Mutational studies performed to address the role of the N-WLNP have found that, generally, the domains closest to the membrane seem to be the most important both for the ultimate incorporation of copper into Fet3 in yeast complementation assays and in the copper-dependent translocation of WLNP from the vesicular to the cytosolic membrane (23)(24)(25). However, the mechanism by which these domains acquire copper and then transport it across the vesicular membrane is unknown.…”