The Role of GMXCXXC Metal Binding Sites in the Copper-induced Redistribution of the Menkes Protein

Abstract

The Menkes protein (MNK or ATP7A) is a transmembrane, copper-transporting CPX-type ATPase, a subgroup of the extensive family of P-type ATPases. A striking feature of the protein is the presence of six metal binding sites (MBSs) in the N-terminal region with the highly conserved consensus sequence GMXCXXC. MNK is normally located in the trans-Golgi network (TGN) but has been shown to relocalize to the plasma membrane when cells are cultured in media containing high concentrations of copper. The experiments described in this report test the hypothesis that the six MBSs are required for this copper-induced trafficking of MNK. Site-directed mutagenesis was used to convert both cysteine residues in the conserved MBS motifs to serines. Mutation of MBS 1, MBS 6, and MBSs 1-3 resulted in a molecule that appeared to relocalize normally with copper, but when MBSs 4 -6 or MBSs 1-6 were mutated, MNK remained in the TGN, even when cells were exposed to 300 M copper. Furthermore, the ability of the MNK variants to relocalize corresponded well with their ability to confer copper resistance. To further define the critical motifs, MBS 5 and MBS 6 were mutated, and these changes abolished the response to copper. The region from amino acid 8 to amino acid 485 was deleted, resulting in mutant MNK that lacked 478 amino acids from the N-terminal region, including the first four MBSs. This truncated molecule responded normally to copper. Moreover, when either one of the remaining MBS 5 and MBS 6 was mutated to GMXSXXS, the resulting proteins were localized to the TGN in low copper and relocalized in response to elevated copper. These experiments demonstrated that the deleted N-terminal region from amino acid 8 to amino acid 485 was not essential for copper-induced trafficking and that one MBS close to the membrane channel of MNK was necessary and sufficient for the copper-induced redistribution.

Clusterin and COMMD1 Independently Regulate Degradation of the Mammalian Copper ATPases ATP7A and ATP7B

“…The generation of stably transfected CHO-K1 cell lines (WT-ATP7B, MBD1-6del, MBD3-5del, wt-ATP7A, ATP7Am4 -6, and ATP7Am1-6) were described previously (42,43). In this study, "stably" and "transiently" expressed proteins refer to proteins expressed from integrated and nonintegrated plasmids, respectively.…”

“…Plasmid Constructs-Plasmid cDNA constructs encoding wild type (WT) ATP7B, ATP7B (MBD1-6del), and ATP7B (MBD3-5del) were generated previously (42), as were plasmid constructs encoding WT-ATP7A, ATP7Am4 -6, and ATP7Am1-6 (43). Clusterin cDNA in pIREShyg1 (Clontech) was kindly provided by Saverio Bettuzzi (University of Parma, Italy).…”

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

“…Another potential function for the metalbinding domains is mediating copper-responsive cellular relocalization. For example, mutations in MNK domains 4 -6 interfere with the ability to traffic to the plasma membrane in response to elevated copper (30,31). Finally, some of the domains may interact specifically with the copper chaperone Atox1.…”

Posttranslational regulation of copper transporters

“…Endogenous as well as overexpressed ATP7A and ATP7B were detected in the TGN in a wide variety of polarized and nonpolarized cell types under basal copper conditions, both in vitro and in vivo [9,11,12,14,[47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62]. In ATP7A, a putative TGN-targeting signal was identified within transmembrane helix 3 [54].…”

“…Barnes et al [68] demonstrated that copperinduced trafficking of endogenously expressed ATP7B in kidney-derived HEK293T cells, MDCK cells, Cos-7 cells, or primary kidney cells was perturbed, whereas copperdependent trafficking of ATP7A did take place in these same cell types. Reversible copper-dependent trafficking of ATP7A to the plasma membrane or post-Golgi vesicles in nonpolarized cells [11,[54][55][56][57][58] and specifically towards the basolateral membrane in polarized cells [59-62, 69, 70] was observed.…”