Functional Analysis of the N-terminal CXXC Metal-binding Motifs in the Human Menkes Copper-transporting P-type ATPase Expressed in Cultured Mammalian Cells

Abstract

The Menkes protein (MNK) is a copper-transporting P-type ATPase, which has six highly conserved metalbinding sites, GMTCXXC, at the N terminus. The metalbinding sites may be involved in MNK trafficking and/or copper-translocating activity. In this study, we report the detailed functional analysis in mammalian cells of recombinant human MNK and its mutants with various metal-binding sites altered by site-directed mutagenesis. The results of the study, both in vitro and in vivo, provide evidence that the metal-binding sites of MNK are not essential for the ATP-dependent copper-translocating activity of MNK. Moreover, metal-binding site mutations, which resulted in a loss of ability of MNK to traffick to the plasma membrane, produced a copper hyperaccumulating phenotype. Using an in vitro vesicle assay, we demonstrated that the apparent K m and V max values for the wild type MNK and its mutants were not significantly different. The results of this study suggest that copper-translocating activity of MNK and its copper-induced relocalization to the plasma membrane represent a well coordinated copper homeostasis system. It is proposed that mutations in MNK which alter either its catalytic activity or/and ability to traffick can be the cause of Menkes disease.

Mechanism of Cu+-transporting ATPases: Soluble Cu+chaperones directly transfer Cu+to transmembrane transport sites

“…6). These observations provide further support for a regulatory mechanism in which a rise in Cu ϩ levels would lead to an increase of the chaperone⅐Cu ϩ pool, subsequent Cu ϩ transfer to N-MBDs, displacement of N-MBD⅐Cu ϩ from a slow turnover conformation (perhaps providing better access of the chaperone to the TM-MBS), and a resulting higher Cu ϩ -ATPase turnover/transport rate (14,16,18,19). However, in the particular case of A. fulgidus CopA, little CopZ⅐Cu ϩ excess saturates the C-MBD-binding sites, indicating a K eq Ͼ Ͼ 1 for CopZ⅐Cu ϩ ϩ C-MBD 7 CopZ ϩ C-MBD⅐Cu ϩ .…”

“…N-MBDs bind Cu ϩ with high affinity (24)(25)(26), and in vivo, they receive Cu ϩ from the corresponding Cu ϩ chaperones (23,27,28). In bacterial Cu ϩ -ATPases, deletion of these domains or mutation of Cu ϩ -binding Cys residues does not prevent metal activation of the ATPase, although they affect enzyme turnover rate (15,16,19). Significantly, N-MBDs appear necessary for ATPase metal-dependent targeting and localization in eukaryote systems (9).…”

“…However, transfer of Cu ϩ from an MBD to the TM-MBS has not been shown. Moreover, considering that in bacterial ATPases, removal of N-MBDs or mutation of their metal-binding Cys residues does not result in loss of transport (15,16,19), an alternative parsimonious model appears plausible. In this model, the chaperone would ''directly'' deliver the metal to the TM-MBS by docking with the exposed metal entrance to the TM-MBS (represented by a green line in Fig.…”

“…The subsequent conformational change allows metal deocclusion and release to the extracellular (vesicular/luminal) compartment followed by enzyme dephosphorylation and return to the E1 form (10). Functional studies of various Cu ϩ -ATPases have characterized the Cu ϩ transport, Cu ϩ -dependent ATPase activity, phosphorylation, and dephosphorylation partial reactions (5,(13)(14)(15)(16)(17)(18)(19).Cu ϩ -ATPases consist of eight transmembrane segments, two large cytosolic loops comprising the A-domain and the ATP-BD, and regulatory metal-binding domains (MBDs) in their N terminus (6, 8-10, 20, 21) (Fig. 1).…”

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.…”