Functional Expression of the Menkes Disease Protein Reveals Common Biochemical Mechanisms Among the Copper-transporting P-type ATPases

Abstract

Menkes disease is a fatal neurodegenerative disorder of childhood caused by the absence or dysfunction of a putative P-type ATPase encoded on the X chromosome. To elucidate the function of the Menkes disease protein, a plasmid containing the open reading frame of the human Menkes disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue. ccc2⌬ yeast are deficient in copper transport into the secretory pathway, and expression of a wild type human Menkes cDNA complemented this defect, as evidenced by the restoration of copper incorporation into the multicopper oxidase Fet3p. Site-directed mutagenesis demonstrated the essential role of four specific amino acids in this process, including a conserved histidine, which is the site of the most common disease mutation in the homologous Wilson disease protein. The expression of Menkes cDNAs with successive mutations of the conserved cysteine residues in the six aminoterminal MXCXXC metal binding domains confirmed the essential role of these cysteine residues in copper transport but revealed that each of these domains is not functionally equivalent. These data demonstrate that the Menkes disease protein functions to deliver copper into the secretory pathway of the cell and that this process involves biochemical mechanisms common to previously characterized members of this P-type ATPase family.

Functional Properties of the Copper-transporting ATPase ATP7B (The Wilson's Disease Protein) Expressed in Insect Cells

“…The ability of MNKP and WNDP to transport copper has been demonstrated (8,(17)(18)(19) and recent studies from Voskoboinik et al (8,20) confirmed that copper transport by these proteins is ATP-dependent. Further characterization of MNKP indicated that this protein formed a phosphorylated intermediate in a manner similar to other P-type ATPases (8).…”

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

“…These domains are responsible for the cooperative effect of copper on WND catalytic phosphorylation activity (28). In contrast to WND, yeast complementation studies on MNK indicate that the first four N-terminal domains are important for copper transport (29). It may be that the domains function differently in the two ATPases.…”

Posttranslational regulation of copper transporters

“…CTR1 is absolutely required for dietary copper absorption and is essential for cellular copper uptake [1][2][3][4][5][6][7]. Cellular copper export is equally important and is dependent on ATP7A and ATP7B [8][9][10][11][12]. In contrast to other organisms, hardly any transcriptional regulation of genes primarily involved in copper homeostasis exists in mammalian cells [13], and it is commonly accepted that regulation of copper homeostasis occurs predominantly by posttranslational mechanisms.…”

“…Structure-function relationship of copper-transporting P-type ATPases Cellular copper export is dependent on the expression of ATP7A or ATP7B [9,10,[14][15][16][17][18]. ATP7A and ATP7B have dual functions depending on the cellular copper status.…”

“…As trans Golgi network (TGN)-resident proteins, they mediate copper transport into the biosynthetic pathway for subsequent incorporation in cuproenzymes. After relocalization to the plasma membrane or to vesicles at the cell periphery under conditions of copper overload, these proteins are essential for cellular copper export [9,10,[14][15][16][17][18].…”