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. ccc2Delta 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 amino-terminal 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.
Highlights
Copper is a trace element that is essential to human physiology and the development of the central nervous system
Saccharomyces cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue [14], are defective in high affinity iron transport due to the lack of copper incorporation into Fet3p, a multicopper oxidase homologous to human ceruloplasmin [15, 16]. ccc2⌬ yeast have been utilized to evaluate the function of the Caenorhabditis elegans P-type ATPase [17] as well as to demonstrate copper transport by the Wilson disease protein and to define specific amino acid residues of the protein involved in the delivery of copper into the yeast secretory compartment [11]
The data presented in this study demonstrate that the Menkes disease protein functions to deliver copper into the secretory pathway of eukaryotic cells
Summary
Menkes Antibody Production—Oligonucleotide primers synthesized with EcoRI linkers were used to amplify the portion of the Menkes cDNA corresponding to amino acids 1046 –1356 in the polymerase chain reaction (PCR) using Klentaq polymerase (CLONTECH) according to manufacturer’s recommendations. The pellet was redissolved in phosphatebuffered saline (PBS) containing 6 M urea and again centrifuged at 16,000 ϫ g for 20 min at 4 °C. The supernatant was diluted to 1 M urea with PBS, pooled with the initial soluble fraction, and applied to glutathione-agarose beads. Bound fusion protein was eluted with 5 mM reduced glutathione in 50 mM Tris, pH 8, and dialyzed extensively against PBS, pH 7.4. Cells were lysed in 1% Nonidet P-40, 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 10% glycerol supplemented with.
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