No abstract

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the transGolgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.

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.

The Menkes protein is a transmembrane copper translocating P-type ATPase. Mutations in the Menkes gene that affect the function of the Menkes protein may cause Menkes disease in humans, which is associated with severe systemic copper deficiency. The catalytic mechanism of the Menkes protein, including the formation of transient acylphosphate, is poorly understood. We transfected and overexpressed wild-type and targeted mutant Menkes protein in yeast and investigated its transient acyl phosphorylation. We demonstrated that the Menkes protein is transiently phosphorylated by ATP in a copper-specific and copper-dependent manner and appears to undergo conformational changes in accordance with the classical P-type ATPase model. Our data suggest that the catalytic cycle of the Menkes protein begins with the binding of copper to high affinity binding sites in the transmembrane channel, followed by ATP binding and transient phosphorylation. We propose that putative copper-binding sites at the N-terminal domain of the Menkes protein are important as sensors of low concentrations of copper but are not essential for the overall catalytic activity.Copper is an essential trace element: its ability to redox cycle between Cu(I) and Cu(II) states is utilized by cuproenzymes participating in redox reactions. However, these same properties make excess copper toxic to biological systems (1). Finely tuned complex mechanisms of copper homeostasis have evolved to allow the regulated uptake of copper, its delivery to target proteins, and detoxification by chelation and/or efflux from the cell (2-4). Copper-translocating P-type ATPases found in a variety of organisms are implicated in the delivery of copper to some cuproenzymes and in the efflux of copper from the cell (2-4).The Menkes (MNK) 1 protein (ATP7A) is a copper-translocating P-type ATPase expressed in most tissues except the liver (5-8). Mutations in the Menkes gene that cause the loss of function of the MNK protein result in Menkes disease in humans, a potentially lethal X-linked disorder associated with severe systemic copper deficiency. Menkes patients suffer from neurological and connective tissue abnormalities as a result of copper deficiency, which reduces the activity of copper-dependent enzymes (9). Through clinical and laboratory studies on Menkes disease patients, the role of the MNK protein in the absorption of dietary copper from gut epithelium, delivery of copper to cuproenzymes, and efflux from the cell were established (9, 10).P-type ATPases are multispanning membrane proteins that translocate ions (e.g. H ϩ , Na, and Cd 2ϩ ) across biological membranes against an electrochemical and concentration gradient using ATP as an energy source (11, 12). The catalytic cycle of P-type ATPases is characterized by the coupled reactions of cation translocation and ATP hydrolysis with a transient aspartyl phosphate formed as a part of the reaction cycle. The phosphorylation results in the enzyme changing its conformation from the high affinity cation and nucleotide binding s...