Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network

We have generated polyclonal antibodies against the amino-terminal third ofthe Menkes protein (ATP7A; MNK) by immunizing rabbits with ahistidine-tagged MNK fusion construct containing metal-binding domains 1-4.The purified antibodies were used in Western analysis of cell lysates andin indirect immunofluorescence experiments on cultured cells. On Westernblots, the antibodies recognized the approximately 165 kDa MNK protein inCHO cells and human fibroblasts. No MNK signal could be detected infibroblasts from a patient with Menkes disease or in Hep3B hepatocellularcarcinoma cells, confirming the specificity of the antibodies.Immunocytochemical analysis of CHO cells and human fibroblasts showed adistinct perinuclear signal corresponding to the pattern of the Golgicomplex. This staining pattern was similar to that of alpha-mannosidase IIwhich is a known resident enzyme of the Golgi complex. Using brefeldin A, afungal inhibitor of protein secretion, we further demonstrated that the MNKprotein is localized to the trans- Golgi network. This data provides directevidence for a subcellular localization of the MNK protein which is similarto the proposed vacuolar localization of Ccc2p, the yeast homolog of MNKand WND (ATP7B), the Wilson disease gene product. In light of the proposedrole of MNK both in subcellular copper trafficking and in copper efflux,these data suggest a model for how these two processes are linked andrepresent an important step in the functional analysis of the MNK protein.