IMMUNOCYTOCHEMICAL LOCALIZATION OF THE MENKES COPPER TRANSPORT PROTEIN (ATP7A) TO THE TRANS-GOLGI NETWORK

We have generated polyclonal antibodies against the amino-terminal thi rd of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a his tidine-tagged MNK fusion construct containing metal-binding domains 1- 4, The purified antibodies were used in Western analysis of cell lysat es and in indirect immunofluorescence experiments on cultured cells, O n Western blots, the antibodies recognized the similar to 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be det ected in fibroblasts from a patient with Menkes disease or in Hep3B he patocellular carcinoma cells, confirming the specificity of the antibo dies, Immunocytochemical analysis of CHO cells and human fibroblasts s howed a distinct perinuclear signal corresponding to the pattern of th e Golgi complex. This staining pattern was similar to that of alpha-ma nnosidase II which is a known resident enzyme of the Golgi complex, Us ing brefeldin A, a fungal inhibitor of protein secretion, we further d emonstrated that the MNK protein is localized to the trans-Golgi netwo rk. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localizat ion of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson dis ease gene product, in light of the proposed role of MNK both in subcel lular copper trafficking and in copper efflux, these data suggest a mo del for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.