The Menkes protein (ATP7A; MNK) cycles via the plasma membrane both in basal and elevated extracellular copper using a C-terminal di-leucine endocytic signal

Menkes disease is an X-linked recessive copper deficiency disorder caused b y mutations in the ATP7A (MNK) gene which encodes a copper transporting P-t ype ATPase (MNK), MNK is normally localized predominantly in the trans-Golg i network (TGN); however, when cells are exposed to excessive copper it is rapidly relocalized to the plasma membrane where it functions in copper eff lux, In this study, the c-myc epitope was introduced within the loop connec ting the first and second transmembrane regions of MNK. This myc epitope al lowed detection of the protein at the surface of living cells and provided the first experimental evidence supporting the common topological model. In cells stably expressing the tagged MNK protein (MNK-tag), extracellular an tibodies were internalized to the perinuclear region, indicating that MNK-t ag at the TGN constitutively cycles via the plasma membrane in basal copper conditions. Under elevated copper conditions, MNK-tag was recruited to the plasma membrane; however, internalization of MNK-tag was not inhibited and the protein continued to recycle through cytoplasmic membrane compartments . These findings suggest that copper stimulates exocytic movement of MNK to the plasma membrane rather than reducing MNK retrieval and indicate that M NK may remove copper from the cytoplasm by transporting copper into the ves icles through which it cycles. Newly internalized MNK-tag and transferrin w ere found to co-localize, suggesting that MNK-tag follows a clathrin-coated pit/endosomal pathway into cells. Mutation of the di-leucine, L1487 L1488, prevented uptake of anti-myc antibodies in both basal and elevated copper conditions, thereby identifying this sequence as an endocytic signal for MN K, Analysis of the effects of the di-leucine mutation in elevated copper pr ovided further support for copper-stimulated exocytic movement of MNK from the TGN to the plasma membrane.