A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK (ATP7A) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans-Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.