Signals regulating trafficking of Menkes (MNK; ATP7A) copper-translocating P-type ATPase in polarized MDCK cells

Abstract

The Menkes protein (MNK; ATP7A) functions as a transmembrane copper-translocating P-type ATPase and plays a vital role in systemic copper absorption in the gut and copper reabsorption in the kidney. Polarized epithelial cells such as Madin-Darby canine kidney (MDCK) cells are a physiologically relevant model for systemic copper absorption and reabsorption in vivo. In this study, cultured MDCK cells were used to characterize MNK trafficking and enabled the identification of signaling motifs required to target the protein to specific membranes. Using confocal laser scanning microscopy and surface biotinylation we demonstrate that MNK relocalizes from the Golgi to the basolateral (BL) membrane under elevated copper conditions. As previously shown in nonpolarized cells, the metal binding sites in the NH2-terminal domain of MNK were found to be required for copper-regulated trafficking from the Golgi to the plasma membrane. These data provide molecular evidence that is consistent with the presumed role of this protein in systemic copper absorption in the gut and reabsorption in the kidney. Using site-directed mutagenesis, we identified a dileucine motif proximal to the COOH terminus of MNK that was critical for correctly targeting the protein to the BL membrane and a putative PDZ target motif that was required for localization at the BL membrane in elevated copper.