FUNCTIONAL-ANALYSIS AND INTRACELLULAR-LOCALIZATION OF THE HUMAN MENKES PROTEIN (MNK) STABLY EXPRESSED FROM A CDNA CONSTRUCT IN CHINESE-HAMSTER OVARY CELLS (CHO-K1)

The Menkes protein (MNK or ATP7A) is an important component of the mam malian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure an d function of this protein and to elucidate its role in cellular coppe r homeostasis, a cDNA construct encoding the full-length MNK protein w as cloned into a mammalian expression vector under the control of the CMV promoter. Transfection of this plasmid construct into CHO-K1 cells yielded clones that expressed MNK at varying levels. Detailed charact erization of four clones showed that an increase in MNK protein expres sion led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the par ental CHO-K1 and the transfected cell lines, MNK was located in a post -Golgi compartment which, based on immunogold electron microscopic ana lyses, most likely represented the trans-Golgi network (TGN), When the extracellular copper concentration was increased, MNK in the clones a s well as in CHO-K1 cells was redistributed to the cytoplasm and plasm a membrane, but returned to the TGN under basal, low copper conditions . This report presents the first ultrastructural evidence for the asso ciation of MNK with vesicles within the cell and with the TGN and plas ma membrane. It also demonstrates the stable expression of a functiona l MNK protein from a cDNA construct in mammalian cells, as well as the copper-induced redistribution of MNK in a cell line (CHO-K1) that was not selected for copper resistance or overexpression of MNK.