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 mammalian copper transport pathway and is defective in Menkes disease, a fatal X-linked disorder of copper transport. To study the structure and function of this protein and to elucidate its role in cellular copper homeostasis, a cDNA construct encoding the full-length MNK protein was 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 characterization of four clones showed that an increase in MNK protein expression led to a corresponding increase in the level of copper resistance of the cells. Subcellular localization studies showed that in the parental CHO-K1 and the transfected cell lines, MNK was located in a post-Golgi compartment which, based on immunogold electron microscopic analyses, most likely represented the trans -Golgi network (TGN). When the extracellular copper concentration was increased, MNK in the clones as well as in CHO-K1 cells was redistributed to the cytoplasm and plasma membrane, but returned to the TGN under basal, low copper conditions. This report presents the first ultrastructural evidence for the association of MNK with vesicles within the cell and with the TGN and plasma membrane. It also demonstrates the stable expression of a functional 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.
La Fontaine
S
S
The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Parkville, Victoria 3052, Australia.
Firth
S D
SD
Lockhart
P J
PJ
Brooks
H
H
Parton
R G
RG
Camakaris
J
J
Mercer
J F
JF
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics*; D027682:Cation Transport Proteins; D003001:Cloning, Molecular; D000073840:Copper-Transporting ATPases; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D007706:Menkes Kinky Hair Syndrome / Q000235:genetics*; D011993:Recombinant Fusion Proteins
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases