Immunocytochemical localization of the Menkes copper transport protein (ATP7A) to the trans-Golgi network.
We have generated polyclonal antibodies against the amino-terminal third of the Menkes protein (ATP7A; MNK) by immunizing rabbits with a histidine-tagged MNK fusion construct containing metal-binding domains 1-4. The purified antibodies were used in Western analysis of cell lysates and in indirect immunofluorescence experiments on cultured cells. On Western blots, the antibodies recognized the approximately 165 kDa MNK protein in CHO cells and human fibroblasts. No MNK signal could be detected in fibroblasts from a patient with Menkes disease or in Hep3B hepatocellular carcinoma cells, confirming the specificity of the antibodies. Immunocytochemical analysis of CHO cells and human fibroblasts showed a distinct perinuclear signal corresponding to the pattern of the Golgi complex. This staining pattern was similar to that of alpha-mannosidase II which is a known resident enzyme of the Golgi complex. Using brefeldin A, a fungal inhibitor of protein secretion, we further demonstrated that the MNK protein is localized to the trans-Golgi network. This data provides direct evidence for a subcellular localization of the MNK protein which is similar to the proposed vacuolar localization of Ccc2p, the yeast homolog of MNK and WND (ATP7B), the Wilson disease gene product. In light of the proposed role of MNK both in subcellular copper trafficking and in copper efflux, these data suggest a model for how these two processes are linked and represent an important step in the functional analysis of the MNK protein.
Dierick
H A
HA
Department of Pediatrics, University of Michigan, Ann Arbor 48109-0618, USA.
Adam
A N
AN
Escara-Wilke
J F
JF
Glover
T W
TW
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D020126:Brefeldin A; D016466:CHO Cells; D002352:Carrier Proteins / Q000032:analysis* / Q000276:immunology; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D005347:Fibroblasts; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000032:analysis; D006056:Golgi Apparatus / Q000737:chemistry*; D006527:Hepatolenticular Degeneration / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008361:Mannosidases / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000276:immunology; D029701:Saccharomyces cerevisiae Proteins; D043323:alpha-Mannosidase
D000906:Antibodies; C092974:CCC2 protein, S cerevisiae; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D003517:Cyclopentanes; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020126:Brefeldin A; D003300:Copper; D008361:Mannosidases; D043323:alpha-Mannosidase; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases