Dynamics of endogenous ATP7A (Menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites.
We report for the first time on the copper-dependent behavior of endogenous ATP7A in two types of polarized intestinal epithelia, rat enterocytes in vivo and filter-grown Caco-2 cells, an accepted in vitro model of human small intestine. We used high-resolution, confocal immunofluorescence combined with quantitative cell surface biotinylation and found that the vast majority of endogenous ATP7A was localized intracellularly under all copper conditions. In copper-depleted cells, virtually all of the ATP7A localized to a post-TGN compartment, with <3% of the total protein detectable at the basolateral cell surface. When copper levels were elevated, ATP7A dispersed to the cell periphery in punctae whose pattern did not overlap with the steady-state distributions of post-Golgi, endosomal, or basolateral membrane markers; only approximately 8-10% of the recovered ATP7A was detected at the basolateral cell surface. These results raise several questions regarding prevailing models of ATP7A dynamics and the mechanism of copper efflux.
Nyasae
L
L
Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21210, USA.
Bustos
R
R
Braiterman
L
L
Eipper
B
B
Hubbard
A
A
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D018938:Caco-2 Cells; D027682:Cation Transport Proteins / Q000378:metabolism*; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology; D019327:Copper Sulfate / Q000494:pharmacology; D000073840:Copper-Transporting ATPases; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000378:metabolism*; D020895:Enterocytes / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007421:Intestine, Small / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D008297:Male; D007706:Menkes Kinky Hair Syndrome / Q000378:metabolism / Q000473:pathology; D010618:Phenanthrolines / Q000494:pharmacology; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D027682:Cation Transport Proteins; D002614:Chelating Agents; D010618:Phenanthrolines; C028559:bathocuproine sulfonate; D003300:Copper; C028419:cuprous chloride; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; C486202:Atp7a protein, rat; D000073840:Copper-Transporting ATPases; D019327:Copper Sulfate