title: Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells.
abstract: Ctr1 (copper transporter 1) mediates high-affinity copper uptake. Ctr2 (copper transporter 2) shares sequence similarity with Ctr1, yet its function in mammalian cells is poorly understood. In African green monkey kidney COS-7 cells and rat tissues, Ctr2 migrated as a predominant band of approximately 70 kDa and was most abundantly expressed in placenta and heart. A transiently expressed hCtr2-GFP (human Ctr2-green fluorescent protein) fusion protein and the endogenous Ctr2 in COS-7 cells were mainly localized to the outer membrane of cytoplasmic vesicles, but were also detected at the plasma membrane. Biotinylation of Ctr2 with the membrane-impermeant reagent sulfo-NHS-SS-biotin [sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate] confirmed localization at the cell surface. Cells expressing hCtr2-GFP hyperaccumulated copper when incubated in medium supplemented with 10 microM CuSO(4), whereas cells depleted of endogenous Ctr2 by siRNAs (small interfering RNAs) accumulated lower levels of copper. hCtr2-GFP expression did not affect copper efflux, suggesting that hCtr2-GFP increased cellular copper concentrations by promoting uptake at the cell surface. Kinetic analyses showed that hCtr2-GFP stimulated saturable copper uptake with a K(m) of 11.0+/-2.5 microM and a K(0.5) of 6.9+/-0.7 microM when data were fitted to a rectangular hyperbola or Hill equation respectively. Competition experiments revealed that silver completely inhibited hCtr2-GFP-dependent copper uptake, whereas zinc, iron and manganese had no effect on uptake. Furthermore, increased copper concentrations in hCtr2-GFP-expressing cells were inversely correlated with copper chaperone for Cu/Zn superoxide dismutase protein expression. Collectively, these results suggest that Ctr2 promotes copper uptake at the plasma membrane and plays a role in regulating copper levels in COS-7 cells.
authors.affiliation: Nutrition Research Division, Health Products and Food Branch, Health Canada, Sir Frederick G. Banting Research Centre, PL 2203C, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, Canada, K1A 0L2.
authors.forename: Jesse
authors.initials: J
authors.lastname: Bertinato
authors.forename: Eleonora
authors.initials: E
authors.lastname: Swist
authors.forename: Louise J
authors.initials: LJ
authors.lastname: Plouffe
authors.forename: Stephen P J
authors.initials: SP
authors.lastname: Brooks
authors.forename: Mary R
authors.initials: MR
authors.lastname: L'abbé
mesh_terms: D000818:Animals; D019556:COS Cells; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003300:Copper / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D009928:Organ Specificity; D051381:Rats; D000080083:SLC31 Proteins
chemical_list: D027682:Cation Transport Proteins; D000080083:SLC31 Proteins; C524078:SLC31A2 protein, human; C000590693:Slc31a2 protein, rat; D003300:Copper