Affiliation:
1. 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
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 ∼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 μM CuSO4, 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 Km of 11.0±2.5 μM and a K0.5 of 6.9±0.7 μM 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.
Subject
Cell Biology,Molecular Biology,Biochemistry
Cited by
90 articles.
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