Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells

被引:87
作者
Bertinato, Jesse [1 ]
Swist, Eleonora [1 ]
Plouffe, Louise J. [1 ]
Brooks, Stephen P. J. [1 ]
L'Abbe, Mary R. [1 ]
机构
[1] Hlth Canada, Sir Frederick G Banting Res Ctr, Hlth Prod & Food Branch, Nutr Res Div, Ottawa, ON K1A 0L2, Canada
关键词
copper chaperone for Cu/Zn superoxide dismutase (CCS); copper transport; copper transporter 2 (Ctr2); kinetics; plasma membrane; RNA interference (RNAi);
D O I
10.1042/BJ20071025
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 similar to 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 mu M 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 K. of 11.0 +/- 2.5 mu M and a K-0.5 of 6.9 +/- 0.7 mu M 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.
引用
收藏
页码:731 / 740
页数:10
相关论文
共 35 条
[1]   Projection structure of the human copper transporter CTR1 at 6-A resolution reveals a compact trimer with a novel channel-like architecture [J].
Aller, SG ;
Unger, VM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2006, 103 (10) :3627-3632
[2]   Ctr6, a vacuolar membrane copper transporter in Schizosaccharomyces pombe [J].
Bellemare, DR ;
Shaner, L ;
Morano, KA ;
Beaudoin, J ;
Langlois, J ;
Labbé, S .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (48) :46676-46686
[3]   Maintaining copper homeostasis:: regulation of copper-trafficking proteins in response to copper deficiency or overload [J].
Bertinato, J ;
L'Abbé, MR .
JOURNAL OF NUTRITIONAL BIOCHEMISTRY, 2004, 15 (06) :316-322
[4]   Copper modulates the degradation of copper chaperone for Cu,Zn superoxide dismutase by the 26 S proteosome [J].
Bertinato, J ;
L'Abbé, MR .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (37) :35071-35078
[5]   Copper deficiency induces the upregulation of the copper chaperone for Cu/Zn superoxide dismutase in weanling male rats [J].
Bertinato, J ;
Iskandar, M ;
L'Abbé, MR .
JOURNAL OF NUTRITION, 2003, 133 (01) :28-31
[6]   Mechanisms of the copper-dependent turnover of the copper chaperone for superoxide dismutase [J].
Caruano-Yzermans, AL ;
Bartnikas, TB ;
Gitlin, JD .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2006, 281 (19) :13581-13587
[7]   The copper chaperone for superoxide dismutase [J].
Culotta, VC ;
Klomp, LWJ ;
Strain, J ;
Casareno, RLB ;
Krems, B ;
Gitlin, JD .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (38) :23469-23472
[8]   MOLECULAR CHARACTERIZATION OF A COPPER TRANSPORT PROTEIN IN SACCHAROMYCES-CEREVISIAE - AN UNEXPECTED ROLE FOR COPPER IN IRON TRANSPORT [J].
DANCIS, A ;
YUAN, DS ;
HAILE, D ;
ASKWITH, C ;
EIDE, D ;
MOEHLE, C ;
KAPLAN, J ;
KLAUSNER, RD .
CELL, 1994, 76 (02) :393-402
[9]  
DARWISH HM, 1983, J BIOL CHEM, V258, P3621
[10]   Copper chaperones: Personal escorts for metal ions [J].
Field, LS ;
Luk, E ;
Culotta, VC .
JOURNAL OF BIOENERGETICS AND BIOMEMBRANES, 2002, 34 (05) :373-379