Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells
Ctr2 is partially localized to the plasma membrane and stimulates copper uptake in COS-7 cells
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DOI：
10.1042/bj20071025
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发表时间：
2008-02-01
影响因子：
4.1
通讯作者：
L'Abbe, Mary R.
中科院分区：
文献类型：
--
作者：
Bertinato, Jesse;Swist, Eleonora;L'Abbe, Mary R.
ctr 1（铜转运蛋白1）介导高亲和力铜摄取。Ctr 2（铜转运蛋白2）与Ctr 1具有序列相似性，但其在哺乳动物细胞中的功能知之甚少。在非洲绿色猴肾COS-7细胞和大鼠组织中，Ctr 2作为类似于70 kDa的优势条带迁移，并且在胎盘和心脏中最丰富地表达。瞬时表达的hCtr 2-GFP（human Ctr 2-绿色荧光蛋白）融合蛋白和内源性Ctr 2在COS-7细胞中主要定位于胞质囊泡的外膜，但也可在质膜上检测到。用膜不渗透试剂磺基-NHS-SS-生物素[磺基琥珀酰亚胺基-2-（生物素酰胺基）乙基-1，3-二硫代丙酸酯]对Ctr 2进行生物素化，证实了在细胞表面的定位。当在补充有10 μ M CuSO 4的培养基中孵育时，表达hCtr 2-GFP的细胞过度积累铜，而通过siRNA（小干扰RNA）耗尽内源性Ctr 2的细胞积累较低水平的铜。hCtr 2-GFP表达不影响铜流出，表明hCtr 2-GFP通过促进细胞表面的摄取来增加细胞铜浓度。动力学分析表明，hCtr 2-GFP刺激饱和铜摄取与K。的11.0 +/- 2.5 μ M和K-0.5的6.9 +/- 0.7 μ M时，数据分别拟合到矩形双曲线或希尔方程。竞争实验表明，银完全抑制hCtr 2-GFP依赖的铜的摄取，而锌，铁和锰的摄取没有影响。此外，hCtr 2-GFP表达细胞中铜浓度的增加与铜/锌超氧化物歧化酶蛋白表达的铜伴侣呈负相关。总的来说，这些结果表明，Ctr 2促进细胞膜上的铜摄取，并在COS-7细胞中调节铜水平中发挥作用。
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.