Molecular characterization of hCTR1, the human copper uptake protein
Molecular characterization of hCTR1, the human copper uptake protein
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DOI：
10.1074/jbc.m203652200
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发表时间：
2002-08-09
影响因子：
4.8
通讯作者：
Kaplan, JH
中科院分区：
文献类型：
--
作者：
Eisses, JF;Kaplan, JH
我们已经表达了hCTR 1，人类铜转运蛋白，在Sf 9细胞使用杆状病毒介导的表达系统，我们观察到大大增强铜的摄取。蛋白质印迹表明，蛋白质被传递到质膜，在那里它介导饱和铜吸收与一个K-m的类似3.5 μ m。我们还表达了功能性转运蛋白，其中N-连接的糖基化位点被取代，我们为氨基末端的细胞外位置提供了证据。氨基末端FLAG表位的抗体透化前和羧基末端FLAG透化后的可及性证实了氨基末端的细胞外位置，并建立了羧基末端的细胞内位置。hCTR 1的胰蛋白酶消化发生在细胞质环内，并产生一个10-Da的羧基末端肽;铜的存在阻止了切割。其中两个天然半胱氨酸Cys-161和Cys-189被丝氨酸取代的hCTR 1突变体也介导铜摄取，表明半胱氨酸残基对于转运都不是必需的。然而，突变体提供了这些残基可以稳定hCTR 1寡聚化的证据。免疫印迹法显示，在Sf 9细胞中的hCTR 1的表达水平比哺乳动物细胞（HepG 2）高100倍。高水平的功能表达和低水平的内源性铜吸收将使未来的结构-功能分析这一重要的蛋白质。
We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K-m of similar to3.5 mum. We also expressed functional transporters where the N-linked glycosylation sites were substituted, and we provided evidence for the extracellular location of the amino terminus. Accessibility of amino-terminal FLAG epitope to antibody prior to permeabilization and of carboxyl-terminal FLAG only after permeabilization confirmed the extracellular location of the amino terminus and established the intracellular location of the carboxyl terminus. Tryptic digestion of hCTR1 occurred within the cytoplasmic loop and generated a 10-Da carboxyl-terminal peptide; cleavage was prevented by the presence of copper. hCTR1 mutants where Cys-161 and Cys-189, the two native cysteines, were replaced with serines also mediated copper uptake, indicating that neither cysteine residue was essential for transport. However, the mutants provided evidence that these residues may stabilize hCTR1 oligomerization. Western blots of hCTR1 in Sf9 cells showed expression levels 100-fold higher than in mammalian (HepG2) cells. The high level of functional expression and the low level of endogenous copper uptake will enable future structure-function analysis of this important protein.