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

Author:

Bertinato Jesse1,Swist Eleonora1,Plouffe Louise J.1,Brooks Stephen P. J.1,L'Abbé Mary R.1

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.

Publisher

Portland Press Ltd.

Subject

Cell Biology,Molecular Biology,Biochemistry

Cited by 90 articles. 订阅此论文施引文献 订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3