High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis.
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Research Article|
September 12 2007
Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake Available to Purchase
Peter V. E. van den Berghe;
Peter V. E. van den Berghe
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Dineke E. Folmer;
Dineke E. Folmer
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Helga E. M. Malingré;
Helga E. M. Malingré
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Ellen van Beurden;
Ellen van Beurden
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Adriana E. M. Klomp;
Adriana E. M. Klomp
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Bart van de Sluis;
Bart van de Sluis
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
†Complex Genetics Section, Department of Medical Genetics, University Medical Center Utrecht, 3508 TA Utrecht, The Netherlands
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Maarten Merkx;
Maarten Merkx
‡Department of Biomedical Engineering, Technical University Eindhoven, 5600 MB Eindhoven, The Netherlands
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Ruud Berger;
Ruud Berger
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
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Leo W. J. Klomp
Leo W. J. Klomp
1
*Department of Metabolic and Endocrine Diseases, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands
1To whom correspondence should be addressed (email [email protected]).
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Publisher: Portland Press Ltd
Received:
May 29 2007
Revision Received:
July 04 2007
Accepted:
July 06 2007
Accepted Manuscript online:
July 06 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2007 Biochemical Society
2007
Biochem J (2007) 407 (1): 49–59.
Article history
Received:
May 29 2007
Revision Received:
July 04 2007
Accepted:
July 06 2007
Accepted Manuscript online:
July 06 2007
Citation
Peter V. E. van den Berghe, Dineke E. Folmer, Helga E. M. Malingré, Ellen van Beurden, Adriana E. M. Klomp, Bart van de Sluis, Maarten Merkx, Ruud Berger, Leo W. J. Klomp; Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake. Biochem J 1 October 2007; 407 (1): 49–59. doi: https://doi.org/10.1042/BJ20070705
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