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Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

van den Berghe, P. V. E., Folmer, D. E., Malingré, H. E. M., van Beurden, E., Klomp, A. E. M., van de Sluis, B., Merkx, M., Berger, R. & Klomp, L. W. J., 1-Oct-2007, In : Biochemical Journal. 407, 1, p. 49-59 11 p.

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  • Department of Medical Genetics, University Medical Center Utrecht

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DOI

  • 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

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 muM CuCl2, consistent with the K(m) of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-luciferase reporter activation in a copper-dependent manner at 40 and 100 microM 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.

Original languageEnglish
Pages (from-to)49-59
Number of pages11
JournalBiochemical Journal
Volume407
Issue number1
Publication statusPublished - 1-Oct-2007
Externally publishedYes

    Keywords

  • Amino Acid Sequence, Biological Transport, Cation Transport Proteins/analysis, Cell Membrane/metabolism, Cells, Cultured, Copper/metabolism, Cytosol/metabolism, Endosomes/chemistry, HeLa Cells, Humans, Lysosomes/chemistry, Molecular Sequence Data, Sequence Alignment, Transfection

ID: 65874559