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

Overview & Synopsis

High affinity cellular copper uptake is mediated by the copper transporter 1 (CTR1) family of proteins. The highly homologous human CTR2 (hCTR2) protein has been identified, but its function in copper uptake is unknown. To characterise the role of hCTR2 in copper homeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2-vsvG predominantly migrated as a 17-kDa band after immunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in detection of higher molecular mass complexes containing hCTR2-vsvG. Furthermore, hCTR2-vsvG was coimmunoprecipitated with hCTR2-flag, suggesting that hCTR2 forms multimers like hCTR1. Transiently transfected hCTR2-eGFP was localized exclusively in 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-Luciferase reporter contained four metal responsive elements (MRE) of the mouse metallothionein 1A promoter upstream of the Firefly Luciferase open reading frame. Thus, the MRE- Luciferase reporter measured bio-available cytosolic copper. Expression of hCTR1 resulted in strong reporter activation, with maximal induction at 1 µM CuCl 2}, consistent with the K m} of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE-Luciferase reporter activation in a copper-dependent manner at 40 µM and 100 µM CuCl 2}. Taken together, these data 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 of endosomal / lysosomal copper pools in maintaining cellular copper homeostasis.