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

Abstract

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.

Figure 1. Biochemical characterization of hCTR2

(A) Sequence alignment between hCTR1 and CTR2 from different species using the AlignX module from Vector NTI (Invitrogen). Identical regions (white text on black background), conserved regions (black text on grey background) and similar regions (white text on grey background) of amino acids are indicated. The transmembrane regions (boxed regions) are indicated by roman numerals. The MXXXM motif, the GXXXG motif involved in intrahelical interactions and the C-terminal His-Cys-His (HCH) motif are indicated. The methionine motifs and histidine-rich regions involved in high-affinity copper transport are underlined, and one conserved methionine residue is indicated by an asterisk. (B) Empty vector (EV), hCTR1–vsvG or hCTR2–vsvG constructs (lanes 1–3) were transiently transfected into HEK-293T cells prior to lysis. Immunoblot analysis was performed on proteins immunoprecipitated by anti-vsvG antibodies. (C) HEK-293T cells were transiently transfected with EV (lanes 1 and 2) or hCTR2–vsvG (lanes 3–5) constructs. Cells were incubated for 30 min at room temperature with increasing concentrations of the chemical cross-linker EGS (lanes 2–5). Immunoprecipitation and subsequent immunoblot analysis was performed to detect hCTR2–vsvG-containing complexes (arrows). The heavy chain of the antibodies is indicated (IgG). (D) HEK-293T cells were transiently co-transfected with hCTR2–FLAG (flag), hCTR2–vsvG, cullin1–FLAG or RABIP4–vsvG. Immunoprecipitation (IP) was performed with either mouse anti-FLAG M2–agarose beads or rabbit anti-vsvG attached to Protein A–agarose beads. Precipitates were washed and resolved by SDS/PAGE (12% gels), and immunoblot (wb) analysis was performed with antibodies as indicated. Molecular masses in kDa are shown on the left-hand side of the immunoblots.

Figure 2. hCTR2 is localized in intracellular vesicles

(A) Localization of hCTR2 was assessed by direct confocal laser-scanning microscopy in HEK-293T, U2OS and HeLa cells after transient transfection with hCTR2–eGFP. (B) HeLa cells were transiently co-transfected with both hCTR2–vsvG and hCTR2–eGFP constructs. hCTR2–vsvG was immunolabelled with rabbit anti-vsvG antibodies, and secondary labelling was performed with Alexa Fluor® 568 conjugated-goat anti-rabbit and goat anti-mouse antibodies. hCTR2–eGFP was visualized by direct confocal microscopy. Co-localization is indicated by arrowheads.

Figure 3. hCTR2 is partially co-localized with hCTR1

(A) Confocal laser-scanning microscopy was performed on HeLa cells which were transiently transfected with hCTR1–eGFP and hCTR2–vsvG constructs. hCTR2–vsvG was immunolabelled with rabbit anti-vsvG antibodies, and secondary labelling was performed using Alexa Fluor® 568-conjugated antibodies. hCTR1–eGFP was visualized by direct confocal microscopy. Plasma membrane localization of hCTR1–eGFP is indicated with arrows, and co-localization with arrowheads. (B) HEK-293T cells were transiently transfected with the hCTR2–eGFP construct. Prior to analysis, cells were incubated for 1 h with either BCS or CuCl₂ at the concentrations indicated.

Figure 4. hCTR2 co-localized with late endosomal and lysosomal vesicles

HEK-293T cells were transiently transfected with hCTR2–eGFP prior to labelling with antibodies against different vesicular markers: the TGN marker p230, TfR (early endosomal marker), CD63 (late endosomal marker) and the lysosomal markers LAMP-1 and LAMP-2. Secondary labelling was performed using Alexa Fluor® 568-conjugated antibodies before being visualized by direct confocal microscopy. Green and red channels were merged to determine the co-localization of hCTR2–eGFP with the different compartmental markers (arrowheads).

Figure 5. Characterization of a novel MRE–luciferase reporter assay

(A) The MRE–luciferase reporter was constructed by cloning four MREs upstream of the firefly luciferase open reading frame of the pGL3-TATA construct as indicated. (B) HEK-293T cells were transiently transfected with the MRE–luciferase reporter or the control vector, pGL3-TATA. After transfection, cells were incubated for 24 h with different concentrations of CuCl₂. (C) Metal toxicity was measured using MTT viability assays after incubation for 24 h with different metal concentrations, and LD₅₀ values were also determined. (D) HEK-293T cells were transiently transfected with the MRE–luciferase reporter, and, 24 h after transfection, cells were incubated with a low or a high sub-lethal metal concentration. (E) HEK-293T cells were transiently transfected with either the MRE–luciferase reporter or a HRE–luciferase reporter. After transfection, cells were incubated with 100 μM CuCl₂ or 100 μM of DFO (to mimic hypoxia). Luciferase reporter activities were measured and normalized for Renilla luciferase activities. Values are expressed as fold induction relative to control incubations ±S.E.M. (see the Experimental section) (n≥3).

Figure 6. Cellular copper uptake is facilitated by hCTR2 and is dependent on the CTR-specific MXXXM motif

The MXXXM motifs were mutated to form IXXXI in pEBB-hCTR1-FLAG and pEBB-hCTR2-FLAG. HEK-293T cells were transiently transfected with the MRE–luciferase reporter in combination with wild-type pEBB-hCTR1-FLAG (A,B), pEBB-hCTR2-FLAG (C,D) or the IXXXI mutant motif constructs. After transfection, cells were incubated for 24 h with increasing amounts of CuCl₂ (A,C) or ZnCl₂ (B,D). MRE–luciferase reporter activities were measured and RLU were calculated after normalization for Renilla luciferase activity. Values are expressed as fold induction relative to control incubations (see the Experimental section)±S.E.M. (n=3). Statistical differences between empty vector (EV)- and hCTR1- or hCTR2-transfected cells are indicated (*P<0.01). (E) Lysates from the MRE–luciferase assay were resuspended in SDS/PAGE sample buffer, resolved by SDS/PAGE and subjected to immunoblot analysis using the mouse anti-FLAG antibody. Molecular masses are indicated in kDa.