Mammalian copper homeostasis requires retromer-dependent recycling of the high-affinity copper transporter 1 (CTR1/SLC31A1)

The mammalian cell surface is decorated with a plethora of integral membrane proteins including those required for the transport of micronutrients, such as copper, which are essential to cellular health. The concentration of micronutrients within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. The sorting and recycling of nutrients transporters within the endo-lysosomal network is recognised as an essential process in regulating nutrient balance. The evolutionarily conserved endosomal sorting complex, retromer, coordinates integral membrane protein recognition and retrieval. Cellular copper homeostasis is regulated primarily by two transporters: the major copper influx transporter copper transporter 1 (CTR1/SLC31A1), which controls the uptake of copper from the extracellular environment and is essential for early embryonic development, and the established retromer cargo, the copper-transporting ATPase, ATP7A. Here, we show that in response to fluctuating extracellular copper the retromer complex controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and in response to decreased extracellular copper retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in platinum uptake. Significantly, the efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug, cisplatin.


INTRODUCTION
Integral membrane proteins are delivered and removed from the cell surface by means of membrane trafficking pathways (Cullen and Steinberg, 2018). The balance between delivery and internalisation defines the steady-state cell surface abundance of these proteins and provides the means to rapidly alter the abundance of individual proteins and larger groups of functionally related proteins in response to environmental cues. For responses triggered by, for example, metabolic cues and toxic stresses, the endocytic network plays an important role in the adaptive response (Corvera et al., 1986;Jenness and Spatrick, 1986;Jones et al., 2012;Mueller et al., 2015;Tanner and Lienhard, 1987). Comprising a number of intracellular membrane bound compartments, this network sorts internalised cell surface integral membrane proteins between two fates: either degradation within the lysosome or retrieval from this fate for subsequent recycling and reuse at the cell surface (Burd and Cullen, 2014;Cullen and Steinberg, 2018). By deciding the fate of proteins that include signalling receptors, nutrient and solute transporters, cell-cell and cell-matrix adhesion molecules, the endocytic network can alter the functionality of the cell surface during a range of extracellular stimuli. However, our understanding of the role of the endocytic retrieval and recycling pathways in adaptive responses has been hampered by a lack of basic understanding of the machinery that regulates these pathways (Bai et al., 2012;Grant and Donaldson, 2009;McNally and Cullen, 2018).
Several protein complexes have now been identified that control the sorting of internalised integral proteins for retrieval and recycling (Arighi et al., 2004;Derivery et al., 2009;Gomez and Billadeau, 2009;McNally et al., 2017;Phillips-Krawczak et al., 2015;Seaman, 2004;Seaman et al., 1998;Simonetti et al., 2019). These include the mammalian retromer, a stable heterotrimeric complex of VPS35, VPS29 and VPS26 of which two isoforms, VPS26A and VPS26B, are expressed in humans (Arighi et al., 2004;Kerr et al., 2005;Seaman, 2004;Seaman et al., 1998). Retromer functions by scaffolding an endosome-associated multiprotein assembly that co-ordinates sequence-dependent cargo selection with the biogenesis of cargo-enriched transport carriers that delivery the sorted cargo back to the cell surface (Burd and Cullen, 2014;Wang et al., 2018). Important retromer accessory proteins include the cargo adaptor sorting nexin-27 (SNX27) and the actin polymerising FAM21-containing WASH (Wiskott-Aldrich syndrome protein and Scar Homology) complex (Gallon et al., 2014;Gomez and Billadeau, 2009;Harbour et al., 2012;Jia et al., 2012;Steinberg et al., 2013;Temkin et al., 2011). Retromer regulates the endosomal retrieval and recycling of numerous integral membrane proteins that include a number of transporters for the uptake and extrusion of essential but also toxic transition metals such as copper (Steinberg et al., 2013).
It is clear retromer is central to regulating cargo traffic through the endosomal network and we recently recognised that retromer itself is tightly regulated in response to extracellular cues (Curnock et al., 2019).
Many retromer-dependent cargoes have been identified (Cullen and Steinberg, 2018). The copper-transporting ATPase, ATP7A is an established retromer cargo and its expression at the cell surface upon copper elevation requires retromer and SNX27 (Steinberg et al., 2013).
Cellular copper homeostasis is regulated by two principle transporters: the copper pumping ATP7A and ATP7B which sequesters copper into the trans-Golgi-network (TGN) and mobilises copper to the cell periphery, and the structurally unrelated cell surface localised copper transporter 1 (CTR1) which controls the uptake of copper from the extracellular environment (Hartwig et al., 2019;Hung et al., 1997;Lee et al., 2002;Petris et al., 1996). Cellular adaptation to elevated extracellular copper is mediated by membrane trafficking pathways that deliver ATP7A to the cell surface to enhance the extrusion of copper, and the endocytosis of CTR1 which serves to prevent further copper uptake and accumulation (Kim et al., 2008). Copper is an essential micronutrient, vital for development and growth but in excess it is toxic (Madsen and Gitlin, 2007). Understandably, homeostatic copper uptake and distribution is tightly controlled. The adverse effects of copper misbalance manifest in the neurological disorders Menkes and Wilson's diseases, resulting from copper deficiency and overload, respectively (Daniel et al., 2004). Abnormal copper accumulation has also been linked with neuronal cell death and disease pathology in neurodegenerative disorders such as Parkinson's disease (PD) (Madsen and Gitlin, 2007). It is now well-established that mutations leading to perturbed retromer function are risk factors for the development of PD (McMillan et al., 2017). In addition to the established role for retromer in ATP7A transport, we previously identified the main copper uptake transporter CTR1 as a putative retromer cargo (Steinberg et al., 2013). Therefore, to fully appreciate whether there is a link between retromer function, copper homeostasis and potentially PD progression, in this study we examined the membrane trafficking of CTR1.
The high-affinity copper transporter CTR1 is evolutionarily conserved mediating the cellular import of copper in eukaryotes from fungi to vertebrates and is the only known mammalian importer of copper (Dancis et al., 1994;Zhou and Gitschier, 1997). CTR1 exists in the plasma membrane as homo-trimer with an extracellular N-terminal region, three transmembrane helices and an intracellular C-terminus (Eisses and Kaplan, 2005;Klomp et al., 2002;Lee et al., 2002;Puig et al., 2002;Ren et al., 2019). Deletion studies in mice have demonstrated that CTR1 is essential for development with early embryonic lethality in homozygous knockout animals. Characterisation of heterozygote mice has revealed a severe deficiency in brain copper concentration indicating a crucial role for CTR1 in copper uptake and distribution in the brain (Kuo et al., 2001;Lee et al., 2001). In addition to the cellular import of copper, CTR1 has also been implicated in the cellular uptake and accumulation of platinum-derived anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin (Holzer et al., 2006). Furthermore, responsiveness to these platinum-based chemotherapeutic agents in cancer patients has been correlated with high levels of CTR1 expression (Song et al., 2004). The occupancy and turnover of CTR1 at the plasma membrane is therefore pertinent to not only cellular copper homeostasis but also the efficacy of platinum-based cancer drugs. In the present study, using RNAi-mediated suppression and CRISPR-Cas9-mediated knockout of VPS35, we have investigated whether the cell surface levels and trafficking of CTR1 in response to changes in extracellular copper concentration are dependent on retromer expression. We find using antibody internalisation in the presence of copper and confocal analysis that internalised CTR1 resides on retromerpositive endosomes. Cell surface biotinylation together with flow cytometry revealed that CTR1 basal cell surface levels and copper concentration-dependent delivery to the cell surface relies on retromer expression. The effects of increasing extracellular copper and exposure to cisplatin, the most commonly used platinum chemotherapy drug, on metabolic activity and cell viability were assessed using colorimetric MTT assays. We demonstrate that retromer is essential for full sensitivity to extracellular copper loading and cisplatin toxicity.

Retromer is required for the cell surface localization of CTR1
A multitude of metal ion transporters were previously identified as SNX27-retromer cargo using a global unbiased approach (Steinberg et al., 2013). A distinct subset of these transporters were identified as being independent of the accessory protein SNX27 and solely retromer-dependent. The main mammalian copper importer, CTR1 was one such transporter (Steinberg et al., 2013). To validate this finding, SNX27 and the core component of the retromer complex, VPS35 were efficiently suppressed using siRNA and cell surface biotinylation followed by fluorescence-based quantitative western blotting was used to assess the cell surface abundance of CTR1. A significant reduction in the cell surface levels of CTR1 was observed in cells suppressed for VPS35 but not SNX27 ( Figure 1A). Notably, as would be anticipated the cell surface levels of ATP7A, an established retromer and SNX27 cargo (Steinberg et al., 2013) was depleted in cells treated with siRNA targeting VPS35 and SNX27 ( Figure 1A). Immunofluorescence analysis revealed a reduction in CTR1 signal intensity at the cell surface in VPS35 suppressed cells ( Figure 1B). Furthermore, flow cytometry in VPS35 KO HeLa cells confirmed a significant reduction in the abundance of cell surface CTR1 in the absence of retromer function ( Figure 1C).
In response to elevated extracellular copper, CTR1 is internalised in a dynamin-and clathrindependent manner to prevent excess copper uptake (Clifford et al., 2016). To visualise the trafficking of CTR1, we performed antibody internalisation in the presence of copper (200 µM CuCl2). Here, cells on ice were surface labelled with an antibody against an exofacial epitope of CTR1 and then allowed to internalise the antibody bound CTR1 by incubation at 37°C in growth media supplemented with 200 µM CuCl2. Cells were subsequently fixed prior to immunofluorescence and colocalization analysis using LAMP1 as a marker of the degradative lysosomal compartment. After 2 hours in the presence of copper, a significant increase in colocalization between CTR1 and LAMP1 was observed in HeLa cells treated with siRNA targeting VPS35 ( Figure 2A) and in VPS35 KO HeLa cells ( Figure 2B). Under these conditions, in control cells and following RNAi-mediated suppression or CRISPR-Cas9-mediated knockout of SNX27 CTR1 localised to the cell peripherally or LAMP1negative intracellular compartments. SNX27 does not therefore make a significant contribution to the copper-dependent sorting of CTR1.
To ensure that the observed mis-sorting and reduction of CTR1 from the cell surface was specific to loss of retromer, we re-expressed VPS35 in VPS35 KO cells and examined the surface levels of CTR1 using flow cytometry. Upon re-expression of VPS35 the cell surface levels of CTR1 were restored to control levels ( Figure 3A). This recovery of CTR1 levels at the cell surface following re-expression of VPS35 was also observed using surface biotinylation ( Figure 3B). Together these data demonstrate that internalised CTR1 is retrieved from entering lysosomes and promoted for recycling back to the cell surface through a retromer-dependent pathway. Retromer is therefore required for controlling the cell surface residency of CTR1.

Internalized CTR1 resides on retromer positive endosomes
Following a 30 minute exposure to extracellular copper, internalised CTR1 has been reported to localise to EEA1 and transferrin receptor positive early endosomes (Clifford et al., 2016). Therefore, we next sought to visualise whether following its copper-induced internalisation CTR1 traffics via a retromer-positive endosomal route. Cells were surface labelled on ice with an endogenous antibody to CTR1 and subsequently incubated at 37°C in excess copper-containing media (200 µM CuCl 2) or standard growth media for 30 minutes prior to immunofluorescence processing. Cells were co-labelled with antibodies against endogenous CTR1 (red) and VPS35 (green). As anticipated, in cells maintained in standard low copper growth media CTR1 remained at the cell surface. Whereas, in cells where CTR1 had undergone copper-induced internalisation we found CTR1 extensively localised to VPS35-positive endosomes. Quantification using Pearson's correlation determined a significant increase in the colocalization between CTR1 and VPS35 in cells exposed to copper ( Figure 4A).
To determine whether this is a ubiquitous trafficking route for CTR1 we performed this CTR1 copper-induced internalisation assay in two different cell lines, H1299 and H1975 cells, both human non-small-cell lung cancer (NSCLC) cell lines. Notably, CTR1 was readily internalised in response to elevated copper in both these cell lines. As observed in HeLa cells ( Figure 4A), in copper treated H1299 and H1975 cells internalised CTR1 localised to VPS35-positive endosomes and this correlated with a significant increase in the Pearson's correlation between CTR1 and VPS35 (Figure 4 B and C). Together, these data provide further supporting evidence that in response to elevated extracellular copper internalised CTR1 is transported through retromer-positive endosomes.

Retromer-dependent CTR1 trafficking is vital for copper homeostasis
Cellular adaptation to the ever-changing extracellular environment is fundamental to cell homeostasis and survival. Through regulating the constant flux of transmembrane proteins, the endosomal network is fundamental to the remodelling of the cell surface in response to extracellular cues (Curnock et al., 2019). To examine the importance of retromer-dependent CTR1 trafficking for copper homeostasis, we established a protocol to periodically incubate cells for 1 hour in high copper containing media (200 μM CuCl 2) followed by a 1 hour incubation in normal growth media to effect a copper washout ( Figure 5). This revealed a tight reciprocal coupling between CTR1 expression at the cell surface and the presence of elevated copper ( Figure 5). Thus, upon addition of high copper CTR1 underwent endocytosis and underwent recycling back to the cell surface following copper washout.
Entirely consistent with retromer regulating the recycling of internalised CTR1, the ability of cells to adapt to periodic exposure to elevated copper was completely lost upon retromer depletion ( Figure 5).
To appreciate the role of retromer-dependent CTR1 trafficking in copper homeostasis we next performed MTT assays to assess the effect of increasing extracellular copper on metabolic activity as an indicator of cell viability. This colorimetric assay measures the ability of NAD(P)H-dependent oxidoreductases and dehydrogenases localized to cytoplasm and mitochondria to reduce MTT, a water-soluble tetrazolium dye, to a purple insoluble formazan (Berridge et al., 2005). Control and VPS35 KO HeLa cells were subjected to nine extracellular copper (CuCl 2) concentrations from 3.125 µM to 800 µM for 48 or 72 hours and MTT reduction was measured by absorbance at 595 nm. In control cells after exposure to 400 µM copper for 48 hours a dramatic decline in metabolised MTT correlating with a decrease in cell viability was observed ( Figure 6A). Strikingly, compared to control cells, VPS35 KO cells showed significantly reduced sensitivity to higher concentrations of extracellular copper. After 48 hours exposure to 400 µM copper, VPS35 KO cells still maintained the ability to metabolise MTT ( Figure 6A). With prolonged exposure to copper for 72 hours, this difference in copper sensitivity between control and VPS35 KO cells became more pronounced ( Figure 6B). After 72 hours, the toxic effects of the copper exposure were observed at lower concentrations in control cells however this was not as evident in VPS35 KO cells ( Figure 6B). Importantly, we found that sensitivity to copper was restored upon reexpression of VPS35, thus demonstrating that the ability of the cell to sense and respond to toxic levels of extracellular copper relies on retromer function ( Figure 6C).

Retromer expression is required for maximal cisplatin toxicity
In addition to its role as the main transporter responsible for the cellular influx of copper, CTR1 has also been linked to the intracellular accumulation of platinum and therefore also platinum-containing cancer drugs (Howell et al., 2010). The efficiency of anti-tumour platinum-based drugs is directly related to the concentration of the drug that enters the cell.
There is evidence that CTR1-dependent uptake controls the cytotoxicity of the widely used chemotherapeutic drugs cisplatin, carboplatin, and oxaliplatin (Holzer et al., 2006). Studies examining ovarian cancer cell lines and NSCLC patients samples identified that changes in CTR1 expression were linked to acquired cisplatin resistance and poor clinical outcome, whilst knockout of CTR1 in a murine model was shown to eliminate the responsiveness of tumours to cisplatin (Holzer et al., 2004;Jandial et al., 2009;Kim et al., 2014;Larson et al., 2009).
We have demonstrated that CTR1 surface abundance relies on retromer expression and knockout of VPS35 leads to a reduction in cellular sensitivity to extracellular copper. Therefore, given the evidence suggesting a dependency on CTR1 for cisplatin uptake and sensitivity, we next examined whether perturbation of retromer function affected cell viability in response to cisplatin exposure. We incubated control and VPS35 KO HeLa cells with nine increasing concentrations of cisplatin (from 0.39 μM to 100 μM). After 48 hours, a significant disparity in the sensitivity of control and VPS35 KO cells to cisplatin concentrations above 25 μM was observed ( Figure 7A). These MTT assays revealed that in the absence of retromer, HeLa cells were partially desensitised to the toxic effects of cisplatin. To exclude that this effect was unique to HeLa cells, we also examined the human NSCLC cell line H1975. We reasoned that this was a suitable cell line to test this hypothesis as platinum-based chemotherapy drugs such as cisplatin and carboplatin form part of standard therapies commonly used to treat NSCLC (Cosaert and Quoix, 2002). Furthermore, our immunofluorescence analysis demonstrated that internalised CTR1 localised to VPS35positive endosomes ( Figure 4C) indicating that CTR1 undergoes an endosomal trafficking route in these cells.
To first validate that retromer is required for CTR1 cell surface residency in this NSCLC cell line, surface biotinylation was performed following population based CRISPR-Cas9mediated knockout of VPS35. Consistent with HeLa cells ( Figure 1A and 3B), a significant reduction in the cell surface levels of CTR1 was observed in VPS35-depleted H1975 cells ( Figure 7B). Notably, a loss of the copper transporting ATPase, ATP7A was also detected indicating that analogous to HeLa cells, in H1975 cells both the major cellular copper transporters responsible for maintaining copper homeostasis are affected by loss of retromer function. We next assessed using MTT assays whether perturbed retromer function effected sensitivity to cisplatin as observed in HeLa cells ( Figure 5A). Following 48 hours in the presence of cisplatin, VPS35-depleted H1975 cells displayed a trend towards reduced sensitivity to cisplatin compared to the control counterparts ( Figure 7C). Across different cell lines retromer is therefore required for cisplatin toxicity, implicating that disruption of retromer function may lead to a reduction in efficacy of platinum-based cancer drugs.

DISCUSSION
Regulated copper homeostasis is fundamental in mammalian cells. Copper is an essential substrate for cell growth and differentiation, a cofactor of many enzymes and even recently identified as necessary for ULK1/2 signalling linking it to autophagy activity (Kim et al., 2008;Tsang et al., 2020). Therefore, it is unsurprising there are severe adverse effects associated with copper dyshomeostasis. Treatment for diseases resulting in acute cellular copper imbalances, such as Menkes disease are limited and prognosis is poor (de Bie et al., 2007).
As the only known mammalian importer of copper, CTR1 could be a prospective pharmacological target. Therefore, gaining better insight into the membrane trafficking of CTR1 will enhance our mechanistic understanding of the regulation of cellular copper import and distribution, leading to a better appreciation for how this may be manipulated in disease.
The endosomal system is fundamental to organising the spatial distribution of integral membrane proteins such as metal transporters (Cullen and Steinberg, 2018). In response to elevated extracellular copper, ATP7A traffics from the TGN to the cell surface where it extrudes excess copper out of the cell (Petris et al., 1996;Polishchuk and Lutsenko, 2013).
Maintaining the cell surface level of ATP7A, under basal conditions and following copper elevation, is reliant upon retromer and SNX27 orchestrating the endosomal retrieval and recycling of the internalised pump (Clairfeuille et al., 2016;Steinberg et al., 2013). This acute regulatory trafficking response requires a direct interaction between the PDZ binding motif present in the carboxy-terminal tail region of ATP7A and the PDZ-domain-containing SNX27 to mediate the endosomal delivery of ATP7A to the cell surface (Steinberg et al., 2013). In the present work we demonstrate that CTR1 is an additional retromer cargo and that retromer is required for the cell to adapt to changes in extracellular copper concentration. In response to elevated extracellular copper clathrin-dependent endocytosis of CTR1 is triggered, this serves to lower the CTR1 levels at the plasma membrane and restrict copper entry (Molloy and Kaplan, 2009). This cellular adaptation is vital to protect the cell from excessive copper accumulation. Here we demonstrate that retromer is required to facilitate the recycling of CTR1 back to the cell surface when extracellular copper levels are reduced ( Figure 5). This acute recovery of CTR1 plasma membrane levels suggests that, in the presence of excess extracellular copper, the internal pool of CTR1 is not readily degraded and remains endosomally associated and accessible for retromer-mediated recycling ( Figure   4A-C and Figure 8). We were unable to biochemically detect a direct interaction between CTR1 and retromer or retromer-associated adaptors (data not shown). We speculate therefore that interactions between CTR1 and retromer or retromer-associated endosomal recycling machinery are likely to be very transient and possibly even copper-dependent. It is interesting to postulate a system where copper modulates the conformation of CTR1 or even possibly key recycling machinery proteins to regulate their binding and dissociation, thereby coupling CTR1 recycling to extracellular copper levels.
Following the import of copper by CTR1, copper is transferred to the cytoplasmic copper chaperone ATOX1 (Kahra et al., 2016). CTR1 has a short 13-residue cytoplasmic C-terminal tail containing a highly conserved copper binding HCH motif that is necessary for relaying copper to the ATOX1 for delivery to copper-dependent enzymes (Kahra et al., 2016). NMR spectroscopy performed by Kahra et al., demonstrated that ATOX1 and the CTR1 tail peptide containing the HCH motif do not interact in the absence of copper, but copper exchange readily occurs from the copper loaded tail peptide to ATOX1. Copper transfer to high-affinity chaperones is therefore tightly controlled (Kahra et al., 2016).
For delivery of copper to the secretory pathway, the cytoplasmic copper chaperone, ATOX1, also interacts with the ATPases, ATP7A and ATP7B in a copper-dependent manner (Hamza et al., 1999;Larin et al., 1999;Pufahl et al., 1997). The copper-ATPases are known to form other copper-dependent protein interactions, such as with the antioxidant enzyme glutaredoxin 1 and the dynactin subunit p62 (Lim et al., 2006a;Lim et al., 2006b).
Phosphorylation of the ATPases have also been identified to provide another level of regulation within the copper transport system with evidence of copper-stimulated phosphorylation and hyperphosphorylation (Vanderwerf et al., 2001;Voskoboinik et al., 2003). Understanding determinants that regulate copper-induced trafficking for CTR1 and the ATPases could provide fundamental information on how to modulate this pathway in disease.
It is established that highly conserved methionine residues within the import pore formed by the CTR1 trimer are essential for copper binding and uptake activity ( Overall, through a variety of approaches we have shown that retromer is required for the cell surface localisation and copper-dependent recycling of CTR1 ( Figure 1A, C and Figure 3A, B and Figure 7B). We propose a working model where upon copper-or cisplatin-dependent internalisation CTR1 enters retromer positive endosomes. Here CTR1 can undergo one of two fates: in sustained elevated copper CTR1 remains associated with the maturing endosome and is degraded within the lysosome (Guo et al., 2004;Liu et al., 2007;Petris et al., 2003) or, as we demonstrate in the present work, following a reduction in extracellular copper levels CTR1 becomes available for retromer-dependent recycling back to the cell surface ( Figure 8). Exploring this model further will reveal greater insight into the regulation and trafficking of CTR1 and may suggest approaches to manipulate cellular copper import and distribution in disease and modulating cellular sensitivity and toxicity to platinum-based treatments.

Antibodies
Antibodies used in the study were: (

Quantitative Western blot analysis
For Western blotting, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4-12% precast gels (Invitrogen). Proteins were transferred from gels to PVDF membrane (Immobilon-FL, pore size 0.45 μm, Millipore, and quantification were carried out using an Odyssey infrared scanning system (LI-COR Biosciences).

Cell surface biotinylation
Cells were surface biotinylated 72 h post siRNA transfection or after the indicated treatment with membrane-impermeable biotin (Thermo) at 4 °C to prevent endocytosis. Postbiotinylation, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor EDTA-free tablets, pH 7.5 and lysates were cleared by centrifugation.
Equal amounts of protein from the control, indicated knockdown, knockout or treatment lysate were then added to streptavidin Sepharose to capture biotinylated proteins.
Streptavidin beads and lysates were incubated for 30 mins at 4 °C before washing in PBS containing 1.2 M NaCl and 1% Triton X-100. Proteins were eluted in 2× NuPAGE LDS Sample Buffer (Life Technologies) by boiling at 95 °C for 10 min, then separated by SDS-PAGE and subjected to quantitative Western blot analysis (as described above).

Immunofluorescence
Cells were washed three times in PBS, fixed in 4% paraformaldehyde (PFA) and then washed in PBS and permeabilized with 0.1% Triton X-100 or saponin. Fixed cells were then blocked in PBS containing 1% BSA and incubated with primary antibodies and appropriate secondary antibodies (Alexa Fluor; Thermo Fisher Scientific) in PBS containing 0.1% BSA.

Antibody uptake assays
For the CTR1 trafficking assays, cells were seeded onto sterile coverslips. The following day, cells were surface labelled by incubation with an antibody against CTR1 for 30 minutes on ice. Cells were then incubated at 37°C in excess copper-containing medium (200 µM CuCl2) or standard growth medium for either 30 minutes or 2 hours as indicated, at which point, cells were fixed in 4% PFA and processed for immunofluorescence staining as described above.

Flow cytometry
Cells were washed three times in PBS (without Ca or Mg), and then incubated for 20 minutes with Accutase to detach cells. 200,000 resuspended cells were transferred to 5 ml round-bottom polystyrene tubes (BD Biosciences). Cells were reconstituted in PBS (without Ca or Mg), containing 1% BSA and incubated with rabbit monoclonal CTR1 antibody (ab129067) or an isotype control antibody (Biolegend, clone Poly29108) for 1 hour on ice.
Cells were washed once in ice-cold PBS (without Ca or Mg) and incubated for 30 min on ice with Alexa Fluor 647 conjugated donkey anti-rabbit secondary antibody (Invitrogen, A31573) in PBS (without Ca or Mg), containing 5% BSA. The cells were washed again and resuspended in PBS (without Ca or Mg) containing 1% BSA and propidium iodide.

MTT assay
Cells were seeded into 96-well plates at a density of 8 × 10 3 cells/well in DMEM without phenol-red supplemented with 10% (v/v) FBS. The following day, cells were treated with serial dilutions of copper (CuCl2 3.125 µm to 800 µm) or cisplatin (0.39 μM to 100 μM). Each condition and an untreated control were performed in quadruplicate. After incubation at 37°C with 5% CO2 atmosphere for either 48 or 72 hours as indicated, 20 μl 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide; Life Technologies M6496) was added to each well, and the plates were incubated for an additional 3.5 hours at 37°C. MTT was removed and 150 μl DMSO was added to each well and mixed thoroughly by pipetting.
MTT absorbance was read at 595 nm using a microplate reader. The average of the quadruplicate reading for each condition was expressed as percentage of the untreated control.

Image acquisition and analysis
Microscopy images were collected with a confocal laser-scanning microscope (SP5 AOBS; Leica Microsystems) attached to an inverted epifluorescence microscope (DMI6000; Thermo Fisher Scientific). A 63× 1.4 NA oil immersion objective (Plan Apochromat BL; Leica Biosystems) and the standard SP5 system acquisition software and detector were used.
Images were captured using Application Suite AF software (version 2.7.3.9723; Leica Microsystems) and then analyzed with Volocity 6.3 software (PerkinElmer). For colocalization studies, Pearson's correlation (measuring the correlation in the variation between two channels) was measured using Volocity 6.3 software (PerkinElmer).

Statistical Analysis
All statistical analyses were performed using Prism 7 (GraphPad Software). For comparing two groups, statistical analysis was performed using student's t test (unpaired). For multiple comparisons, one-way ANOVA followed by Dunnett's multiple comparison test were used.
Data distribution was assumed to be normal, but this was not formally tested. All quantified Western blot and confocal colocalization data are the mean of at least three independent experiments. Graphs represent means and SEM.

Funding
This work was supported from awards to PJC from the MRC (MR/P018807/1), Wellcome Trust (  In response to elevated copper or exposure to platinum-based drugs, such as cisplatin, CTR1 is endocytosed from the plasma membrane and sequestered on retromer-positive endosomes. In prolonged high copper or cisplatin conditions, CTR1 remains associated with the maturing endosome. Upon removal of extracellular copper or cisplatin, CTR1 is recycled back to the plasma membrane in a retromer-dependent manner.