© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1711-1721 doi:10.1242/jcs.173351

RESEARCH ARTICLE Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu+ uptake system Rebecca J. Clifford, Edward B. Maryon and Jack H. Kaplan*

ABSTRACT superoxide dismutase (SOD), as a co-factor for cytochrome c Cu ion (Cu) entry into human cells is mediated by CTR1 (also known oxidase, in neurons for neurotransmitter and brain peptide as SLC31A1), the high-affinity Cu transporter. When extracellular Cu biosynthesis, and in the extracellular matrix as a co-factor for lysyl is raised, the cell is protected against excess accumulation by rapid oxidase in collagen cross-linking (Iakovidis et al., 2011). However, internalization of the transporter. When Cu is lowered, the transporter because of its ready non-specific reaction with many amino acid side returns to the membrane. We show in HEK293 cells overexpressing chains and ability to undergo Fenton chemistry and generate reactive CTR1 that expression of either the C-terminal domain of AP180 oxygen species, Cu can be damaging at excessive levels. The cellular (also known as SNAP91), a clathrin-coat assembly protein that homeostasis of Cu is accomplished by a Cu proteome consisting of sequesters clathrin, or a dominant-negative mutant of dynamin, uptake transporter(s), specific metallochaperones, and glutathione decreases Cu-induced endocytosis of CTR1, as does a dynamin (GSH) that deliver cellular Cu to its target proteins and exporters that inhibitor and clathrin knockdown using siRNA. Utilizing imaging, protect against excess accumulation (Harrison et al., 2000; Maryon siRNA techniques and a new high-throughput assay for endocytosis et al., 2013b). The main high-affinity Cu uptake protein, CTR1, employing CLIP-tag methodology, we show that internalized CTR1 delivers Cu to the intracellular milieu, whereas under low (normal) accumulates in early sorting endosomes and recycling compartments cellular Cu the export proteins ATP7A and ATP7B, Cu-activated P- (containing Rab5 and EEA1), but not in late endosomes or lysosomal type , are responsible for delivering Cu to the proteins in the pathways. Using live cell fluorescence, we find that upon extracellular secretory pathway. When intracellular Cu builds up, these trans- Cu removal CTR1 recycles to the cell surface through the slower- Golgi-resident proteins bud off in the membranes of vesicles into recycling Rab11-mediated pathway. These processes enable cells to which the excess Cu has been pumped and fuse with the plasma dynamically alter transporter levels at the plasma membrane and membrane to expel excess cellular Cu (Hung et al., 1997; Petris et al., acutely modulate entry as a safeguard against excess cellular Cu. 1996). This is of clinical significance as Menkes disease and Wilson disease are associated with inherited dysfunction of ATP7A and KEY WORDS: CTR1, Regulatory endocytosis, Metal ion ATP7B, respectively (Daniel et al., 2004). Elevated Cu levels have homeostasis, Cu transporter recycling been implicated in tumor growth and have made Cu systems promising targets for anticancer therapy (Gupte and Mumper, 2009; INTRODUCTION Safi et al., 2014). The plasma membrane content of a transporter determines the total Cellular Cu homeostasis and protection against the potential toxic flux of its substrate into (or out of) the cell. There is a need to regulate effects of excess intracellular Cu are achieved by relocation of the cellular composition to either protect against the toxic effects of Cu-ATPases. Just as the Cu exporters ATP7A and ATP7B respond certain substrates, as with elevation of P-glycoprotein and drug to elevated intracellular Cu, the Cu uptake protein CTR1 (also resistance (Bradley et al., 1989), or to ensure substrate is absorbed known as SLC31A1) can respond to elevated extracellular Cu. We under low substrate conditions, as in the amino acid permease in showed previously that excess extracellular Cu caused a loss of yeast (Ghaddar et al., 2014). In these cases, transporter synthesis or plasma membrane CTR1, and proportional lowering of Cu entry degradation is regulated. There are many hormone-regulated rate. This internalization protects against Cu toxicity (Molloy and transporters, among them the action of insulin on the trafficking of Kaplan, 2009). When extracellular Cu was returned to normal GLUT4 (Klip et al., 2014). In the present work, we levels, CTR1 recycled to the plasma membrane. We termed this provide evidence on the uptake mechanism and recycling pathway process regulatory endocytosis and proposed that it was a defense utilized by mammalian cells to regulate the uptake of Cu ions against acute elevations of extracellular Cu. The process could be (hereafter referred to as Cu), an essential but toxic micronutrient. The repeated several times, was seen in a wide range of cells, and was an regulation is unique among transporters in that it is controlled by the acute regulatory response to elevated medium Cu (Molloy and extracellular level of the substrate and acts directly on the transporter in Kaplan, 2009). CTR1 is a small protein of 190 amino acid residues a dynamic, acutely reversible mechanism that depends upon medium with three transmembrane segments, a variable extracellular amino Cu and not alterations in synthesis or degradation of the transporter. terminus and a highly conserved intracellular carboxyl terminus of Cu is essential for normal mammalian cell physiology. It is about 15 residues (Kaplan and Maryon, 2016). During a recent utilized as a co-factor in many enzymatic processes including: functional mutational analysis, we observed that mutations in the protection against as a co-factor in final three highly conserved residues (His-Cys-His) resulted in high rates of transport and the mutant CTR1 did not undergo regulatory Department of Biochemistry and Molecular Genetics, University of Illinois College of endocytosis in elevated Cu (Maryon et al., 2013a). The basis for Medicine, Chicago, IL 60607, USA. this loss of regulatory endocytosis is not understood. In order to *Author for correspondence ([email protected]) adequately describe cellular Cu regulation it was necessary to learn more about the internalization process, and the intracellular pathway

Received 23 April 2015; Accepted 2 March 2016 for internalization and recycling of CTR1. Journal of Cell Science

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In the present work, we have investigated the Cu-dependent binds to CTR1 proteins at the plasma membrane. Cells were then CTR1 internalization and recycling process, and sought to identify treated with or without excess Cu (30 min) before imaging. Without the trafficking pathway in this acute regulatory endocytosis and added Cu, CLIP-labeled CTR1 (green) was at the plasma recycling. We employed an expression system in which HEK cells membrane, whereas Tf (red) was internalized into vesicular overexpress CTR1 under the control of a tetracycline-sensitive structures within the cell (Fig. 1A, left panel). After Cu treatment promoter to minimize effects of long-term growth with excessive (Fig. 1A, right panel), CLIP-labeled CTR1 was internalized into Cu uptake. CLIP-tagged CTR1 was also utilized, providing the punctate structures throughout the cytoplasm. Tf was internalized in ability to view cells in real-time using live cell imaging or to utilize a both low and excess Cu-treated cells (Fig. 1A, left and right panels). novel fluorescent plate assay technique for quantitating endocytosis. Cu-treated cells showed internalized CTR1 in vesicle structures also We find that CTR1 is internalized in a clathrin-mediated endocytic containing Tf (merged images as yellow). Tf binds to transferrin (CME) pathway when cells are exposed to excess Cu. Vesicles receptor (TfR) at the surface and the ligand–receptor complex is carrying CTR1 fuse with the early endosome, where CTR1 is then endocytosed into clathrin-coated vesicles. To determine if the effect sorted into recycling compartments and returned to the cell surface of Cu treatment is specific to CTR1, or whether other plasma upon Cu removal. Depletion of Rab11 GTPases, known to regulate membrane proteins are also internalized, we compared the the slow recycling pathway, prevents the return of endocytosed localization of CTR1 with that of either TfR (Fig. 1B) or β- CTR1, establishing its role in CTR1 recycling. This substrate- catenin (Fig. 1C). HEK-FLAG–CTR1 cells were treated with dependent regulatory recycling is unique among metal ion transferrin with or without 30 µM Cu (30 min), fixed, and probed transporters and provides new information on the cell of for anti-FLAG (for CTR1) and TfR (Fig. 1B) or β-catenin (Fig. 1C). Cu transporters and their roles in the regulation of Cu homeostasis. Confocal imaging shows that CTR1 follows a pathway like that of TfR when cells are treated with Cu. DAPI staining was used for cell RESULTS counts, and DAPI stain intensity varied somewhat because of Internalized CTR1 co-localizes with early sorting endosomes reagent incubation duration, manufacturer product lot, and length of and endocytosed transferrin time prior to imaging slides. Quantification of CTR1 co-localization In elevated extracellular Cu, cell surface CTR1 is removed from with TfR was determined using ImageJ software (NIH) with the the plasma membrane (Molloy and Kaplan, 2009; Petris et al., Coloc2 plug-in, and displayed in Table 1 as Manders’ coefficients. 2003). To visualize CTR1 trafficking, we labeled CTR1 proteins Similar co-localization patterns of CTR1 and TfR after rapid in HEK-CLIP–CTR1 cells using CLIP-Surface fluorescent internalization suggest a common pathway of cargo sorting and/or antibodies and then visualized the localization of CTR1 after internalization. The effect of Cu on internalization is specific to 30 min with or without 30 µM excess Cu. The CLIP tag is a 20 kDa CTR1 and not a result of internalization of whole membrane sheets, mutant of the human DNA repair protein O6-alkylguanosine-DNA as β-catenin is not internalized in response to Cu (Fig. 1C). Co- alkyltransferase (AGT) that reacts specifically and rapidly with localization of CTR1 with β-catenin is more prominent before Cu benzylcytosine derivatives covalently labeling the CLIP-tag with a treatment, when CTR1 is mainly at the plasma membrane. Upon Cu synthetic probe. For live cell imaging (Fig. 1A), HEK-CLIP–CTR1 addition, CTR1 internalizes and co-localization with β-catenin is cells were loaded with fluorescent transferrin (Tf), which binds to significantly decreased (Table 1). the transferrin receptor and enters the cell via clathrin-dependent To determine the nature of the vesicles containing internalized endocytosis (Mayle et al., 2012), and CLIP-Surface label that only CTR1, we treated HEK-FLAG–CTR1 cells with or without Cu,

Fig. 1. CTR1 co-localization with membrane or endosome markers after Cu treatment. (A) HEK-CLIP–CTR1 cells were incubated with CLIP-Surface–488 (green), followed by treatment with or without 30 µM Cu and with fluorescent transferrin (red) (30 min), and live cell imaging was performed. (B–D) For fixed cell immunofluorescence, HEK-FLAG–CTR1 cells were treated with or without Cu, fixed cells were labeled with anti-FLAG for CTR1 and either anti-TfR (B), anti-β-catenin (C), or antibody against early endosome marker EEA1 (D). (E) MDCK-FLAG–CTR1 cells treated with or without Cu were double-labeled with anti-FLAG for CTR1 and anti-EEA1. (F) SKCO15 cells transiently transfected to express FLAG- tagged CTR1 were treated with or without Cu (30 min), fixed, probed with anti-FLAG (CTR1). (G) HEK-CLIP–CTR1 cells treated with or without 30 µM Cu (30 min), fixed, and labeled with anti-TfR, anti-EEA1 and DAPI. Stained nuclei are visible in blue and merged fluorescence is displayed in yellow. Scale bars: 10 µm. Journal of Cell Science

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Table 1. Quantification of co-localization between CTR1 and specified that increasing treatment duration with 10 µM Cu had a profound proteins during no-excess-Cu treatment or with 30 µM Cu treatment for effect on CTR1 internalization, with ∼30% of surface CTR1 30 min internalized after 30 min and over 60% after one hour, as visualized Level of co-localization in plate reader well scan images (Fig. S1C) and quantified (Fig. (Manders’ coefficient) S1D). Cu concentrations greater than 20 µM resulted in nearly 90% Direction *P<0.05 CTR1 internalization (Fig. S1E). Protein 1 Protein 2 −Cu +Cu of change **P<0.01 Endocytosis is dependent on clathrin and dynamin CTR1 β-catenin 0.630±0.062 0.413±0.012 ↓ * CTR1 TfR 0.201±0.076 0.588±0.057 ↑ * Excess Cu addition causes internalization of CTR1 (Fig. 1). The CTR1 EEA1 0.119±0.026 0.321±0.030 ↑ ** effect of Cu on CTR1 localization occurs in multiple cell types TfR EEA1 0.226±0.057 0.240±0.050 ↔ (Fig. 1E,F) and is specific to CTR1, as it does not cause increased CTR1 Rab5 0.118±0.022 0.319±0.047 ↑ * endocytosis of surface proteins such as β-catenin or TfR CTR1 Rab4 0.128±0.051 0.289±0.058 ↑ * (Fig. 1C,G). The endocytic route of TfR is well-studied as a ↑ CTR1 Rab11 0.209±0.040 0.518±0.159 * model for CME. After Tf binds to the TfR, the ligand–receptor CTR1 Rab7 0.164±0.027 0.151±0.059 ↔ CTR1 Rab9 0.133±0.011 0.171±0.007 ↔ complex is endocytosed into vesicles that are transported to the early endosomes where TfR continues along the recycling pathway back Manders’ coefficient values were determined from background-subtracted confocal merge images obtained from >3 independent experiments, and to the cell surface (Mayle et al., 2012). To determine if the CTR1 displayed as mean±standard error. Asterisks denote P-values <0.05. that is endocytosed after Cu treatment follows a clathrin-dependent Arrows indicate increased (↑), decreased (↓) or unchanged (↔) levels of endocytic pathway similar to that of TfR, we expressed dominant- co-localization between protein 1 and protein 2 after Cu treatment. negative forms of proteins dynamin-1 or carboxyl-domain- truncated clathrin coat assembly protein AP180 (also known as fixed and double-labeled with antibodies against FLAG (CTR1, SNAP91). These mutant proteins, designated DynK44A and red) and early endosome marker EEA1 (green, Fig. 1D) (Mu et al., AP180c, prevent endocytosis by disrupting clathrin vesicle 1995). Much of the CTR1 was localized in the early endosome after formation and budding (Damke et al., 1994; Hill et al., 2001) or 30 min Cu treatment (Fig. 1D, right panel). Quantification of CTR1 by sequestering clathrin (Zhao et al., 2001), respectively. In Fig. 2, co-localization with EEA1 is displayed in Table 1. The early HEK-CLIP–CTR1 cells were transiently transfected for three days endosomes are distributed in the cytoplasm and serve a sorting with plasmids containing DNA for either empty control (Fig. 2A), function, as proteins accumulate in their tubular extensions and AP180c (Fig. 2B), wild-type dynamin (Fig. 2C), or mutant vesicular regions that bud off and proceed to either recycling or DynK44A (Fig. 2D). Cells were then CLIP-Surface-labeled, and lysosomal pathways (Mellman, 1996). To determine if the treated with 30 µM Cu and Tf (30 min) before cell fixation, internalization of CTR1 is specific to HEK cells or whether other permeabilization and immunofluorescence. Transfection efficiency cells types also display a similar pattern of localization, we was determined by probing with either anti-HA for dynamin employed two different cell types, MDCK and SKCO15 cells. expression or anti-FLAG for AP180c expression (Fig. 2, third MDCK cells are derived from canine kidney, and SKCO15 cells column panels, shown in purple). Approximately 30% of all cells from a metastatic site of a human colon. Internalization of CTR1 expressed the exogenous mutants, and imaging fields containing during Cu treatment was observed in both MDCK (Fig. 1E) and both transfected and non-transfected cells were selected for analysis. SKCO15 cells (Fig. 1F). We also determined whether Cu affects the Cells expressing the exogenous dynamin or AP180c protein were amount of TfR endocytosis. We treated HEK-CLIP–CTR1 cells identified and marked with asterisks for easy visualization in CTR1 with or without 30 µM Cu (30 min), and then fixed cells were and TfR panels. In empty vector control or wild-type-dynamin- probed with anti-TfR and anti-EEA1 (Fig. 1G). Merged images expressing cells (Fig. 2A,C), TfR (red) was present mostly in show co-localization in yellow. No significant change in TfR perinuclear and cytosolic regions, as previously observed in Fig. 1B, co-localization with EEA1 occurred with Cu treatment (Table 1). and CLIP–CTR1 (green) was localized mainly in intracellular To quantify CTR1 internalization during various Cu treatments, vesicles in cells transfected with either empty vector control or wild- we devised a novel and flexible high-throughput CLIP assay that type dynamin and treated with excess Cu. These cells show co- detects only surface CTR1. This assay requires constitutive localization of CTR1 and TfR in cytosolic regions (Fig. 2A,C, expression of CLIP-epitope-tagged CTR1 and the use of a fourth column panels). In Fig. 2B and D, AP180c- or DynK44A- fluorescent CLIP-Surface label. The CLIP-Surface label is expressing cells (asterisks) show TfR (red) located mainly at the impermeable, and will only label CLIP-tagged CTR1 proteins that surface membrane, demonstrating that dynamin- and clathrin- are exposed to the extracellular medium. After treatment with Cu to dependent endocytosis was impaired. Cells labeled for mutant initiate endocytosis, the CLIP label is added and allowed to bind to expression (asterisks) also cause CTR1 to be retained at the plasma CTR1 proteins at the cell surface. Endocytosed CTR1 is not labeled. membrane (Fig. 2B,D, left panels). CTR1 did not endocytose As we observed in previous experiments using cell surface during excess Cu treatment when either clathrin- or dynamin- biotinylation (Molloy and Kaplan, 2009), endocytosis was dependent pathways were impaired. Zoomed images demonstrate dependent on extracellular Cu concentration, incubation time and that CTR1 in these mutant-expressing cells co-localized with TfR at temperature. Biotinylation assays used to pull down surface CTR1 the plasma membrane (Fig. 2B,D, far right panels). It should be proteins after HEK-FLAG–CTR1 cells were treated with excess noted that although mutant expression of DynK44A or AP180c Cu at either 25°C or 37°C demonstrate that CTR1 endocytosis impairs clathrin-mediated endocytosis, the presence of endogenous is permissible at 37°C but is inhibited at room temperature clathrin and/or dynamin might still provide a route, though limited, (Fig. S1A,B). This temperature inhibition of CTR1 endocytosis of clathrin-mediated endocytosis. enabled us to incubate HEK-CLIP–CTR1 cells for sufficient times Using immunofluorescence imaging, we found CTR1 with CLIP label during the CLIP plate assay without causing endocytosis can be prevented by disrupting clathrin-mediated increased endocytosis. Using the CLIP plate assay, we determined pathways with mutant DynK44A or AP180c (Fig. 2). To quantify Journal of Cell Science

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Fig. 2. Inhibiting dynamin-1 or clathrin assembly prevents CTR1 endocytosis: imaging analysis. CTR1 endocytosis was prevented in HEK-CLIP–CTR1 cells expressing dominant-negative DynK44A–HA (D) or AP180c–FLAG (B), but not in cells transfected with control empty vector (A) or expressing wild-type dynamin-1 (DynWT, C). Surface CTR1 proteins were fluorescently labeled with CLIP-Surface label (CTR1), and then cells were treated with Tf and 30 µM Cu for 30 min. Cells were fixed and probed with anti-TfR and either anti-FLAG or anti-HA antibodies. Confocal images at 100× are displayed, and 4× zoomed sections are shown in far right panels. Asterisks indicate cells that contain exogenous expression of dynamin or AP180c. CTR1 and TfR merge is displayed in yellow. Scale bars: 20 µm in first four columns, 10 µm in far right column. the inhibition, we employed a high-throughput CLIP assay. The Dynasore. Dynasore inhibits dynamin-1, dynamin-2, and CLIP assay was used to determine how much CTR1 was retained at mitochondrial dynamin-like protein Drp1 (Macia et al., 2006). We the after 0, 30 or 100 µM Cu treatment for 30 min. tested whether Dynasore disrupts CTR1 endocytosis by treating Fig. 3A displays an example of a CLIP assay well scan visual HEK-FLAG–CTR1 cells with or without Dynasore for 24 h and readout using dual colorimetric analysis. Threshold values for the then with or without 30 µM Cu (30 min). Using biotinylation, we colorimetric display are user-defined (Fig. 3A), whereas quantified determined that Dynasore inhibits essentially all Cu-induced CTR1 fluorescence values of CTR1 internalization compared with endocytosis, thus, Cu-activated endocytosis of CTR1 occurs untreated control cells in histograms (Fig. 3B) were calculated and through a dynamin- and clathrin-dependent pathway (Fig. S2). displayed as mean percentage±standard error. Fig. 3B shows that We also employed siRNAs targeted for clathrin. Clathrin-targeted ∼55% of CTR1 was endocytosed in control cells. This is siRNA transfection decreases the expression of clathrin compared comparable with experiments using cell surface biotinylation with control siRNA, as shown by a reduction in clathrin (Fig. 3C,D). (Molloy and Kaplan, 2009). Cells that express mutant DynK44A To determine the effects of clathrin knockdown on CTR1 or AP180c had 20–30% more CTR1 retained at the cell surface than endocytosis, we employed cells transfected with control or controls. Because not all of the cells expressed DynK44A or clathrin siRNA, and then labeled CTR1 with CLIP-Surface AP180c (Fig. 2) and endogenous clathrin and/or dynamin might reagent, treated with 30 µM Cu (30 min), fixed, and probed with retain limited function, the extent of inhibition of endocytosis is anti-clathrin antibodies. Images of cells transfected with control somewhat underestimated in this assay. siRNA show internalized CTR1, and zoomed inset shows partial co- Selection of stable cells lines expressing DynK44A or AP180c is localization with clathrin after Cu treatment (Fig. 3E, left panel). difficult because of reduced cell viability over time, as functioning When clathrin levels were decreased by clathrin-targeted siRNA clathrin and dynamin are vital for cell health (Damke et al., 1994). (Fig. 3E, right panel), most CTR1 proteins are restricted to the Thus, we had performed short-term transient transfection of plasma membrane, suggesting that clathrin is required for DynK44A or AP180c over three days for studies described in Cu-induced CTR1 internalization. Figs 2 and 3, reducing the negative effects of clathrin and/or dynamin depletion. The amount of mutant versus endogenous CTR1 internalized after Cu treatment enters early, but not expression varied from cell to cell, making it difficult to accurately late, endosomes measure effects of CME inhibition. For more reliable quantification, During endocytosis, protein clusters formed in clathrin-coated pits we utilized a small molecule inhibitor that affects all of the cells, are pinched off from the plasma membrane by dynamin. The Journal of Cell Science

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resulting clathrin-coated vesicle is uncoated in the cytosol and fuses to early endosomes containing Rab5 (Mellman, 1996). Live cell imaging shows internalized CTR1 was present in Rab5-positive large early endosomes (Fig. 4A). Quantification of co-localization of CTR1 with Rab5 is displayed in Table 1. Early endosomes are the site for sorting cargo into one of two major pathways; lysosomal or recycling. Cargo trafficking through the lysosomal pathway is transported to the late endosome, and then to the lysosome. Rink et al. (2005) suggest that early endosomes can be converted into late endosomes by a process in which Rab7 replaces Rab5. Rab7 GTPases are known markers for late endosomes (Barbero et al., 2002). We double-labeled Cu-treated HEK-FLAG–CTR1 cells with anti-FLAG and anti-Rab7 antibodies to determine if co-localization occurs between CTR1 and Rab7. Co-localization of Rab7 with CTR1 was not seen in intracellular compartments after Cu treatment (Fig. 4B, left panel; quantification of co-localization in Table 1). Another GTPase, Rab9, is important for retrograde trafficking from endosomes to the trans-Golgi network (Barbero et al., 2002).

Fig. 3. Cu-induced CTR1 internalization is mediated by clathrin and dynamin pathways: plate assay. HEK-CLIP–CTR1 cells transfected with empty vector control, DynK44A, or AP180c were treated with 30 or 100 µM Cu for 30 min, and surface CTR1 was labeled with fluorescent CLIP- Surface–547. (A) An example of a plate scan from the CLIP plate assay using a 10×10 scan matrix for each well. Red indicates CLIP fluorescence Fig. 4. Internalized CTR1 co-localizes with early endosomes, but not with on surface CTR1, and green indicates lack of fluorescence. (B) Histogram late endosomes. (A) HEK-CLIP–CTR1 cells expressing GFP–Rab5 were showing the relative surface expression of CTR1 after Cu treatment labeled with CLIP-Surface–547, treated with 30 μM Cu for 30 min, and subject compared with untreated control cells, mean±s.e.m., n=6. (C–E) HEK-CLIP– to confocal microscopy. Intracellular compartments containing co-localized CTR1 cells were transiently transfected with siRNA against either scramble Rab5/CTR1 are visible in yellow (arrows). (B) HEK-FLAG–CTR1 cells treated control or clathrin-targeted siRNA. (C) Lysates from siRNA-transfected cells with 30 μM Cu for 30 min were fixed and double-labeled with anti-FLAG treated with or without excess Cu were collected and subject to western blot (CTR1) and either anti-Rab7 or anti-Rab9 antibodies. (C) HEK-CLIP–CTR1 analysis using anti-clathrin antibodies. (D) Cells were probed using anti- cells were transiently transfected with plasmids to express either GFP–Rab4, clathrin antibodies to show sufficient knockdown of clathrin (right panel) GFP–Rab5, or GFP–Rab11. Expression in total cell lysates was determined by compared with control (left panel). (E) siRNA-transfected cells were labeled western blot. (D,E) For live cell confocal fluorescence, transfected cells were with Surface-CLIP (red) and then treated with or without 30 µM Cu for labeled with CLIP-Surface and treated with 30 μM Cu for 30 min. CTR1 is 30 min prior to fixation and anti-clathrin (green) immunofluorescent probing. internalized from the plasma membrane during Cu treatment and then co- Zoomed insets of selected regions are displayed. Scale bars: 20 µm, 5 µm localizes (arrows) with Rab4 (D) and Rab11 (E) GTPases. Scale bars: 5 μmin in insets. A,D,E; 10 μminB. Journal of Cell Science

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Co-localization of Rab9 and CTR1 was present in few intracellular visualized in small punctate compartments positive for Rab11 compartments but the majority of CTR1 was not co-localized with near the plasma membrane. Similar to the recycling pattern Rab9 (Fig. 4B, right panel; Table 1). This suggests that CTR1 does described earlier (Molloy and Kaplan, 2009), we find that not enter lysosomal degradation pathways after short-term Cu- removal of excess Cu causes internalized CTR1 to begin its return mediated endocytosis. Longer Cu (16 h) treatment times were not to the cell surface in as little as 5 min (Fig. 5A, middle panel). Upon tested in this study, but have been previously reported to cause Cu removal, CTR1 is co-localized less abundantly with globular degradation (using 100 μM Cu) and some degradation after 3 h endosomes, and a large portion of CTR1 is present with Rab11 near (Petris et al., 2003). the plasma membrane (Fig. 5A), where Rab11 has been shown to regulate vesicle exocytosis (Takahashi et al., 2012). Time-resolved Cu-induced CTR1 endocytosis is reversed via recycling live cell fluorescence visualizes trafficking of CTR1/Rab11- pathways containing compartments to the cell surface within 5 min of Cu Cu-induced CTR1 endocytosis is reversible upon Cu removal removal from the extracellular growth media (Fig. 5B). Trafficking (Molloy and Kaplan, 2009). As early as 15 min after the removal of of CTR1/Rab11 recycling compartments to the plasma membrane extracellular Cu, CTR1 reintegration into the surface membrane can occurred over ∼90 s from the time of vesicle arrival at the plasma be detected. The rapid re-insertion of CTR1 after Cu removal membrane to apparent lateral dispersion of CTR1 cargo. Initial suggests that endocytosed CTR1 proteins are stored in intracellular time-resolved images were collected at 5 min post-Cu-removal. endocytic compartments until Cu depletion occurs. This, in To better visualize the return of CTR1 to the cell surface via combination with a lack of evidence supporting late endosomal Rab11 recycling pathways, we employed TIRF imaging. In Fig. 6A, trafficking (Fig. 4B), suggests that CTR1 is transported from the control cells not treated with Cu showed CTR1 in a diffuse pattern early sorting endosome to a recycling pathway. There are two main near the cell surface with few small vesicular structures co- routes for recycling proteins back to the cell surface after localizing with Rab11. When 30 μM Cu was added (30 min), CTR1 endocytosis; fast recycling is mediated by Rab4 GTPase and accumulated in larger punctate structures co-localized with Rab11 involves vesicle budding and tubule formation from the early (Fig. 6B). At 15 min after Cu removal, CTR1 was retained in endosome, whereas slower recycling is mediated by Rab11 GTPase. Rab11-positive endosomes near the cell surface (Fig. 6C) Proteins are trafficked from the early endosome to Rab11-positive suggesting CTR1 recycles through a Rab11-dependent reinsertion compartments and then to the cell surface (Takahashi et al., 2012). pathway. Endocytosed TfR recycles to the cell surface via Rab11- (Mayle Rab4 is often found in early endosomes containing Rab5, a et al., 2012) or Rab4-positive compartments (Sönnichsen et al., GTPase also located in sorting endosomes responsible for 2000). controlling endosomal fusion (Jopling et al., 2014; McCaffrey We utilized live cell fluorescence to visualize CTR1 localization et al., 2001). Fusion is frequently accompanied by the formation of with GTPase markers of recycling pathways. HEK-CLIP–CTR1 tubules that facilitate cargo recycling by developing into recycling cells were transiently transfected with GFP–Rab4, GFP–Rab5, or vesicles that can enable either fast recycling directly back to the GFP–Rab11, and Rab expression was assessed (Fig. 4C). plasma membrane or can translocate to perinuclear regions in the Transfected cells were labeled with CLIP-Surface, and treated cytoplasm and regulate the slower return to the plasma membrane with 30 μM Cu (30 min). Live cell images show CTR1 was via Rab11 (Mellman, 1996). The latter process has been shown to internalized into large sorting endosomes positive for Rab4 transport TfR back to the cell surface (Skjeldal et al., 2012). We (Fig. 4D, white arrows). Rab11 GTPases are found (Fig. 4E) in observed a fusion event of homotypic endosomes within ∼1 min of subdomains of the early endosome and in small compartments initial contact (Fig. S3). Upon fusion, the formation of Rab4- consistent with recycling carriers (Sönnichsen et al., 2000). Several positive tubules developed (Fig. S3, small arrow), although no Rab11-positive compartments that contain CTR1 (Fig. 4E, white CTR1 was detected in the tubule. Concurrently, a Rab4-negative arrows) reside in punctate structures localized at or near the plasma subdomain of the fusing endosome contained CTR1, and vesicular membrane. Quantification of CTR1 co-localization with Rab budding was observed during homotypic endosome fusion (Fig. S3, GTPases is displayed in Table 1. arrowhead). During Cu treatment, a large proportion of CTR1 aggregates in Based on immunofluorescence co-localization imaging, globular Rab4- and Rab5-positive endosomes, and some are trafficking of endocytosed CTR1 occurs through Rab11 recycling

Fig. 5. CTR1 is returned to the plasma membrane in Rab11- positive compartments. (A) Confocal images of cells treated with 30 μM Cu for 30 min and then Cu removed for 5 min show that endocytosed CTR1 is co-localized in small vesicles with Rab11 (arrows) after Cu removal. (B) Compartments containing co- localized Rab11 and CTR1 (arrows) are dispersed near the surface membrane at 5 min post-Cu-removal. Scale bars: 5 μm in A, 2.5 μm in B. Journal of Cell Science

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micronutrient in the diet. A balance is normally maintained between the uptake (predominantly by CTR1), the intracellular system responsible for its distribution to target proteins (through GSH and the metallochaperones), and the efflux transporters, Cu-activated ATPases ATP7A and ATP7B (Kaplan and Lutsenko, 2009). Internalization of CTR1 in the presence of excess extracellular Cu is reversible and CTR1 returns to the plasma membrane when extracellular Cu levels are reduced (Molloy and Kaplan, 2009). This regulatory endocytosis serves to protect the cell against excessive Cu accumulation by lowering the plasma membrane content of CTR1 and hence Cu entry. In the present work, we have utilized a range of imaging and siRNA approaches to characterize the trafficking process. There are several forms of endocytosis, classified as either clathrin-mediated or clathrin-independent endocytosis pathways (CIE) (Doherty and McMahon, 2009). In CME, cargo is recruited by adaptor proteins, such as the AP2 complex, which also bind clathrin and enable clathrin pit formation at the membrane. Scission of clathrin pits is performed by dynamin, and clathrin vesicles are then transported to distinct endocytic compartments (Hill et al., 2001). TfR is a well-studied that follows CME, a process enhanced by the addition of transferrin to extracellular Fig. 6. CTR1 is co-localized with Rab11 at the cell surface during media (Mayle et al., 2012). TfR is often used as a marker for CME exocytosis after Cu removal. TIRF fluorescence was used to visualize the and in recycling to the plasma membrane. CIE includes a multitude surface localization of CTR1 and Rab11 after no excess Cu treatment (A), 30 μM Cu treatment for 30 min (B), and return of CTR1 to the cell surface in of pathways that do not require the involvement of clathrin (Doherty Rab11-positive recycling vesicles after excess Cu was removed for 15 min (C). and McMahon, 2009). Scale bars: 20 μm. CME requires several steps for vesicle formation and cargo internalization; initiation of pit formation, plasma membrane protein pathways, but not through Rab4-positive tubules. To confirm this, cargo selection, clathrin coat assembly, dynamin-mediated scission, we employed siRNAs for Rab4 or Rab11. Fig. S4A shows the extent and clathrin uncoating (Mousavi et al., 2004). TfR binds diferric of siRNA-mediated knockdown of Rab4 or Rab11 using transferrin at the cell surface and the ligand–receptor complex is quantitative PCR. Approximately 80% of Rab mRNA was then internalized into clathrin vesicles (Mayle et al., 2012). These knocked down compared with scramble control siRNA. We vesicles fuse to sorting endosomes, where iron is released from the demonstrated that the return of internalized CTR1 to the cell Tf–TfR complex. Tf–TfR then enters Rab4-positive (van Dam and surface after 1 h of Cu removal was inhibited by ∼35% when cells Stoorvogel, 2002) or Rab11-positive (Takahashi et al., 2012) were transfected with Rab11 siRNAs, compared with control recycling compartments and recycles to the plasma membrane. We siRNA transfection (Fig. S4B). Silencing Rab4 did not have a have shown that impairing the function of clathrin at the plasma significant effect on CTR1 return. This suggests that endocytosed membrane inhibits both TfR and CTR1 endocytosis (Figs 2,3). In CTR1 is recycled from intracellular compartments to the plasma addition to clathrin, dynamin is necessary for CME as it allows membrane via the Rab11 recycling pathway. scission of the clathrin-coated pit from the plasma membrane (Hill et al., 2001). Expressing a non-functioning mutant DynK44A or DISCUSSION employing Dynasore also prevents CTR1 endocytosis (see Fig. S2). The modulation of plasma membrane transporter activity is an Thus, endocytosis of CTR1, like TfR, is clathrin- and dynamin- essential aspect of cellular regulation. The varying requirements for 1-dependent. CTR1 internalization occurs in several cell types substrates and metabolites during cell growth and differentiation (Fig. 1E,F), and based on our previous work, we hypothesize that and the fluctuations in substrate availability make this regulation the route of CTR1 endocytosis in those cell types is also clathrin- essential for homeostasis. The internalization phenomena mediated. we describe here that acts acutely on CTR1, the human Cu Clathrin vesicle formation and selection of cargo occurs by AP2 transporter, is triggered rapidly by substrate in the extracellular recruitment to the plasma membrane (Collins et al., 2002). The μ2 media and is rapidly reversed by lowering of extracellular Cu, and/or β2 subunits of AP2 recognize YXXØ or dileucine motifs on without the involvement of new transporter synthesis or degradation the cytoplasmic tail of transmembrane cargo proteins. CTR1 (Molloy and Kaplan, 2009). The internalization of CTR1 in contains a potential μ2-binding motif, YNSM, in its cytoplasmic response to excess Cu correlates with a decrease in Cu uptake into loop. Mutations in this motif of CTR1 showed decreased CTR1 the cell. internalization (Tsai et al., 2014). It seems likely that the YNSM Appropriate Cu homeostasis is vital in mammalian cells. Normal motif in CTR1 might be the site for μ2 binding. The affinity of μ2to serum contains about 1–2 μM total Cu (McMillin et al., 2009) and bind YXXØ motifs is regulated by phosphorylation of the Thr156 internalization of CTR1 is caused by an addition as low as 2–5 μM residue of μ2. Binding sites of μ2 are blocked when the subunit is Cu after exposure for 2 h, whereas the response to 20 μMCuis not phosphorylated preventing cargo protein recognition, but its complete in about 10 min (Molloy and Kaplan, 2009). Cu- phosphorylation results in a conformational change that allows concentration-dependence time courses show complex kinetics. binding to motifs on ligands (Jackson et al., 2010). The potential The acute regulatory response described in the present work might interactions between CTR1 and μ2, and the regulation of such be expected to play a role in the pulsatile appearance of the interactions by AP2 phosphorylation are currently being Journal of Cell Science

1717 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1711-1721 doi:10.1242/jcs.173351 investigated. In initial experiments we have attempted (unsuccessfully) to detect direct interactions between CTR1 and clathrin utilizing biotinylation. This might be because the interactions are transient or require greater sensitivity in their detection. Once cargo proteins are endocytosed, they are transported through a series of vesicles and tubules, regulated by Rab GTPases, which are involved in nearly every step of endocytic trafficking and govern the destination of cargo-carrying compartments (Stenmark, 2009). Rab5 GTPases are involved in the initial stages of endocytosis and are localized primarily in the large early sorting endosome. Cargo located in endocytosed CIE or CME vesicles enters the early endosome, where it is sorted into compartments targeted to either the degradation or recycling pathway. The late endosome is involved in the degradation pathway and contains Rab7 and Rab9 GTPases (Rink et al., 2005). Cargo in Rab7- or Rab9-enriched domains is typically degraded in the lysosome. Cargo that enters the Rab4- or Rab11- enriched compartments is recycled to the plasma membrane Fig. 7. Proposed model of endocytosis and recycling pathways of CTR1. (McCaffrey et al., 2001; Takahashi et al., 2012). Under conditions of elevated extracellular Cu, CTR1 is endocytosed from the Upon internalization, CTR1 resides in specific Rab-GTPase- plasma membrane by a clathrin- and dynamin-mediated (CME) process. enriched compartments. Rab5 and Rab4 are enriched in early Internalized CTR1 is then localized in early endosomes and, when Cu is removed from the external media, it is transported back to the plasma endosomes (Stenmark, 2009), and CTR1 co-localizes with Rab5 membrane in a Rab11-dependent recycling pathway. CIE, clathrin- and Rab4 in these large intracellular compartments during Cu independent pathway; EE, early endosome; Late endo, late endosome; treatment (Fig. 4A,D). Rab4 is involved in rapid cargo recycling Lyso, lysosome; RE, recycling endosome. (McCaffrey et al., 2001), which occurs by cargo-filled tubule and vesicle formation from the early endosome and transport directly to the plasma membrane. CTR1 was found in large Rab4-positive model found support in the observation that a mutant of the endosomes (Fig. 4D), but Rab4-postive tubules extending from the permease that had a higher intrinsic Vmax than the wild-type early endosome did not contain CTR1 (Fig. S3). This, along with transporter is largely resistant to substrate-induced endocytosis. our data showing that Rab4 knockdown does not impair CTR1 This effect and its interpretation show a remarkable similarity to the recycling (Fig. S4), suggests that Rab4 is likely not involved in the substrate-induced endocytosis of CTR1, the human Cu transporter direct trafficking and insertion of recycled CTR1 to the cell surface. (Molloy and Kaplan, 2009). The distribution of CTR1 between the Instead, a slowly recycling pool of CTR1 proteins is sorted from plasma membrane and intracellular sites can be modeled by a simple early endosomes to recycling endosomes, and is returned near the equilibrium where the rate constant governing the inward transition membrane by Rab11. Rab11 accompanies CTR1 in recycling from the plasma membrane is stimulated by elevated extracellular endosomes located in perinuclear regions, and in compartments Cu and the return steps from intracellular sites are Cu-independent. destined for the cell surface (Fig. 5). CTR1/Rab11-positive This simple model and the distributions at normal basal Cu (about compartments nearing the plasma membrane can be observed by 8% CTR1 is detected internally; Molloy and Kaplan, 2009) and at confocal and TIRF microscopy (Fig. 4E, Figs 5,6). When Rab11 100 μM Cu (about 80% is internal), suggests that there is an ∼40- function is impaired using siRNAs, the return of CTR1 to the fold increase in the rate of internalization at elevated Cu. The plasma membrane is largely inhibited (Fig. S4B). Thus, we propose quantitative aspects of this model are under investigation. a model in which Rab11 mediates transport of the CTR1 proteins We have previously reported that in a series of CTR1 mutants, via a slower recycling pathway (Fig. 7). truncations of the C-terminus or mutations in the terminal His-Cys- The regulation of a metal ion transporter by means of alterations His triad have a higher Vmax for transport than the wild-type in its abundance in the plasma membrane has been reported transporter. Furthermore, these mutants fail to undergo Cu- previously in yeast. However, the endocytosis of the Mn transporter dependent endocytosis (Maryon et al., 2013a). This recapitulates in response to the presence of Cd is a stress response, resulting in the observations on the yeast amino acid permease system, but with degradation of the transporter (Nikko et al., 2008). Similarly, CTR1 the substrate-dependent internalization is a dynamic endocytosis of the boron transporter in Arabidopsis thaliana,to reversible process involving endocytosis and recycling without avoid excess boron accumulation, occurs through transfer from the transporter degradation or ubiquitylation. Our recent mutation membranes to the vacuoles for degradation (Takano et al., 2005). studies on CTR1 have begun to define structural elements that are The general amino acid permease, Gap1, of yeast also undergoes important for the regulatory endocytosis. It is clear from our own endocytosis in response to elevated levels of substrates via a and earlier work (Guo et al., 2004) that mutations (M to L) in the ubiquitylation pathway (Ghaddar et al., 2014). It was concluded that important methionine triads (M150, 154) at the entry to the CTR1 the transporter engaged a conformation (caused by substrate pore, which render such CTR1 mutants unable to transport Cu, also binding) that would interact more efficiently with the endocytic are not internalized by excess extracellular Cu. Finally, the removal machinery (Cain and Kaiser, 2011). In later work, the endocytosis by substitution (188HCH to AAA) or truncation of the last seven or was assumed to occur by means of a transient permease nine residues also produce CTR1 proteins that transport Cu more conformation that allowed recognition by arrestin-like adaptors. rapidly and fail to internalize in the face of excess Cu (Maryon et al., This was interpreted as requiring that this conformation be stable 2013a). This led us to propose that either structural elements at the enough to increase its probability to interact with the adaptors. The cytoplasmic face of the protein must interact with the internalization Journal of Cell Science

1718 RESEARCH ARTICLE Journal of Cell Science (2016) 129, 1711-1721 doi:10.1242/jcs.173351 machinery (identified in the present work) in a Cu-dependent Antibodies fashion, or alternatively a specific Cu-dependent conformation Antibody dilutions used for immunoblotting were as follows: 1:10,000 of of the protein that is a part of the normal transport cycle interacts mouse anti-β-catenin (610153; BD Transduction Laboratories, San Jose, with the internalization machinery and, in mutants with elevated CA, USA), 1:5000 of mouse anti-GFP (11814460001; Roche, transport rates, the crucial protein conformation has an insufficient Indianapolis, IN, USA), 1:3000 rabbit anti-flag (A00170-40; Genscript, Piscaraway, NJ, USA). Peroxidase AffiniPure goat anti-mouse (115-035- dwell-time for productive interaction. 146; Jackson Immunoresearch, West Grove, PA, USA) or goat anti-rabbit The current study provides a model for the endocytosis of CTR1 (111-035-144; Jackson Immunoresearch) secondary antibodies were and regulation of CTR1 abundance at the cell surface (Fig. 7). CTR1 diluted 1:5000. Antibodies used for immunofluorescence were diluted at is internalized in clathrin-coated pits, and fission occurs by means of 1:1000; rabbit anti-EEA1 (ab2900; Abcam, Cambridge, MA, USA), dynamin-1. Disruption of either of these processes results in the mouse anti-transferrin-receptor (TfR; ab84036; Abcam), mouse anti-HA retention of CTR1 at the membrane. CTR1 proteins that are (sc-7392; Santa Cruz, Dallas, TX, USA), mouse anti-FLAG (A00013; internalized during Cu treatment are trafficked to compartments in Genscript), mouse anti-Rab9 (MA3-067; Affinity Bioreagents, Golden, the cytosol that are mediated by Rab GTPases. CTR1 endocytosis is CO, USA), mouse anti-Rab7 (ab50533; Abcam), mouse anti-Rab11 reversible and the removal of extracellular Cu enables CTR1 to (ab170134; Abcam), rabbit anti-Rab4 (PA3-912; Thermo Scientific, – – relocalize at the plasma membrane via Rab11-mediated recycling Rockford, IL, USA), and Surface-CLIP 547 or 488 (S9233S, S9232S; New England Biolabs, Ipswich, MA, USA). pathways to once again mediate Cu entry. This work characterizes the pathways used in regulatory endocytosis of CTR1. The Western blot analysis mechanism by which CTR1 internalization is triggered (by Protein samples were subject to SDS-PAGE and western blot analysis Cu acting at sites of lower affinity than transport sites) and the (Clifford and Kaplan, 2008). To obtain total cell lysates, cells were washed structural aspects of CTR1 essential to this process now need to be twice with PBS, harvested by scraping, and whole cells pelleted at 1000 g determined. for 5 min. Cell pellets were resuspended in cold lysis buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris pH 7.5). Suspensions MATERIALS AND METHODS were incubated end-over-end at 4°C for 1 h with protease inhibitors, and Cell lines and constructs cell extracts were cleared by centrifugation at 10,000 g for 10 min. Protein HEK293 and MDCK Flp-In T-REx cells overexpressing CTR1 are isogenic contents of lysates were determined by the Bradford Protein Assay and a tetracycline-regulated promoter allows CTR1 expression to be induced (BioRad, Hercules, CA, USA). Lysates were incubated at room in the presence of tetracycline in growth media (Maryon et al., 2007). temperature with 2× Laemmli sample buffer containing 10% β- Growth in maximal tetracycline results in a maximally ∼20-fold increase in mercaptoethanol (1 h). Samples were separated by 12% SDS-PAGE, CTR1 expression per cell (Maryon et al., 2013a), and over the course of transferred to polyvinylidene difluoride membranes and blocked with 5% several days growth in basal media does not result in re-distribution milk in PBS (1 h). Antibodies were diluted in PBS containing 1% milk of ATP7A or ATP7B or evidence of cytotoxicity. CTR1 sequences +0.1% Tween-20 and used for immunoprobing overnight (4°C). Following contain either a CLIP or FLAG epitope tag at their amino terminus, referred immunoprobing with antibodies, blots were washed 3 times with PBS to as HEK-CLIP–CTR1, HEK-FLAG–CTR1, MDCK-FLAG–CTR1, +0.1% Tween-20. Chemiluminescent Western Blotting Substrate (Thermo or SKCO15-FLAG–CTR1. All cell lines were grown in Dulbecco’s Scientific) was used for peroxidase detection and signal intensity was Minimal Essential Medium (DMEM) (Mediatech, Manassas, VA, USA) quantified on BioRad Chemidoc XRS using BioRad Quantity One Version supplemented with 25 mM HEPES buffer (Mediatech) and 10% 4.6.2 software and corrected for protein loading using anti-β-catenin tetracycline-free fetal bovine serum (Atlanta Biologicals, Flowery Branch, antibodies when appropriate. GA, USA). HEK293- and MDCK-Flp-In-T-REx-derived cell lines were also supplemented with 400 μg/ml Hygromycin B (Mediatech) and 6 μg/ml Microscopy Blasticidin (Research Products International Corp, Mt. Prospect, IL, USA). Cells were trypsinized and re-plated on either glass coverslips for fixed To induce expression of CTR1 in Flp-In T-REx cells, 1 μg/ml of tetracycline cell immunofluorescence, or on glass-bottom plates for live cell imaging. (Sigma, St. Louis, MO, USA) was added to the growth media for 48 h. Cell CTR1 expression was induced with tetracycline (48 h). For fixed cell lines were maintained by washing twice with phosphate-buffered saline imaging, cells were fixed at room temperature (10 min) using 4% (PBS) and incubating with 0.05% trypsin-EDTA (GE Healthcare Life paraformaldehyde, then washed with PBS, permeabilized and blocked Sciences, Logan, UT, USA) until cell detachment, then cells were re-plated (1% bovine albumin, 0.1% Triton X-100 in PBS). Cells were double- with fresh media at a 1:5–1:10 dilution. Cells were maintained in a labeled by probing with primary antibodies, consecutively, followed humidified incubator with 5% CO2 at 37°C. pEGFP-C1-Rab4, pEGFP-C1- by washing with PBS, and fluorescent secondary antibody incubation. Rab5, and pEGFP-C1-Rab11 plasmids were a kind gift from Dr B. Yue Goat anti-mouse and goat anti-rabbit fluorescent antibodies were (Park et al., 2010). pcDNA3.1/Dynamin1K44A-HA was a gift from obtained as FITC and Cy3 conjugates (Jackson Immunoresearch). Dr S. Schmid (Damke et al., 1994), and pFlag-CMV-2/AP180c construct Slides were covered with Vectashield mounting medium with DAPI was a kind gift from Dr J. Donaldson (National Institutes of Health, (Vector Laboratories, Burlingame, CA, USA), and sealed with a glass Bethesda, MD, USA). The pcDNA4/TO plasmid (Invitrogen, Grand Island, coverslip. Images were detected and analyzed using Zeiss LSM 5 Pascal NY, USA) containing the FLAG-tagged CTR1 gene insert was used to microscope (Carl Zeiss Microscopy, Thornwood, NY, USA) and transiently transfect SKCO15 cells, as recommended by the manufacturer’s quantification of co-localization was determined using ImageJ software protocols. For transfection of plasmids, Lipofectamine 2000 Reagent (National Institutes of Health) with Coloc2 plug-ins. Fluorescent live cell (Invitrogen) was used according to the manufacturer’s recommendations. imaging was performed using CLIP-Surface–488 or –547 (New England Cells were incubated with transfection reagent mixture for 24 h and then Biolabs) diluted 1:1000 in growth media to covalently label CTR1 at the media was replaced with fresh growth media for 48 h prior to selection, plasma membrane, as described in the manufacturer’s recommendations. antibiotic addition for stable cell line creation, or experimental usage for Cells were washed three times in growth media and then treated with transient transfections. Cell lines transfected with pEGFP plasmids were or without Cu and transferrin (Sigma) as indicated. Cell nuclei were selected using G418 antibiotic (Thermo Fisher, Waltham, MA, USA). stained with Hoescht (Thermo Scientific) for 5 min and cells were siRNAs against negative control, clathrin, Rab4A, or Rab11A (Qiagen, imaged with either standard confocal microscopy or total internal Valencia, CA, USA) were transiently transfected into HEK-FLAG–CTR1 reflection fluorescence (TIRF). TIRF imaging was performed using the cells using RNAiMAX Reagent (Invitrogen) according to the Zeiss Laser TIRF imaging system fitted with alpha Plan-Fluar 100×/1.45 manufacturer’s procedures, and then cells were incubated in fresh growth objective and Pecon XL TIRF S incubation system, and images analyzed media for 72 h prior to assay. with Zeiss AxioVision software. Journal of Cell Science

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CLIP plate assay Doherty, G. J. and McMahon, H. T. (2009). Mechanisms of endocytosis. Annu. HEK-CLIP–CTR1 cells were treated with tetracycline (1 μg/ml, 48 h) and Rev. Biochem. 78, 857-902. used in the CLIP assay when 80% confluent. Cells are treated with Cu (0– Ghaddar, K., Merhi, A., Saliba, E., Krammer, E. M., Prevost, M. and Andre, B. μ – (2014). Substrate-induced ubiquitylation and endocytosis of yeast amino acid 100 M) for the specified time at 37°C. CLIP-Surface 547 was added to the permeases. Mol. Cell. Biol. 34, 4447-4463. cells at a 1:500 dilution in growth media (30 min at room temperature), and Guo, Y., Smith, K., Lee, J., Thiele, D. J. and Petris, M. J. (2004). Identification of nuclei stained with Hoescht 33342 (Thermo Fisher). Cells were washed methionine-rich clusters that regulate -stimulated endocytosis of the three times with growth media and fluorescence was measured using a human Ctr1 copper transporter. J. Biol. Chem. 279, 17428-17433. FluoStar Omega Plate Reader (BMG Labtech, Cary, NC, USA). Individual Gupte, A. and Mumper, R. J. (2009). Elevated copper and oxidative stress in well scans of cells were performed using a 10×10 scan matrix. Relative cancer cells as a target for cancer treatment. Cancer Treat. Rev. 35, 32-46. fluorescence was determined using a standard curve of CLIP fluorescence Harrison, M. D., Jones, C. E., Solioz, M. and Dameron, C. T. (2000). Intracellular copper routing: the role of copper chaperones. Trends Biochem. from known cell numbers and individual wells were corrected for cell loss Sci. 25, 29-32. using Hoescht fluorescence. CLIP-Surface–547 is cell impermeable. Thus, Hill, E., van der Kaay, J., Downes, C. P. and Smythe, E. (2001). The role of CTR1 that remains on the cell surface after Cu treatment fluoresces, whereas dynamin and its binding partners in coated pit invagination and scission. J. Cell internalized CTR1 proteins do not. Biol. 152, 309-324. Hung, I. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D. and Gitlin, J. D. (1997). Biochemical characterization of the and qPCR functional expression in the yeast saccharomyces cerevisiae. J. Biol. Chem. 272, To measure knockdown of endogenous Rab4 and Rab11 mRNA, total RNA 21461-21466. from HEK-CLIP–CTR1 cells transfected with siRNA oligos against Rab4a, Iakovidis, I., Delimaris, I. and Piperakis, S. M. (2011). Copper and its complexes in Rab11a, or control scramble were prepared. Cell pellets from 60-mm tissue medicine: a biochemical approach. Mol. Biol. Int. 2011, 594529. culture dishes were used for RNA isolation using RNeasy/QIAshredder Jackson, L. P., Kelly, B. T., McCoy, A. J., Gaffry, T., James, L. C., Collins, B. M., ̈ (Qiagen, Valencia, CA, USA) as instructed by the manufacturer. First-strand Honing, S., Evans, P. R. and Owen, D. J. (2010). A large-scale conformational ® change couples membrane recruitment to cargo binding in the AP2 clathrin cDNA was made with SuperScript III Reverse Transcriptase (Thermo adaptor complex. Cell 141, 1220-1229. Scientific), and qPCR was performed with human Rab4a, Rab11a, or Jopling, H. M., Odell, A. F., Pellet-Many, C., Latham, A. M., Frankel, P., GAPDH primers purchased from Qiagen and SYBR Green Mastermix Sivaprasadarao, A., Walker, J. H., Zachary, I. C. and Ponnambalam, S. (Qiagen), using a BioRad CFX96 Touch™. The Livak method for relative (2014). Endosome-to-plasma membrane recycling of VEGFR2 receptor gene expression was used to quantify the expression of Rab4a and Rab11a tyrosine regulates endothelial function and blood vessel formation. in cells treated with siRNA or control [ΔΔCT=ΔCT (rab)-ΔCT(control)]. Cells 3, 363-385. Reactions and calculations were performed according to the manufacturer’s Kaplan, J. H. and Lutsenko, S. (2009). Copper transport in mammalian cells: special care for a metal with special needs. J. Biol. Chem. 284, 25461-25465. recommendations. Kaplan, J. H. and Maryon, E. B. (2016). How mammalian cells acquire copper: an essential but potentially toxic metal. Biophys. J. 110, 7-13. Acknowledgements Klip, A., Sun, Y., Chiu, T. T. and Foley, K. P. (2014). Signal transduction meets We would like to thank Dr N. Segev (UIC) and J. Donaldson (NIH) for helpful vesicle traffic: the software and hardware of GLUT4 translocation. Am. J. Physiol. discussions and our anonymous referees for their constructive input. Cell Physiol. 306, C879-C886. Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C. and Kirchhausen, T. Dev. Cell Competing interests (2006). Dynasore, a cell-permeable inhibitor of dynamin. 10, 839-850. Maryon, E. B., Molloy, S. A. and Kaplan, J. H. (2007). O-linked glycosylation at The authors declare no competing or financial interests. threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells. J. Biol. Chem. 282, 20376-20387. Author contributions Maryon, E. B., Molloy, S. A., Ivy, K., Yu, H. and Kaplan, J. H. (2013a). Rate and R.J.C. conducted most of the experiments and evaluated the data. E.B.M. designed regulation of copper transport by human copper transporter 1 (hCTR1). J. Biol. the new endocytosis assay and advised on the manuscript. J.H.K. conceived and Chem. 288, 18035-18046. directed the project and evaluated data. R.J.C. and J.H.K. wrote and revised the Maryon, E. B., Molloy, S. A. and Kaplan, J. H. (2013b). Cellular glutathione plays a manuscript. key role in copper uptake mediated by human copper transporter 1. Am. J. Physiol. Cell Physiol. 304, C768-C779. Mayle, K. M., Le, A. M. and Kamei, D. T. (2012). The intracellular trafficking pathway Funding of transferrin. Biochim. Biophys. Acta 1820, 264-281. This work was partially supported by the National Institutes of Health [grant 5P01 McCaffrey, M. W., Bielli, A., Cantalupo, G., Mora, S., Roberti, V., Santillo, M., GM067166]. Deposited in PMC for release after 12 months. Drummond, F. and Bucci, C. (2001). Rab4 affects both recycling and degradative endosomal trafficking. FEBS Lett. 495, 21-30. Supplementary information McMillin, G. A., Travis, J. J. and Hunt, J. W. (2009). Direct measurement of free Supplementary information available online at copper in serum or plasma ultrafiltrate. Am. J. Clin. Pathol. 131, 160-165. http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.173351/-/DC1 Mellman, I. (1996). Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12, 575-625. Molloy, S. A. and Kaplan, J. H. (2009). Copper-dependent recycling of hCTR1, the References human high affinity copper transporter. J. Biol. Chem. 284, 29704-29713. Barbero, P., Bittova, L. and Pfeffer, S. R. (2002). Visualization of Rab9-mediated Mousavi, S. A., Malerød, L., Berg, T. and Kjeken, R. (2004). Clathrin-dependent vesicle transport from endosomes to the trans-Golgi in living cells. J. Cell Biol. 156, endocytosis. Biochem. J. 377, 1-16. 511-518. Mu, F.-T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, Bradley, G., Naik, M. and Ling, V. (1989). P-glycoprotein expression in multidrug- R. G., Campbell, P. L., McCluskey, J., Yeo, J.-P., Tock, E. P. C. and Toh, resistant human ovarian carcinoma cell lines. Cancer Res. 49, 2790-2796. Cain, N. E. and Kaiser, C. A. (2011). Transport activity-dependent intracellular B.-H. (1995). EEA1, an early endosome-associated protein. EEA1 is a sorting of the yeast general amino acid permease. Mol. Biol. Cell 22, conserved alpha-helical peripheral membrane protein flanked by cysteine “ ” J. Biol. Chem. 1919-1929. fingers and contains a -binding IQ motif. 270, Clifford, R. J. and Kaplan, J. H. (2008). {Beta}-subunit overexpression alters the 13503-13511. stoicheometry of assembled na-K-ATPase subunits in MDCK cells. Nikko, E., Sullivan, J. A. and Pelham, H. R. (2008). Arrestin-like proteins mediate Am. J. Physiol. Renal Physiol. 295, F1314-F1323. ubiquitination and endocytosis of the yeast metal transporter Smf1. EMBO Rep. 9, Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. and Owen, D. J. (2002). 1216-1221. Molecular architecture and functional model of the endocytic AP2 complex. Cell Park, B., Ying, H., Shen, X., Park, J.-S., Qiu, Y., Shyam, R. and Yue, B. Y. J. T. 109, 523-535. (2010). Impairment of protein trafficking upon overexpression and mutation of Damke, H., Baba, T., Warnock, D. E. and Schmid, S. L. (1994). Induction of mutant optineurin. PLoS ONE 5, e11547. dynamin specifically blocks endocytic coated vesicle formation. J. Cell Biol. 127, Petris, M. J., Mercer, J. F., Culvenor, J. G., Lockhart, P., Gleeson, P. A. and 915-934. Camakaris, J. (1996). 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