The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification. Electronic supplementary material The online version of this article (10.1007/s00018-019-03105-5) contains supplementary material, which is available to authorized users.

drug resistance by reducing the concentration of chemotherapeutics below a cell-killing threshold. In addition, MDR transporters are also expressed in pharmacological barriers such as the blood-brain barrier, where they modulate the passage of drugs [3].
ABCB6 is widely expressed in many tissues, especially in the heart, liver, skeletal muscles [4], the red blood cells [5,6], and skin [7]. ABCB6 is a half transporter of 842 amino acids, containing a unique N-terminal region followed by the ABC core consisting of a transmembrane domain and a cytoplasmic nucleotide-binding domain. ABCB6 forms homodimers [8,9] and was shown to possess ATPase and transport activities after purification and functional reconstitution into liposomes [10]. At present, the subcellular localization of ABCB6 remains a matter of debate. In 2006, ABCB6 was described as a mitochondrial porphyrin transporter with an essential role in heme biosynthesis [8]. Subsequent studies have found ABCB6 to be dispensable for erythropoiesis [5,9], suggesting that mitochondrial porphyrin import may not depend on ABCB6. In addition, several research groups have identified ABCB6 in extramitochondrial compartments, challenging the paradigm linking the expression and function of ABCB6 to mitochondria. ABCB6 was detected in the plasma membrane of cells [11], the red blood cell membrane [5,9], melanosomes [12] and throughout the endolysosomal continuum [13][14][15][16][17]. However, the physiological function of ABCB6 in the endolysosomal compartment has remained elusive.
ABCB6 exhibits topological and sequential similarity to HMT (Heavy Metal Tolerance) family proteins (Supplementary Table 1). HMT-1 proteins in fission yeast (Schizosaccharomyces pombe), nematode (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster) fulfill a conserved role in conferring heavy metal resistance [18][19][20][21]. In fission yeast, SpHMT-1 mediates the vacuolar sequestration of metal adducts including phytochelatin, glutathione or metallothionein complexes of heavy metal ions [18]. An elegant study from the Vatamaniuk laboratory has shown that HMT-1 proteins in C. elegans (CeHMT-1) and D. melanogaster (DmHMT-1) can also mediate the sequestration and elimination of Cd complexes. In particular, heterologously expressed DmHMT-1 or CeHMT-1 were shown to suppress the cadmium hypersensitivity of S. pombe hmt-1 mutants, concomitant with the localization of CeHMT-1 to the vacuolar membrane. These results clearly indicated that the HMT-1-mediated detoxification of heavy metals is preserved during evolution, extending to some invertebrate species lacking the ability to synthesize phytochelatin (PC) [20,21]. Given the similarity of HMT-1 and ABCB6 sequences, the major aim of this study was to test if ABCB6 can complement the function of HMT-1 proteins. We show that ABCB6 can be functionally expressed in the vacuolar/ endosomal membrane, resulting in a rescue of the cadmium sensitivity phenotype of HMT-1-deficient S. pombe and C. elegans strains. Our findings reveal ABCB6 as a functional orthologue of the HMT-1 proteins, linking ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification. Consistent with our previous findings showing extramitochondrial localization, these results provide functional evidence supporting the endolysosomal function of ABCB6.

Heterologous expression of human ABCB6 restores cadmium tolerance of S. pombe hmt-1Δ mutants
To test whether ABCB6 and SpHMT-1 have overlapping functions, we expressed the wild-type human ABCB6 protein, a catalytically inactive mutant variant (ABCB6-KM [9]) and SpHMT-1 in a hmt-1-deleted mutant S. pombe strain showing increased cadmium (Cd) sensitivity (Fig. 1a). SpHMT-1-GFP was also localized to the vacuoles, matching the staining of the vacuolar membrane by FM 4-64 [22]. Confocal microscopy analysis of cells expressing ABCB6-GFP or SpHMT-1-GFP revealed a similar intracellular pattern, indicating that the human ABCB6 protein is targeted to the yeast vacuoles (Fig. 1b). As expected, expression of SpHMT-1 fully eliminated the increased cadmium sensitivity of the hmt-1Δ mutant strain. Expression of wild-type ABCB6 also restored cadmium tolerance, allowing transformed S. pombe colonies to grow in the presence of Cd(II) (Fig. 1c, Supplementary Figure 1). Rescue of hmt-1-deleted strains depended on the functionality of the heterologously expressed transporter, since an inactivating mutation affecting a conserved Walker A lysine of ABCB6 prevented the growth of hmt-1-deleted colonies in the presence of cadmium. Rescue was also observed in liquid medium (Fig. 1d). Cytotoxicity assays revealed that the expression of ABCB6 in hmt-1Δ S. pombe cells conferred resistance to As(III), but not to As(V), Sb(V), Hg(II), or Zn(II) (Supplementary Figure 2).

Determination of vacuolar cadmium content
SpHMT-1 reduces the intracellular concentrations of cadmium by catalyzing the vacuolar sequestration of Cd-PC complexes [18]. To verify that the ability of ABCB6 to suppress the Cd hypersensitivity of HMT-1-deficient S. pombe mutants relies on an orthologous function, we assayed the Cd contents of intact vacuoles isolated from CdCl 2 -treated yeast cells. The integrity of the purified vacuoles was confirmed by acridine-orange (AO) staining (Supplementary Figure 3). Graphite furnace atomic absorption spectrometry (GFAAS) analysis showed . Expression of SpHMT-1 was revealed using anti-HA antibody; ABCB6 was labeled by the ABCB6-567 antibody; EGFP tagged proteins were labeled by an anti-EGFP antibody. b ABCB6-GFP (green) localizes to vacuoles (red) of S. pombe. Hmt-1-deleted S. pombe was transformed with pREP1-HMT-1-GFP or ABCB6-GFP; vacuoles were stained with FM 4-64. Insets show individual cells. Scale bar 10 μm. c Wild-type S. pombe cells transformed with empty pREP1 vector (WT); hmt-1Δ mutant cells transformed with empty pREP1 vector (hmt-1Δ), pREP1-HMT-1-HA (hmt-1Δ/SpHMT-1-HA), pREP1-ABCB6 (hmt-1Δ/ABCB6) or pREP1-ABCB6-KM (hmt-1Δ/ABCB6-KM) were grown overnight to an A 600nm of 1.8. Aliquots of the cell suspensions were then serially diluted and spotted onto solid EMM supplemented with adenine, uracil and the indicated concentrations of CdCl 2 . Colonies were visualized after incubating the plates for 8 days at 30 °C. d Transformants were grown overnight to an A 600nm of 0.8-1. Aliquots of 100-μL were inoculated into 2 mL of the same medium containing CdCl 2 at the indicated concentrations. A 600nm was measured after growth at 30 °C for 72 h. Values, expressed as viability (%), were normalized to untreated control (n = 3)
Since the HMT-1 proteins in S. pombe and C. elegans have been shown to share an orthologous function [20], we investigated whether ABCB6 could also rescue the Cd-sensitive phenotype of an HMT-1-deficient C. elegans strain. Adult hermaphrodites were allowed to lay eggs onto NGM plates supplemented with the indicated concentrations of CdCl 2 , and the progeny reaching adulthood was counted 3 days after hatching at 20 °C (Fig. 4a). Whereas wild-type and HMT-1-deficient worms were indistinguishable in the absence of heavy metals, the latter were markedly more sensitive to Cd, showing developmental delay, larval arrest and death at early larval stages. As expected, expression of CeHMT-1 provided a full rescue. Remarkably, ABCB6 also restored tolerance to Cd exposure (Fig. 4a, b).

Human ABCB6 confers Cd tolerance to SNB-19 glioblastoma cells
The functional relevance of ABCB6 in Cd sensitivity was further evaluated in SNB-19 glioblastoma cells. ABCB6 was overexpressed or silenced by lentiviral transduction (Fig. 5a, Supplementary Figure 5). Immunocytochemical analysis of SNB-19 cells by confocal microscopy confirmed the localization of the endogenous ABCB6 protein in the lysosomal compartment (labeled by LAMP1), and its absence in mitochondria (labeled by AIF) (Fig. 5b, upper panels). Overexpression of ABCB6 also resulted in endolysosomal expression that was clearly distinct from the mitochondrial pattern ( Fig. 5b, lower panels). Attenuation of ABCB6 expression sensitized SNB-19 cells to Cd, as compared to cells stably transfected with the scrambled shRNA construct. In line with these results, ABCB6 overexpression conferred resistance to Cd, showing that ABCB6 effectively modulates the cadmium tolerance of SNB-19 cells (Fig. 5c).

Discussion
Cadmium is a nonessential divalent metal ion, posing significant health concerns. Chronic exposure to cadmium is associated with increased mortality and cancer risk [26]. By displacing essential biological metals, cadmium induces oxidative stress and eventual cell death. Organisms have evolved several mechanisms to detoxify and eliminate cadmium from the cells [27]. In Saccharomyces cerevisiae, sequestration of cadmium-glutathione complexes is mediated by ScYCF1, which belongs to the ABCC subfamily [28]. In other species, vacuolar sequestration of cadmium complexes is mediated by HMT-1 proteins, which belong to the ABCB subfamily. Phylogenetic analysis shows that HMT-1s from S. pombe, Arabidopsis thaliana, C. elegans and D. melanogaster cluster with ABCB6, together with mitochondrial ATM proteins that are involved in iron-sulfur enzyme biogenesis. ABCB6 was initially named MTABC3, because it was considered to be the functional orthologue of Atm1P [4], leading to the erroneous classification of ABCB6 as a mitochondrial protein. Later studies have convincingly demonstrated that the functional orthologue of Atm1P is in fact ABCB7, which is a canonical mitochondrial ABC transporter localized to the inner mitochondrial membrane [29,30].
The high degree of sequence and topological similarity between the HMT-1 proteins and ABCB6 suggests an evolutionary conserved function, implying ABCB6 in heavy metal resistance [19]. Circumstantial evidence including increased copy numbers, or increased expression in resistant cells [31][32][33], as well as correlation of gene expression to chemotherapy outcome has implicated ABCB6 in resistance to chemotherapeutic agents [34][35][36]. Overexpression of rat Abcb6 in LoVo cells conferred tolerance toward copper, suggesting an involvement of rAbcb6 in transition metal homeostasis [16]. There is a direct correlation between arsenic resistance and ABCB6 expression in various human and mouse cell lines, which was interpreted to be based on an ABCB6-mediated increase of cytosolic heme levels, resulting in the reduction of arsenite-induced oxidative stress [37][38][39]. However, models relying on the mitochondrial function of ABCB6 are difficult to reconcile with the endolysosomal expression pattern shown here and reported by several groups [12][13][14][15][16][17]. Cell fractionation experiments, images obtained of fixed cells with confocal and electron microscopy, and live cell imaging have repeatedly demonstrated that the endogenous ABCB6 protein is expressed in the endolysosomal system, and not in mitochondria.
Schizosaccharomyces pombe and C. elegans have served as important models for elucidating conserved pathways and processes relevant to human biology and disease. In particular, rescue of mutant phenotypes have established the function of several orthologous human proteins. In the past several years, well-developed genetic, genomic, biochemical and cell biological tools have provided fresh insights into vacuolar protein sorting, organelle homeostasis, autophagy, and stress-related functions of the yeast vacuole, and these insights have often found parallels in mammalian lysosomes [23,40]. In this paper, we show that ABCB6 localizes to the same intracellular compartment as HMT-1, performing an overlapping function linked to the intracellular sequestration of metal complexes in both model organisms. Vacuolar localization in yeast was revealed by the expression of differently tagged ABCB6 and SpHMT-1 (Fig. 1b). In C. elegans, ABCB6 was expressed under the control of the endogenous CeHMT-1 promoter, offering an opportunity to study ABCB6 localization in an intact organism without the burden of artifacts associated with overexpression. In complete agreement with a recent report [41], we find that CeHMT-1 is localized to the endosomal compartment in the intestinal cells of the nematode (Fig. 3). Importantly, ABCB6 was found in the same intracellular compartment (Fig. 3). Our results confirm recent studies establishing the relevance of the N-terminal domain in the localization of ABCB6 and CeHMT-1 [13,41]. Determining the subcellular localization of a protein is a key step toward understanding the cellular function of a protein. Although we find that the endogenous ABCB6 protein is confined to the endolysosomal compartment of SNB-19 glioblastoma cells, it may be argued that the precise intracellular localization can only be established with the discovery of a matching physiological function. In addition to evidence based on imaging of ABCB6 in native organisms, in this paper we provide functional proof supporting the role of ABCB6 in the vacuolar/ endosomal sequestration of cadmium. First, we show that ABCB6 rescues the Cd-sensitive phenotype of HMT-1-deficient S. pombe and C. elegans strains. Second, we show that ABCB6 function is required for the sequestration of cadmium into HMT-1-deficient yeast vacuoles. Third, we provide evidence that ABCB6 modulates the cadmium sensitivity of human glioblastoma cells. Taken together, these results clearly establish ABCB6 as the human orthologue of HMT-1 proteins.
SpHMT-1 and CeHMT-1 confer cadmium resistance by sequestrating Cd-phytochelatin complexes. Phytochelatins (PCs) are (γ-Glu-Cys)n Gly polymers that are restricted to plants and fungi, with the notable exception of C. elegans. In animal cells and S. cerevisiae, cytoplasmic cadmium is complexed with glutathione (GSH), which is a common chelator involved in cellular response, transport and excretion of metal cations. Importantly, detoxification by CeHMT-1 does not depend on PC synthesis [21], and SpHMT-1 was shown to confer cadmium tolerance in the absence of phytochelatins, but depending on the presence of GSH and ATP, demonstrating that a common, highly conserved mechanism has been selected during evolution [20,42]. Given the conservation of HMT-1 proteins, we suggest that the ABCB6mediated increase of vacuolar cadmium levels shown in Fig. 2 can be explained by the direct transport of (Cd-GS 2 ) complexes. Remarkably, overexpression of ABCB6 conferred resistance to cadmium in human SNB-19 cells, suggesting that the HMT-1 detoxification pathway is preserved from yeast to human. The contribution of GSH to cadmium detoxification was further suggested by experiments in which SNB-19 cell overexpressing ABCB6 depleted of GSH showed an increased cadmium sensitivity (not shown here). However, no resistance against cadmium has been observed in HeLa cells overexpressing ABCB6 (not shown here). In that respect, ABCB6 is similar to ABCC1 (MRP1), whose role in cadmium detoxification appears to be cell specific [42], even though it can functionally complement ScYCF1 in yeast [43]. The reason why the orthologous function of ABCB6 (and ABCC1) does not uniformly manifest in all mammalian cell models is not clear. In mammals, cadmium detoxification relies primarily on metallothioneins, which bind Cd and also scavenge free radicals generated in oxidative stress [44]. Also, we cannot rule out that the role of ABCB6 in cadmium detoxification of mammalian cells is indirect. Whereas mitochondrial uptake of porphyrins seems improbable, ABCB6 may mediate the sequestration of toxic by-products of Cd-heme interactions into the endolysosomal system [41]. In all examined organisms, overexpression of SpHMT-1 conferred tolerance to cadmium, but not to Sb(III), Ag(I), As(III), As(V), Cu(II), or Hg(II) [42]; whereas substrates of CeHMT-1 also include As(III) and Cu(II) [21], indicating how subtle changes in the primary sequence of transporters can fine-tune substrate specificity through evolution [45]. In fission yeast, ABCB6 conferred resistance to Cd(II), As(III), but not to As(V) or Cu(II) (Supplementary Figure 2). Preliminary experiments using purified ABCB6 protein have failed to demonstrate stimulation of the ABCB6 ATPase activity by cadmium-GSH complexes (not shown here). Future work, using reconstituted, transport-competent ABCB6 will be needed to verify the exact nature and extent of ABCB6 substrates.
It also remains to be determined how the evolutionary conserved role in detoxification is manifested in pathological conditions associated with impaired ABCB6 function. Interestingly, lack of ABCB6 in mice does not result in an overt phenotype [12,46], and ABCB6 deficiency in humans, as observed in Lan-negative individuals, is also without any clinical consequences [5]. On the other hand, disruption of the ABCB6 gene in mice exacerbates porphyria phenotypes in the Fech(m1Pas) mouse model [47], and ABCB6 is a genetic modifier of porphyria [47]. Mutations in the ABCB6 gene were implied in several hereditary diseases ranging from pseudohyperkalemia, coloboma [48], or dyschromatosis universalis hereditaria (DUH) [7,49,50]. The pathogenic role of ABCB6 in these conditions is not understood, as there is no obvious overlap between these phenotypes. Pseudohyperkalemia is a dominant red cell trait characterized by increased serum [K + ] in whole blood stored at, or below room temperature (RT), without additional hematological abnormalities [51]. Coloboma is a developmental disorder affecting the eyes, whereas DUH is characterized by asymptomatic hyper-and hypopigmented macules distributed over the body. Based on the results presented in this study, it is tempting to speculate that a common theme in these phenotypes may be disturbed endolysosomal metal homeostasis due to the impaired sequestration of glutathione adducts. The relevance of the endolysosomal compartment in the metabolism/homeostasis of metals is well-known [52]. Thus, the coloboma phenotype may be related to the pathophysiological consequences associated with cadmium exposure, which was shown to alter visually guided behavior in zebrafish as a result of toxicity occurring at the cellular level [53]. Similarly, the ultrastructural abnormalities observed in MNT-1 cells expressing DUH mutant ABCB6 variants may be explained by the impaired intraluminal homeostasis of the maturing early melanosome [12].
The identification of ABCB6 as an HMT-1 orthologue links ABCB6 to heavy metal-related diseases, such as neurodegenerative conditions, dysfunction of the digestive tract and cancer [19]. The pathophysiological relevance of ABCB6 in these conditions remains to be confirmed by studies using relevant disease models. In parallel, heterologous expression of ABCB6 in hmt-1-deficient S. pombe cells may be used as a tool for better understanding the structure and function of ABCB6.

Cell lines
The SNB-19 glioblastoma cell line was obtained from DSMZ (Germany), HeLa cells were from ATCC. Cells were grown in high glucose DMEM (Gibco 521000-47) supplemented with 10% FBS, 2 mmol/L glutamine, and 100 units/ mL penicillin and streptomycin (Life Technologies) at 37 °C in 5% CO 2 . Cells were periodically tested for mycoplasma contamination with the MycoAlert mycoplasma detection Kit (Lonza, Basel, Switzerland).

Schizosaccharomyces pombe
Plasmid constructs were amplified in E. coli strain Top10 (Invitrogen, Carlsbad, CA, USA) grown at 37 °C in liquid Luria-Bertani (LB) medium supplemented with appropriate antibiotics. Hemagglutinin-tagged S. pombe hmt-1 (Z14055) cDNA was synthesized by GenScript (Piscataway, NJ, USA). Site-specific mutation was engineered using the QuikChange site-directed mutagenesis kit (Stratagene, San Diego, CA, USA); the mutation was confirmed by sequencing. The cDNAs encoding hmt-1 and ABCB6 variants were subcloned into the pREP1 fission yeast expression vector; pEGFP-N1 (BD Biosciences, Franklin Lakes, NJ, USA) was used for the N-terminal EGFP-tagging of the transporters.

Yeast transgenic strains
Yeast cells were grown at 30 °C in Edinburgh minimal medium (EMM). At an A 600nm (OD600) of 1, cells were grown in EMM containing minimal glucose (5 g/L). Cells were transformed using the standard lithium acetate procedure [55]. S. pombe transformants were selected for leucine prototrophy in EMM.

C. elegans transgenic strains
Transgenic C. elegans strains were generated by biolistic transformation using the Biolistic PDS-1000/He particle delivery system (BioRad, Hercules, CA, USA) according to standard methods described by Rieckher et al. [56].

Human cell lines with enhanced or silenced ABCB6 expression
ABCB6 knock-down and overexpression were achieved using a self-inactivating lentiviral system, as described previously in [9]. To induce the expression of the shRNA constructs, IPTG (1 mM) was added to the cells for 6 days before additional treatments.

Schizosaccharomyces pombe
Transformed cells were grown in EMM complemented with appropriate supplements. To characterize the chemosensitivity of yeast strains in liquid medium, 100-μL overnight cultures (A 600nm of 0.8) were diluted into 2-mL EMM containing different concentrations of metal compounds (Cd(II) As(III) As(V), Sb(III), Sb(V), Hg(II), Cu(II), or Zn(II)). In case of Sb(III) and Cu(II), we could not detect toxic concentrations in EMM medium. Cells were then grown at 30 °C. The extent of growth after 72 h was determined by measuring absorbance at 600 nm (A 600nm ). Viability curves were fitted with Graph Pad Prism 5 software using the sigmoidal dose-response model. To characterize chemosensitivity on agar plates, overnight cultures were diluted in EMM (A 600nm of 0.7). Colonies were spotted onto plates containing different concentrations of metal compounds and incubated for 6-7 days at 30 °C.

Caenorhabditis elegans
Heavy metal tolerance of C. elegans strains was assayed as described in [19]. Briefly, 8-10 adult worms were allowed to lay eggs for 2 h at 20 °C on NGM plates supplemented with the indicated concentrations of CdCl 2 . CdCl 2 tolerance was quantified by determining the ratio of adult worms and larvae after 3 days at 20 °C. At least 60 animals, derived from 3 parallel plates containing at least 20 animals/category, were counted by light microscopy in 3 independent trials.

Human cell lines
In cytotoxicity experiments, cells were seeded in 100-μL DMEM medium at a density of 4000 cells/well in 96-well plates, and serially diluted drugs were added on the following day in 100-μL medium to give the indicated final concentration. Cells were then incubated for 72 h at 37 °C in 5% CO 2 . Cytotoxicity assays were performed in triplicate. Cell survival was assessed by the PrestoBlue assay (Life Technologies), according to the manufacturer's instructions. Viability curves were fitted with Graph Pad Prism 5 software using the sigmoidal dose-response model.

Assessment of integrity of vacuole preparations
The integrity of the vacuoles was assessed by measuring fluorescence as described in [57]. Acridine orange (AO, Sigma-Aldrich) fluorescence (Supplementary Figure 3) was measured using an Attune Acoustic Focusing cytometer (Applied Biosystems, Life Technologies, Carlsbad, CA, US).

Determination of vacuolar Cd contents
Vacuolar Cd content was determined by graphite furnace atomic absorption spectrometry (GFAAS In these steps, the maximum flow (2 dm 3 /min) of the GF sheath gas (5.0 Ar, supplier: Messer, Hungary) was applied, except the atomization step, being set to stopped gas flow. Integrated, 3D-absorbance signals were recorded with integration time of 3 s, using iterative spectral background correction. Each measurement data corresponds to an average of three replicate determinations. For quantitative determinations, external standardization was applied by means of setting up five-point calibration curves (range: 0.5-50 ng/ mL Cd; solutions preserved in 2.6% (v/v) HNO 3 ). Recovery was checked by spiking selected samples with 5 µL of a Cd standard solution at a concentration of 5 ng/mL and 50 ng/ mL. The precision of the determinations, expressed as relative standard deviation (RSD), was typically below 2.1%, but never worse than 5.3%. All Cd concentration data were normalized to the protein content of the samples.

Localization of ABCB6 in S. pombe
For the evaluation of intracellular localization of the transporters, hmt-1-deleted S. pombe was transformed with pREP1-HMT-1-GFP or ABCB6-GFP. Cells were grown to mid-log phase (A 600nm of 0.5-0.8) and stained with FM 4-64 as described in [58] with the following modifications. FM 4-64 (T3166 ThermoFischer Scientific Waltham, MA, USA) was dissolved in dimethyl-sulphoxide at a concentration of 1.64 mM. Cells were harvested and incubated with 1-μL FM 4-64 in 50-μL EMM medium at 30 °C for 20 min. An aliquot of 1-mL EMM was added and cells were centrifuged at 5000×g for 5 min at RT. The cell pellet was resuspended in 5-mL EMM, and the suspension was shaken at 30 °C for 90 min. The total volume was transferred to a centrifuge tube and spun for 5 min at 5000×g at RT. The cell pellet was resuspended in 1-mL sterile water, and centrifuged at 5000×g for 5 min at RT. Cells were resuspended in 25-μL EMM. An aliquot of 7 μL was spotted on ConA/polyKcoated (1:1 mixture of 2 mg/mL concanavalin A and 0.1% poly-l-lysine) glass slides covered with an 18 × 18 mm 2 cover slip. Confocal images were obtained using a LSM 710 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a Plan-Apochromat 63 ×/1.4 Oil DIC M27 objective. Noise reduction and deconvolution of the images were performed with Huygens Essential (Scientific Volume Imaging B.V.).

Localization of ABCB6 in human cells
Monoclonal antibodies, dyes and their sources were as follows: Rabbit monoclonal Anti-AIF [D39D2] antibody (#5318) to apoptosis inducing factor, rabbit monoclonal Anti-EEA1 [C45B10] antibody (#3288) to early endosome antigen 1, rabbit monoclonal Anti-LAMP1 [D2D11] antibody (#9091) to lysosome-associated membrane protein 1, secondary goat anti-mouse IgG (H + L) F(ab′) 2 fragment conjugated to Alexa Fluor 647 (#4410) and secondary goat anti-rabbit IgG (H + L) F(ab′) 2 fragment conjugated to Alexa Fluor 647 (#4414) were from Cell Signaling Technology. Secondary goat polyclonal antibody to human IgG conjugated to DyLight 488 (ab96907) was purchased from Abcam. Hoechst 33342 (R37605) nuclear counterstain was from Thermo Fisher Scientific. The OSK43 antibody was a kind gift from Dr. Yoshihiko Tani (Japanese Red Cross Osaka Blood Center, Osaka, Japan). SNB-19 cells expressing ABCB6 variants were plated in an Eppendorf 8-well imaging coverglass (#0030742036). Hoechst 33342 was applied to the cells for 20 min prior to fixation; subsequently, cells were rinsed in PBS and fixed for 30 min in 4% Paraformaldehyde/PBS at RT. Fixed cells were quenched for 10 min in PBS/100 mM glycine (quenching buffer), washed with PBS and blocked and permeabilized in PBS containing 0.2 mg/mL BSA/0.1% Triton X-100/10% Normal Goat Serum (blocking buffer). Primary antibody was diluted in PBS containing 0.2 mg/mL BSA/0.1% Triton X-100/3% normal goat serum (incubation buffer, IB). Cells were incubated with the primary antibody overnight at 4 °C in a humified chamber, washed five times in IB, and incubated with the corresponding secondary anti-human, anti-rabbit and anti-mouse antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 diluted in IB for 90 min at RT. Samples were washed five times with PBS and subsequently imaged. Confocal images were obtained using a LSM 700 confocal laser scanning microscope (Carl Zeiss, Inc.) equipped with a Plan-Apochromat 63×/1.4 NA Oil DIC M27 objective. Images were acquired in three channels (blue (Hoe-chst33342), green (Alexa Fluor 488), red (Alexa Fluor 647)), blue emitting Hoechst 33342 was excited using the 405 nm laser line, green emitting Alexa Fluor 488 was excited using the 488 nm laser line and infrared emitting Alexa Fluor 647 was excited using the 633 nm laser line. Noise reduction and deconvolution of the images were performed with Huygens Essential (Scientific Volume Imaging B.V.).