Supporting Information

Supplemental Figure Legends

Fig. S1. SLC39A13 is detected in various tissues. Expression of SLC39A13 was assessed using a human multiple tissue Northern blot (Clontech) following the manufacturer’s instructions. The 32P-labeled probe used contained nucleotides 125-596 of the SLC39A13 open reading frame. The expected transcript size of SLC39A13 is ~2.3 kb.

Fig. S2. Detection of ZIP13 using anti-ZIP13 antibodies. (A) Specific immunoblot detection of ZIP13 in

HeLa cells. HeLa cells expressing endogenous ZIP13 or transiently overexpressing human ZIP13 were grown in basal medium. Total membrane preparations were collected for immunoblot analysis with an anti-ZIP13 antibody or anti-ZIP13 antibody in competition with an excess of antigenic peptide. (B)

Immunofluorescence microscopy of endogenous ZIP13 (red) in untransfected HeLa cells grown in basal conditions. HeLa cells incubated with Alexa Fluor 594 secondary antibody alone (2° only, left), with anti-

ZIP13 antibody and the secondary antibody (middle), or with anti-ZIP13 antibody in competition with excess peptide (right). The cell nuclei were stained with DAPI (blue). (C) Immunofluorescence localization of ZIP13 in HEK293 and HepG2 cells. Untransfected HEK293 and HepG2 cells were grown in basal conditions, immunostained with anti-ZIP13 antibody, and the cell nuclei were stained with DAPI

(blue). ZIP13 (red) was detected in intracellular vesicles in both cell types, consistent with its localization observed in HeLa cells and primary fibroblasts.

Fig. S3. Vesicular localization of ZIP13 under different zinc conditions. (A) Untransfected HeLa cells

were grown in basal media and treated with 5 µM TPEN (low zinc) or 100 µM ZnCl2 (high zinc) for 12 hr

prior to ZIP13 immunofluorescence (red). Nuclei were stained with DAPI (blue). (B) Primary fibroblasts

1 of an unaffected parent (M2/II) were grown in basal medium or treated with 5 µM TPEN (low zinc) or 100

µM ZnCl2 (high zinc) for 12 hr in basal medium prior to ZIP13 immunofluorescence (green).

Fig. S4. Immunofluorescence co-localization of ZIP13 (red) and various organellar markers (green or blue). Despite extensive efforts, ZIP13 did not co-localize with any of the organellar markers tested.

Images are shown for experiments with the following markers: cytochrome oxidase subunit IV (CoxIV), biogenesis factor 19 (Pex19), lysosomal-associated membrane (LAMP1, LAMP2), microtubule-associated 1 light chain 3 (LC3-GFP), adaptor proteins (AP1, AP2, AP3), transferrin receptor (TfR), mannose-6-phosphate receptor (M6P-R), Rab proteins (Rab4-GFP, Rab5-GFP, Rab7- cerulean, Rab9-YFP and Rab11-YFP), early endosomes antigen 1 (EEA1), melanoma 1 antigen (CD63).

Fig. S5. Validation of ZIP13-specific knockdown by siRNA treatment. (A) Expression of SLC39A1 and

SLC39A7 is not affected by the siRNA treatment. Total RNA was isolated from HeLa cells transiently transfected with NT or siZIP13 siRNA, and qRT-PCR was conducted to assess SLC39A1 and SLC39A7 transcript levels. Mean values are shown (n=3; ± SE). (B) SLC39A13 expression is significantly reduced by individual siRNA treatments. HeLa cells were transiently transfected with single siRNAs from the pool of siRNAs targeting ZIP13 used in this study. Total RNA was extracted from these cells and qRT-PCR was conducted to assess SLC39A13 transcript levels. (C) Immunofluorescence microscopy with HeLa cells treated with single siRNAs. Transfectants were grown in basal media, and probed for ZIP13 with anti-ZIP13 antibody. Greatly reduced level of ZIP13 is detected in cells transfected with each siRNA targeting ZIP13 (siZIP13), compared to the cells treated with the control non-targeting siRNA (NT). (D)

Labile zinc accumulated in HeLa cells transfected with single siRNAs as detected by FluoZin-3 staining.

The cells were treated with 100 µM ZnCl2 for 12 hr prior to FluoZin-3 staining.

2 Fig. S6. Vesicular labile zinc in primary fibroblasts (M2/II and P3/II) and siRNA-transfected HeLa cells

(NT and siZIP13) grown under basal conditions. Cells were stained with FluoZin-3 without 100 µM ZnCl2 treatment.

Fig. S7. Labile zinc staining with Newport Green DCF in primary fibroblasts and siRNA-treated HeLa cells. The accumulation of labile zinc in intracellular vesicles in cells with reduced ZIP13 function (i.e.

P3/II and siZIP13) is consistent with that observed using FluoZin-3 staining. All cells were treated with

100 µM ZnCl2 for 12 hr before staining with Newport Green DCF. Compared to images of cells stained with FluoZin-3, the overall resolution of fluorescently stained vesicles was significantly lower in Newport

Green DCF stained images. This most likely reflects the difference between the affinities of these labile

2+ zinc probes (Newport Green DCF: Kd for Zn ~1 µM; FluoZin-3: Kd of ~15 nM).

Fig. S8. Total zinc content is not increased in cells with loss of ZIP13 function. Primary fibroblasts (A) and siRNA-treated HeLa cells (B) were grown in basal media, and zinc content in these cells were determined by ICP-MS analysis. Zinc content in cells with reduced ZIP13 function, i.e. SCD-EDS patient fibroblasts (P3/II) and HeLa cells transfected with ZIP13-targeting siRNA (siZIP13), showed a decreasing trend but was not significantly different from that compared to their controls. Mean values are shown

(n=6) with error bars representing ± SE.

Fig. S9. Validation of Zap1-based luciferase assays in HeLa cells. HeLa cells were transiently

UAS transfected with GAL1 -luciferase (firefly), Renilla luciferase and pGal4DBD-Zap1 or pGal4DBD alone, and treated with various combinations of TPEN and/or supplementary zinc (100 µM ZnCl2) as indicated

12 hr prior to dual luciferase assays. The luciferase activity is significantly induced under zinc limiting conditions, i.e. upon higher levels of TPEN treatment, and declined as more zinc was added to the media.

3 Zap1-based luciferase activity was normalized with Renilla luciferase and total protein. Mean values are shown (n=3) with error bars indicating ± SE.

Fig. S10. Endogenous ALP activities of siRNA-treated HeLa cells. Non-targeting (NT) or ZIP13- targeting (siZIP13) siRNA transfectants were grown in basal conditions, treated with TPEN or supplementary zinc (100 µM ZnCl2) as indicated 12 hr prior to the assay. Mean values are shown (n=3) with error bars indicating ± SE.

Fig. S11. Labile zinc staining of pyrithione-treated cells with FluoZin-3. Primary fibroblasts (M2/II and

P3/II) and siRNA-transfected HeLa cells (NT and siZIP13) were incubated with supplemental zinc (25 µM

ZnCl2 for M2/II and P3/II, 100 µM ZnCl2 for NT and siZIP13) in the presence of 5 µM pyrithione for 30 min prior to FluoZin-3 staining.

Supplemental Experimental Procedures

Cell lines and cell culture

HeLa, HEK293, HepG2, and primary dermal fibroblasts from an SCD-EDS patient (P3/II) and an asymptomatic heterozygous parent (M2/II) (Giunta et al. Am. J. Hum. Genet. 82:1-16, 2008) were used in this study. All of these cells were grown in a humidified incubator at 5% CO2 and 37 °C. HEK293 and primary fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1 x non-essential amino acids (NEAA;

Invitrogen). HeLa cells and HepG2 cells were grown in Minimum Essential Medium (MEM; Invitrogen) supplemented with 10% FBS and 1x NEAA.

Zinc uptake assays

A human SLC39A13 cDNA (accession number NM_001128225) was cloned into the pcDNA3.1 Puro(+)

4 plasmid (Invitrogen) using the NheI and EcoRI restriction sites and verified by sequencing. Transiently transfected HEK293 cells expressing pcDNA 3.1 Puro(+) (vector) or pcDNA-ZIP13 were used in 65Zn uptake assays as described. After 48 hr post transfection, cells were washed with uptake buffer (15 mM

HEPES, pH 7.0, 100 mM glucose, 150 mM KCl) and incubated in pre-warmed (37 °C) uptake buffer

65 containing varying concentrations of zinc with the radioactive tracer ZnCl2 (PerkinElmer). For

65 competition assays, excess non-radioactive metal ions were added to the uptake buffer with ZnCl2.

Sodium ascorbate (1 mM) was used to reduce Fe3+ to Fe2+. The cells were then incubated at 37 °C for

15 min over which time uptake is linear, and then the uptake reaction was quenched with an equal volume of ice-cold stop buffer (15 mM HEPES, pH 7.0, 100 mM glucose, 150 mM KCl, 1 mM EDTA).

Cells were collected on 0.45 µm nitrocellulose filters (Millipore), washed with stop buffer, and radioactivity of 65Zn was measured using a Packard Auto-Gamma 5650 γ-counter. Zinc accumulation was normalized to total protein.

siRNA sequences

The pool of siRNA targeting SLC39A13 (siGENOME SMART pool, Dharmacon) consisted of four siRNAs targeting the following sequences: siZIP13#1: 5’-aagatgttcctgg acagcaag-3’; siZIP13#2: 5’- agcatcaaagtcagcggctac-3’; siZIP13#3: 5’-ctggccaacaccatcgataac-3’; siZIP13#4: 5’- gccagcttccttgtgagcaag-3’.

Immunoblotting

HeLa cells were washed with ice-cold PBS, scraped from the plates, and harvested in MHTB (10 mM

HEPES, pH 7.5, 0.2 M mannitol, 50 mM sucrose, 1 mM EDTA, 10 mM KCl) containing EDTA-free protease inhibitor cocktail tablets (Roche). The cells were lysed by sonication and the crude membrane fraction enriched after centrifugation of the cell lysate at 100,000 x g in a TLA55 rotor (Beckman Coulter).

5 The enriched membrane fraction was resuspended in MHTB, quantified, separated by SDS-urea PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 1% Bio-Rad milk in PBST, probed with anti-ZIP13 antibody (1:2000) and a goat-anti-rabbit HRP conjugated secondary (Pierce). The antibody complex was detected by chemiluminescence (Roche).

Immunofluorescence microscopy

Localization of endogenous ZIP13 was determined using an anti-ZIP13 antibody. Cells were grown on coverslips in 6-well plates, washed with PBS and fixed in 4% formaldehyde (Pad Tella) in PBS for 15 min at room temperature, blocked and permeabilized with 1% bovine serum albumin, 1% cold water fish gelatin (Sigma), 0.1% Tween-20 in PBS for 30 min. Endogenous ZIP13 was probed with anti-ZIP13 antibody (1:100) and visualized with donkey anti-rabbit Alexa Fluor 594 antibody (1:500, Invitrogen). Co- localization experiments of human ZIP13 were performed with various subcellular marker proteins. The marker proteins were visualized with appropriate secondary antibodies, and markers tagged with a fluorescence protein were examined 40-44 hr post-transfection. Stained coverslips were mounted on to slides in medium containing DAPI (Vectashield) or with Fluoromount-G (SouthernBiotech). Fluorescence and brightfield images were obtained using a Nikon TE 2000 epifluorescence microscope. Images were deconvolved using Volocity software and processed with Volocity and ImageJ software.

The source of the organellar marker antibodies used are as follows: mouse anti-Golgin-97 (Invitrogen) and mouse anti-KDEL receptor (Invitrogen); mouse anti-COX-IV (Abcam), mouse anti-mannose-6- phosphate receptor (M6PR, Abcam); mouse anti-AP-1, mouse anti-AP-2, mouse anti-AP-3 and mouse anti-EEA1 (BD Biosciences); mouse anti-Lamp1 and mouse anti-Lamp2 (University of Iowa Hybridoma

Bank); mouse anti-transferrin receptor 1 (TfR clone H68.4, Zymed Laboratories), anti-Vamp8 (gift from

Guy Groblewski, University of Wisconsin); anti-ZnT9 and anti-ZnT10 (gift from Shannon Kelleher, Penn

State University). The mammalian subcellular expression vectors Rab4-YFP, Rab5-YFP, Rab7-cerulean,

6 Rab9-YFP, Rab11-YFP, Pex19-YFP, Lgp120-YFP, Cd3Δ-GFP, GPI-GFP were kindly provided from

Jennifer Lippincott-Schwarz (NIH).

Quantitative reverse transcriptase-PCR (qRT-PCR)

Total RNA was extracted from cells using TRIzol reagent (Invitrogen) and reverse transcription of RNA was performed with 0.5 μg total RNA using qScript™ cDNA SuperMix (Quanta Biosciences) following manufacturers’ instructions. qRT-PCR was conducted using SYBR® Green PCR master mix (Applied

Biosystems) and monitored in an ABI Prism 7000 Sequence Detection System (Applied Biosystems).

The thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min, 40 cycles at

95°C for 15 s, and 60°C for 1 min. Relative expression of the examined was determined using

2−ΔCt method and normalized to 18S rRNA.

Sequences of primers used for qRT-PCR

Specific primers used for qRT-PCR were as follows: for SLC39A13: 5’-agccaagctgcaactctcaacag-3’

(forward) and 5’-agcgggagaacacccaactactc-3’ (reverse); 18S rRNA: 5'-gaggtagtgacgaaaaataacaat-3'

(forward) and 5'-ttgccctccaatggatcct-3' (reverse); MT1a: 5'-cactggctcctgcaaatg-3' (forward) and 5’- acttctctgatgcccctttg-3' (reverse); MT2a: 5’- ctccaagtcccagcgaac-3’ (forward) and 5’-gagcagttgggatccatgg-

3’ (reverse); SLC39A1: 5’-tcccactgcaagagttcatcc-3’ (forward) and 5’-accattcactgttcccagcag-3’ (reverse);

SLC39A7: 5'-tgccaaaggaaggaactgtagc-3' (forward) and 5'-atgcaacatggtcagacagtgc-3' (reverse).

7 Supplemental Figures

Fig. S1. Fig. S2.

A ZIP13

ZIP13

B 2° only anti-ZIP13 anti-ZIP13+peptide

C HEK293 HepG2 Fig. S3.

A

Basal 5 µM TPEN 100 µM Zn

B Fig. S4.

ZIP13 Cox-IV merged

ZIP13 Pex19 merged

ZIP13 LAMP1 merged

ZIP13 LAMP2 merged

ZIP13 LC3-GFP merged Fig. S4 (continued).

ZIP13 AP1 merged

ZIP13 AP2 merged

ZIP13 AP3 merged

ZIP13 TfR merged

ZIP13 M6P-R merged Fig. S4 (continued).

ZIP13 Rab4-GFP merged

ZIP13 Rab5-GFP merged

ZIP13 Rab7-cerulean merged

ZIP13 Rab9-YFP merged

Zip13 Rab11-YFP merged Fig. S4 (continued).

0 ZIP13 EEA1 merged

ZIP13 CD63 merged

0 Fig. S5.

A 2 B 100

NT10NT SLC39A13

1.8 siZip13siZIP13 1.6 80 change) 1.4 1.2 60 1 0.8 40 0.6 Relative transcript level transcript Relative 0.4 20 0.2 Relative transcript level (fold- 0 0 SLC39A1ZIP1 SLC39A7ZIP7 NTNT siZIP13#2#2 siZIP13#3#3

C

ZIP13

NT siZIP13#2 siZIP13#3

D

FluoZin-3

NT siZIP13#2 siZIP13#3 Fig. S6.

M2/II NT

P3/II siZIP13 Fig. S7.

M2/II NT

P3/II siZIP13 Fig. S8.

A 16 B 45

14 40 35 12 30 10 25 8 20 6 ng Zn/mg protein ng Zn/mg protein 15 4 10

2 5

0 0 M2/II P3/II NTNT siZip13siZIP13 Fig. S9.

12

Zap1pGal4-lucDBD -Zap1

10 pcDNA3pGal4DBD

8

6

4

Normalized luciferase activity luciferase Normalized 2

0 basal TPEN3 TPEN5+Zn3 TPEN5+Zn20 Zn100 TPEN (μM) 0 1 3 5 5 5 5 5 0 Zinc (μM) 0 0 0 0 3 5 20 100 100 Fig. S10.

3.5 NT

3 siZip13

2.5

2

1.5

1

Normalized ALP Normalized ALP activity 0.5

0 basalBasal 5 μTPENM TPEN 50 Zn50μM Zn

Fig. S11.

M2/II P3/II

NT siZIP13