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1 Supplementary Figure 1 sirna and shrna mediated depletion of ATP7A results in loss of melanosomal ATP7A staining. a-h, sirna mediated ATP7A depletion. Immunofluorescence microscopy (IFM) analysis of ATP7A expression (ATP7A) and bright field microscopy (BF) of wild-type melan-ink4a melanocytes transfected with either control sirna or either of three different sirnas (Atp7a-1, -2, and -3 sirna) directed to mouse Atp7a. Arrows denote cells with reduced or absent ATP7A labeling in pigmented cells. i-p, shrna mediated ATP7A depletion. IFM analysis of wild-type melan-ink4a melanocytes transiently transfected with either control shrna vector or either of three different shrna constructs (Atp7a-1, -2, and -3 shrna) directed to mouse Atp7a. Scale bars, 10 µm. q, Quantitative analysis of ATP7A knock down. Loss of ATP7A staining both in peripheral puncta (including melanosomes) and the Golgi was quantified by IFM in sirna- or shrna-transduced melan-ink4a cells. Because transduction efficiencies varied from experiment to experiment, the data shown are from a single representative experiment in which ~200 cells were analyzed for each sample. Over three experiments, ATP7A staining was nearly or completely absent in an average of 34%, 30% and 28% of cells transduced with Atp7a-1, -2 and -3 sirnas respectively compared to 6% of cells transduced with the control sirna. Similarly, in three experiments ATP7A staining was reduced or absent in 29%, 39% and 36% of cells transduced with Atp7a-1, -2 and -3 shrna constructs respectively compared to 14% of cells transduced with control shrna; the high background in the shrna transduced cells is likely a consequence of a high rate of transfectioninduced cell death. 1

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3 Supplementary Figure 2 ATP7A is mislocalized to early endosomes in BLOC-1-deficient melanocytes and localized to melanosomes in rescued BLOC-1 and AP-3-deficient melanocytes. a-o, IFM analysis of BLOC-1 - melan-mu (a-c) and melan-rp (g-i) cells, BLOC-1 R melan-mu:muha (d-f) and melan-rp:blos3 (j-l) cells stably expressing the missing BLOC-1 subunits Muted and BLOS3 respectively, and AP-3-deficient melan-pe (m-o) melanocytes. Cells were labeled for ATP7A (a, d, g, j, m) and TYRP1 (b, e, h, k, n). Right (c, f, i, l, o), merged images. Insets, boxed regions magnified X3. Arrowheads, colocalized ATP7A and TYRP1. Scale bar, 10 µm. 3

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5 Supplementary Figure 3 ATP7A is mislocalized to early and recycling endosomes but not to striated melanosomes in BLOC-1-deficient melanocytes. A, C, IFM analysis of BLOC-1 - melan-mu (a-c, m-o) and BLOC-1 R melan-mu:muha cells (d-f, p-r). Cells were labeled for ATP7A (a, d, m, p) and the endosomal marker EEA1 (b, e) or the melanosome marker PMEL17 (n, q). Right, merged images (c, f, o, r). Insets, boxed regions magnified X3. Arrowheads, ATP7A localization. Scale bars, 10 µm. B, IFM analysis of transiently transfected BLOC-1 - melan-mu (g-i) or BLOC-1 R melan-mu:muha cells (j-l) expressing myc-syntaxin 13 (STX13). Cells were labeled for ATP7A (g, j) and the myc-epitope (h, k) and cells expressing low levels of myc-stx13 were analyzed. Right, merged images (i, l). Insets, boxed regions magnified X3. Arrowheads, ATP7A localization. Scale bars, 10 µm. 5

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8 Supplementary Figure 4 ATP7A is localized to the Golgi and to early endosomes in both BLOC-1 - and BLOC-1 R melanocytes. a-e, Ultrathin cryosections of BLOC-1 - melan-mu (a, b) and BLOC-1 R melan-mu:muha cells (c, d, e) were immunogold labeled with antibodies to ATP7A (15 nm gold) and tyrosinase (TYR; 10 nm gold) and analyzed by electron microscopy. Arrows, labeling for ATP7A. Note the labeling within the golgi (GA) and endosomal tubules in both cells (a-d). Also note the absence of ATP7A labeling in tyrosinase-containing striated melanosomes (asterisks) in BLOC-1 - cells (b) and the presence of ATP7A in melanosomes of BLOC-1 R cells (c, e). GA, Golgi apparatus; End, vacuolar endosome; III and IV, melanosome stages. Scale bars, 200 nm. f, This figure shows the entire subcellular fractionation experiment, a portion of which is highlighted in Fig. 2. Whole cell homogenates of BLOC-1 - (melan-mu) and BLOC-1 R (melan-mu:muha) cells were fractionated by ultracentrifugation on sucrose step gradients. Eluted fractions (1-12), collected from bottom to top, and lysates (L, as an input loading control) were probed by western blot with antibodies against ATP7A, TYRP1 and TRF receptor (early endosome marker). Left, molecular weight markers (kda). Arrows, relevant bands. Note the presence in fraction 2 and relative enrichment in fraction 4 of bands for ATP7A and TYRP1, but not TRF receptor (TRF R), from BLOC-1 R but not BLOC-1 - cells. These fractions from BLOC-1 R contained pigment. 8

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10 Supplementary Figure 5 ATP7A is associated with TYRP1 in BLOC-1 - and BLOC-1 R melanocytes but localizes to melanosomes in TYRP1 - melanocytes. a-c, BLOC-1 - melan-mu or BLOC-1 R melan-mu:muha cells were lysed in the presence of 1 mm DSP cross-linker as described in methods, and then subjected to immunoprecipitation (IP) with antibodies to TYRP1 (a-c), or to TRF receptor (TRF R) or LAMP1 as negative controls (b). IP fractions and input lysates (input amount compared to material subjected to IP are: [a] 2.5% for ATP7A and LAMP1, and 8.5% for TYRP1; [b] 3.1% for ATP7A, 10.4% for TRF receptor and LAMP1, and 14% for TYRP1; [c] 1.7% for ATP7A and 8.5% for TYRP1 in TX-100 lanes, 2.5% for ATP7A and 8.5% for TYRP1 in CHAPS lanes) were fractionated by SDS-PAGE and immunoblotted with antibodies to ATP7A, TYRP1 (a-c) and either TRF receptor (b) or LAMP1 (a, b). Note that an immunoreactive band comigrating with ATP7A in cell lysates was observed with anti- TYRP1 IP, but not with anti-trf receptor or anti-lamp1 IP (b). c, BLOC-1 - melan-mu or BLOC-1 R melan-mu:muha cells were lysed with either Triton X-100 (TX-100) or CHAPS detergent in the presence of the indicated concentration of DSP cross-linker, and then the lysates were immunoprecipitated with anti-tyrp1 antibody. Note the coimmunoprecipitation of ATP7A without DSP using CHAPS. d, TYRP1-deficient melan-b cells were labeled for ATP7A and analyzed by IFM. Corresponding bright field (BF, pseudocolored as red) and merged images are shown separately. Insets, boxed regions magnified X2.5. Arrowheads, colocalization of ATP7A with pigmented melanosomes. Scale bar, 10 µm. 10

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12 Supplementary Figure 6 Copper increases tyrosinase activity in wild-type melanocytes. a-d, DOPA cytochemistry of BLOC-1 R melan-mu:muha cells in the absence or presence (+ copper) of 20 µm copper sulphate in the reaction buffer for 4 h. Cells were analyzed by bright field microscopy. Scale bar, 10 µm. 12

13 Supplementary Figure 7 Copper restores tyrosinase activity in vacuolar endosomes and in a fraction of striated melanosomes of BLOC-1 - melanocytes that is similar to the fraction of pigmented melanosomes in BLOC-1 R melanocytes. a-f, h, i, BLOC-1 - melan-mu (c-f, h, i) and BLOC-1 R melan-mu:muha (a, b) cells were lightly fixed and incubated for 2 h with L- DOPA in the absence (a, c, e) or presence (+ copper; b, d, f) of 20 µm copper sulphate, or without L-DOPA or copper sulfate (h) or with copper sulfate alone (i). Cells were subsequently fixed, processed for thin sectioning, and analyzed by EM. Tyrosinase activity is indicated by melanin deposition in the presence of L-DOPA. Note melanin deposits in the trans-most Golgi cisternae but not in endosomes (inset) and stage II melanosomes of BLOC-1 - melan-mu cells in the absence of excess copper, and the additional deposition of melanin in endosomes (inset) in the presence of copper. Melanin deposition is not observed in endosomes of BLOC-1 R cells regardless of the addition of copper sulphate (a, b). Also note that melanin deposits are not observed at all in the absence of L-DOPA, regardless of the addition of copper sulphate. GA, golgi apparatus; TGN, trans-golgi network; End., endosomes; II, III, IV, different stages of melanosome. Scale bars, 500 nm and inset, 200 nm. g, Quantification of melanin deposits in striated melanosomes with or without copper. The percentages of melanosomes that contained melanin deposits in BLOC-1 - melan-mu and BLOC-1 R melan-mu:muha cells were calculated from the total oblong melanosomal compartments counted for each set (indicated in parentheses): melan-mu:muha with L-DOPA alone (239) or L-DOPA plus copper (228) and melan-mu with L-DOPA alone (303) or L-DOPA plus copper (297). The change in the counts for BLOC-1 - cells with and without copper is emphasized by bold type. 13

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15 Supplementary Figure 8 Copper cannot restore tyrosinase activity to BLOC-1-deficient melanocytes at acid ph. a-l, Bright field microscopy analysis of tyrosinase activity in BLOC-1 - melan-mu cells. Cells were incubated at 37 C for 2 h with L-DOPA or D-DOPA either in the absence or presence (+ copper) of 20 µm copper sulphate in isotonic buffers at ph 7.4 (a-d), ph 6.0 (e-h) or ph 5.0 (i-l). Tyrosinase activity is indicated by melanin deposition in the presence of L-DOPA but not D-DOPA. Scale bars, 10 µm. Note that the addition of copper significantly increased melanin deposits at ph 7.4 (compare panels a and c), but only mildly increased melanin deposits at ph 6.0 (compare panels e and g) and did not restore tyrosinase activity at all at ph 5.0 (compare panels i and k). 15

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