Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Gross localization of Pmel17, LAMP-1, tyrosinase, AP-3 and syn13 is not altered by BLOC-1 deficiency. IFM analyses of primary melanocytes from wild-type C57BL/6J (a-c, g-i, m-o) or pallid mice (d-f, j-l, p-r) double labeled for Pmel17 and LAMP-1 (a-f), tyrosinase and EEA1 (g-l), or AP-3 and syntaxin 13 (syn13; m-r). Both individual and merged images are shown. Insets, 2.5-fold magnification of the boxed region. Arrowheads point to puncta labeled for the green marker, and arrows point to puncta labeled for the red marker. Note the paucity of overlap for the indicated markers in both sets of cells. A slight increase in Pmel17 overlap with LAMP-1 in pallid melanocytes likely results from the increase in detectable Pmel17 labeling due to the lack of pigment, which normally obscures Pmel17 immunoreactivity in LAMP-1-containing mature melanosomes. Bar, 10 μm.
• Supplemental Figure 2 - Tyrp1 is not mislocalized to LAMP1-positive late endosomes in BLOC-1 deficient primary melanocytes. IFM analyses of primary melanocytes from wild-type C57BL/6J (a-c) or pallid (d-i) mice that were uninfected (d-f) or infected (g-i) with recombinant retroviruses expressing PaHA and double labeled for Tyrp1 (a, d, g) and LAMP-1 (b, e, h); merged images are shown in c, f, i. Arrows indicate presence or absence of LAMP-1 and arrowheads indicate Tyrp1. Bars, 10 μ ·.
• Supplemental Figure 3 - Localization of Tyrp1 and BLOC-1 in BLOC-1-competent cells. a, ultrathin cryosections of melan-a cells (derived from C57BL/6J mice) that had been incubated for 15 min with Tf-FITC prior to fixation were immunogold labeled for Tyrp1 (PAG10) and for FITC (PAG15). Note labeling of stage IV melanosomes (IV) for Tyrp1, and of closely apposed tubular membranes (arrows) for both Tyrp1 and FITC. GA, Golgi apparatus. Inset, labeling of a stage IV melanosome (IV) for anti-Tyrp1, with a FITC-positive tubular membrane emanating from it (arrow). b, c, ultrathin cryosections of melan-mu:MuHA cells were immunogold labeled with anti-HA antibody. Arrowheads point to immunogold labeling on tubulovesicular structures near the Golgi (GA) and a stage IV melanosome (IV). Bars, 0.2 μm.
• Supplemental Figure 4 - Pigmentation and Tyrp1 distribution relative to late endosomes and stage II melanosomes in BLOC-1-, BLOC-2- and AP-3-deficient melanocytes. a-d. Bright field (BF) images of melan-pa (a), melan-pa reconstituted with PaHA (mel-pa:PaHA; b), melan-coa (c) and melan-pe (d) cells imaged with identical light and camera settings to emphasize the degree of pigmentation. Inset, 2.5X magnification of the boxed region. e-p. IFM analyses of melan-coa (e-g, k-m) and melan-pe (h-j, n-p) cells labeled for Tyrp1 (red; e, h, k, n) and either the stage II melanosome marker Pmel17 (green; f, i) or the late endosome/ lysosome marker LAMP-1 (green; l, o). Panels g, j, m, and p show corresponding merged images, and insets show 2.5X magnifications of the boxed regions. Arrows point to the same position in each of the three corresponding panels to aid in orientation. Bar, 10 μm.
• Supplemental Figure 5 - Tyrp1 distribution to tubulovesicular endosomes and to melanosomes, respectively, in BLOC-2- and AP-3-deficient melanocytes. IEM analyses of melan-coa (a, b) immunogold labeled for Tyrp1 (PAG10) or melan-pe (c)cells immunogold labeled for Tyrp1 (PAG10) and tyrosinase (Tyr, PAG15). MVB, vacuolar endosomes (End), plasma membrane (PM), a mitochondrion (m) and stage IV melanosomes (IV) are indicated. Arrows indicate labeling for Tyrp1. Bars, 0.2ƒnμm.