Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - AP-3 localization to clathrin-coated buds near endosomes in MNT-1 and melan-a cells.

  A. Ultrathin cryosections of MNT-1 cells were immunogold labeled for AP-3 (PAG10) or for AP-3 (PAG15) and Hrs (PAG10 - A inset). Shown are representative endosomal regions (A and A inset). Arrows highlight AP-3 labeling on buds and tubules that are double labeled for AP-3 and Hrs. Note electron dense tubules bearing AP-3 positive buds that occasionally label for Hrs (A inset) close to endosomal vacuoles (stars; tangentially cut in A inset). B. Ultrathin cryosections of melan-a cells were immunogold labeled for AP-3 (PAG15) and clathrin light chain (PAG 10). Note the AP-3 positive buds that are also labeled for clathrin (arrows) close to endosomal vaccuoles (star). Clathrin positive/AP-3 negative buds are also observed (arrowhead). C. MNT-1 cells were incubated at 37°C for 1 hr with Tf-HRP conjugate and then processed for whole mount cytochemistry. Preparations were then immunogold labeled for the cytoplasmic domain of Pmel17 (PAG10). Arrows point to clusters of AP-3 labeling on buds that are linked to electron-dense endosomes. These endosomes are labeled for Pmel17 (stars). Bars : 200 nm

• Supplemental Figure 2 - Immunofluorescence analysis of the distribution of AP-1, AP-3 and TGN46 in MNT-1 cells.

  MNT-1 cells were triple-labeled for TGN46, γ-adaptin (AP-1), and δ-adaptin (AP-3) after fixation with 2% PFA and permeabilisation. AP-1 localises to vesicular structures distributed in the cytoplasm and in the perinuclear Golgi area labeled for TGN46. In comparison to AP-1, AP-3 appears less concentrated in the perinuclear region. Overlays indicate that despite the presence of AP-1 in the TGN region, it does not appear to be on the same membrane domains. The same holds true for AP-3, though this adaptor is present in this area to lesser extent. AP-1 and AP-3 appear to be in different vesicular domains that are often very close. Insets of merged channels, 2.5x magnification of the indicated region. Bar 20 μm.

• Supplemental Figure 3 - Tyrosinase antibody characterization for immunoblotting and indirect immunofluorescence of mouse melan-a and AP-3-deficient melan-pe melanocytes.

  A. Immunoblot of melan-a cell lysates with anti-tyrosinase antibody. Note that only a single band of ~70 kDa is labeled. Polyclonal anti-tyrosinase antiserum can immunoprecipitate murine tyrosinase under native and denaturing conditions but does not recognize human tyrosinase (data not shown). B-C. Immunofluorescence microscopy analysis of melan-a (B.) or melan-pe (C.) cells using antibodies to tyrosinase and either Tyrp1 (upper panels) or Pmel17 (lower panels). At right, both labels are overlaid. Inset shows a 2.5X magnification of the indicated region of the cell. Note the minimal overlap with labeling for either Tyrp1 or Pmel17. The absence of extensive colocalization of tyrosinase with Tyrp1 on mature melanosomes is consistent with immunofluorescence microscopy analyses using other antibodies on human melanocytic cells (data not shown), and likely reflects epitope inaccessibility or fluorescence quenching by melanin. Bars, 16 μm.

• Supplemental Figure 4 - Tyrosinase localization in melan-a cells.

  Ultrathin cryosections of melan-a cells were double immunogold labeled for tyrosinase (PAG10) and either AP-3 (PAG15) (A) or AP-1 (PAG15) (B). A. Note the AP-3-positive vesicles containing tyrosinase (arrowhead in B) that are close to stage IV melanosomes. Note the labeling for tyrosinase in the melanosomal lumen associated to internal membranes (arrows). B. Near the Golgi (GA) the bulk of tyrosinase is present in uncoated vesicles that do not contain AP-1 (arrows). C and inset in C. Ultrathin cryosections of melan-a cells were single immunogold labeled for tyrosinase. Tyrosinase is present in stage IV melanosomes, tubulovesicular structures close to the Golgi (GA) but it is not consistently detected in multivesicular bodies (MVB). D. Thick sections of resin-embedded melan-a cells were analyzed by DOPA cytochemistry for tyrosinase activity. The electron dense reaction product is detected in melanosomes and in tubulovesicular elements in the Golgi area. N: Nucleus. Bars, 200 nm.