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First published online 21 February 2006
doi: 10.1242/jcs.02804

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Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

¹ Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA
² Chemistry Department, University of Virginia, Charlottesville, VA 22904-4319, USA
³ Image Analysis Laboratory, National Cancer Institute, Frederick, MD 21702-1201, USA
⁴ Pathology Department, Health Sciences Center, University of Virginia, Charlottesville, VA 22908, USA

View larger version (39K): [in a new window]   Fig. 1. Protein components of melanosomes. (A,B) Early melanosomes and CCV isolated from M14 or MNT-1 cells were analyzed by IB with antibodies against Pmel17, AP1, AP2 and TfR, as indicated. Experiments were performed twice with comparable results. (C) Pmel17 was immunoprecipitated using PEP13h and was then immunoblotted with antibodies as noted. (D) Melanoma cells as noted were fixed and double stained for Pmel17 (red) and AP1 or AP2 (green) as indicated. Representative images for each combination are shown. Bars, 10 µm.

View larger version (146K): [in a new window]   Fig. 2. AP1 and AP2 colocalization with Pmel17. Melanoma cells were fixed, embedded in resin and double immunostained for Pmel17 with 25 nm gold (25) and AP1 or AP2 with 10 nm gold (10). Colocalization of Pmel17 (black arrows) with AP1 or AP2 (white arrows) was observed in CCV (A and D), stage I melanosomes (B and E) and stage II melanosomes (C and F). Note that AP2-vesicles with or without Pmel17 were observed in stage I melanosomes (E, inset) and inside vesicles in stage II melanosomes (F, inset). Bars, 0.1 µm.

View larger version (46K): [in a new window]   Fig. 3. Expression of the µ1A subunit in melanocytic cells. (A) µ1A transcript levels determined by real-time PCR. Note the expression of µ1A in 1106c normal human melanocytes was higher than in other melanocytic cells or in NHEK. Values are mean ± s.d. of two experiments. (B) Melanoma cells as noted were transiently transfected with a plasmid containing µ1A tagged with HA, or without a plasmid as a control (Ctrl). Comparison of AP1 and µ1A-HA subunits was performed by immunoblotting in those cell lines. Positions of molecular size markers are indicated on the left in kDa. (C-H) Melanoma cells as noted were fixed 48 hours after transfection with µ1A-HA, and stained with a combination of PEP13h (red) and anti-HA (green) antibodies. Specimens were analyzed by confocal microscopy, and yellow fluorescence indicates colocalization. Nuclei were counterstained with DAPI (blue). Representative images are from at least two independent experiments. Bars, 10 µm (C,F); 1 µm (D,E,G,H).

View larger version (48K): [in a new window]   Fig. 4. Expression of the µ1B subunit in melanocytic cells. (A) µ1B transcript levels determined by semi-quantitative real-time PCR. Note the expression of µ1B in NHEK was significantly higher than in melanocytic cells (*). (B) Melanoma cells as noted were pulse-labeled for 30 minutes, and extracts were immunoprecipitated under denatured and reduced conditions with an anti-µ1B antibody or with normal rabbit serum as a control. Immune complexes were denatured and reduced before separation on gels and visualization by fluorography. (C-H) Cells were fixed 48 hours after transfection with µ1B-HA, and stained with a combination of PEP13h (red) and anti-HA (green) antibodies (arrows indicate complexes). Specimens were analyzed by confocal microscopy. Nuclei were counterstained with DAPI (blue). Representative images are shown. Bars, 10 µm (C,F); 1 µm (D,E,G,H).

View larger version (51K): [in a new window]   Fig. 5. Cytoplasmic sorting is restored by µ1B in SKMel-28 cells. (A) M14, MNT-1 and SKMel-28 cells were fixed and double stained for Pmel17 (red) and LDLR (green). Note the restricted distribution of Pmel17 and LDLR in SKMel-28 cells. (B) Z-scan analysis shows localization of LDLR with Pmel17 at the upper plasma membrane of MNT1 cells. Note, LDLR and Pmel17 were retained in the perinuclear area (arrows). (C) SKMel-28 cells were transfected with µ1A or µ1B and were cultured for 2 days. Cells were processed for staining of Pmel17 (red) and LDLR (green) by dual immunofluorescence and were analyzed by confocal microscopy. Note that transfection of µ1B restored cytoplasmic and plasma membrane localization (arrows) for both Pmel17 and LDLR. Representative images from each experiment are shown. Bars, 20 µm.

View larger version (56K): [in a new window]   Fig. 6. Localization of Pmel17 in the plasma membrane. (A,B) Plasma membrane (PM) proteins recovered by crosslinking and SIG purification were analyzed by immunoblotting. All samples were processed in parallel with negative controls. Arrowheads indicate the fully glycosylated 100 kDa band (white), the partially glycosylated 85 kDa band (black), and the cleaved 26 kDa band of Pmel17 (gray). The black arrow identifies a 70 kDa band. (A) Pmel17 was recovered using a biotinylated reagent from the plasma membrane in M14 cells and in MNT1 cells. Recovery of the resident plasma membrane protein, I5, was used as control for the extraction method (see bottom panel). (B) Plasma membrane fraction after SIG purification (left). Using immunoblot analysis, Pmel17 was detected with the PEP13h antibody both in total extracts (TE) and in the SIG fraction. (C) Plasma membrane fractions were analyzed for the presence of markers as noted. Images are representative of two independent experiments.

View larger version (121K): [in a new window]   Fig. 7. Detection of µ1B by TISH in the skin. Skin biopsies were examined by TISH as reported in the Methods. (A,B) Skin before UV stimulation shows Pmel17 and µ1B expression in melanocytes (black arrows) in the epidermis, respectively. (C) Skin 1 day after UV stimulation, shows a moderate increase in the number of µ1B-positive cells and intensity of µ1B expression. (D) Staining with sense probe showed no specific staining. (E,F) Specific expression of µ1B in the cytoplasm of polarized epithelial cells in the skin (sebaceous glands) and kidney (tubular cells) as positive controls. Bars, 100 µm.

View larger version (48K): [in a new window]   Fig. 8. Scheme for Pmel17 trafficking. Pmel17 is sorted from the Golgi to stage I melanosomes directly or indirectly according to whether µ1B is expressed. In melanocytic cells expressing µ1B (left side, black arrows), Pmel17 (85 kDa) is sorted from the TGN either to recycled endosomes or directly to a localized region in the plasma membrane via CCV containing µ1B. Pmel17 is then endocytosed and sorted to stage I melanosomes via AP2. In melanocytic cells that do not express µ1B (right side, gray arrows), default sorting of Pmel17 to the entire surface of the plasma membrane may be mediated by glycosylation or by other unknown determinants. Again, endocytosis and sorting to stage I melanosomes is via AP2. In both cases, the mPmel17 (100 kDa) resulting from its rapid processing and minimal presence in the plasma membrane, is sorted directly to melanosomes, probably via AP1A (white central arrow).

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