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. 2001 Jul;12(7):2075-85.
doi: 10.1091/mbc.12.7.2075.

AP-3 mediates tyrosinase but not TRP-1 trafficking in human melanocytes

M Huizing  1 , R Sarangarajan, E Strovel, Y Zhao, W A Gahl, R E Boissy
Affiliations
Free PMC article

AP-3 mediates tyrosinase but not TRP-1 trafficking in human melanocytes

M Huizing et al. Mol Biol Cell. 2001 Jul.
Free PMC article

Abstract

Patients with Hermansky-Pudlak syndrome type 2 (HPS-2) have mutations in the beta 3A subunit of adaptor complex-3 (AP-3) and functional deficiency of this complex. AP-3 serves as a coat protein in the formation of new vesicles, including, apparently, the platelet's dense body and the melanocyte's melanosome. We used HPS-2 melanocytes in culture to determine the role of AP-3 in the trafficking of the melanogenic proteins tyrosinase and tyrosinase-related protein-1 (TRP-1). TRP-1 displayed a typical melanosomal pattern in both normal and HPS-2 melanocytes. In contrast, tyrosinase exhibited a melanosomal (i.e., perinuclear and dendritic) pattern in normal cells but only a perinuclear pattern in the HPS-2 melanocytes. In addition, tyrosinase exhibited a normal pattern of expression in HPS-2 melanocytes transfected with a cDNA encoding the beta 3A subunit of the AP-3 complex. This suggests a role for AP-3 in the normal trafficking of tyrosinase to premelanosomes, consistent with the presence of a dileucine recognition signal in the C-terminal portion of the tyrosinase molecule. In the AP-3-deficient cells, tyrosinase was also present in structures resembling late endosomes or multivesicular bodies; these vesicles contained exvaginations devoid of tyrosinase. This suggests that, under normal circumstances, AP-3 may act on multivesicular bodies to form tyrosinase-containing vesicles destined to fuse with premelanosomes. Finally, our studies demonstrate that tyrosinase and TRP-1 use different mechanisms to reach their premelanosomal destination.

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Figures

Figure 1
Figure 1
LAMP-3 and β3A expression in normal and HPS-2 cells. Fibroblasts (A–D) and melanocytes (E–H) were fixed on coverslips and stained with mouse monoclonal antibodies to LAMP-3 (A, C, E, and G) or rabbit polyclonal antibodies to β3A (B, D, F, and H). In each normal cell type, the normal distribution patterns of LAMP-3 (green in A and E) and β3A (red in B and F) are shown. In HPS-2 cells, by comparison, the LAMP-3 pattern was normal (green in C and G) but β3A immunofluorescence was significantly reduced (red in D and H). Arrows in G and H denote the site of minimal β3A expression in the HPS-2 melanocytes.
Figure 2
Figure 2
Western blots of AP-3 subunits in melanocytes and fibroblasts. Protein extracts (25 mg) of cultured melanocytes and fibroblasts were electrophoresed on a 4–20% polyacrylamide gel, blotted onto nitrocellulose, and treated with antibodies to β3, μ3, ς3, δ3, and Lamp-1. N-1, N-2, normal cell extract from two different individuals; Pt, HPS-2 cell extract. Expression of β3, μ3, ς3, and δ3 was deficient in HPS-2 fibroblasts and melanocytes; Lamp-1 was normal for the patient and demonstrates a comparable amount of protein loaded in each lane.
Figure 3
Figure 3
TRP-1 and tyrosinase distribution in normal and HPS-2 melanocytes. Melanocyte monolayers were fixed in 2% formaldehyde and stained with anti–TRP-1 monoclonal (green) and anti-tyrosinase polyclonal (red) antibodies. In normal melanocytes (A–C), TRP-1 (A) and tyrosinase (B) exhibited a similar distribution throughout the melanocyte (C = merged image). In HPS-2 cells (D–F), TRP-1 (D) had a normal distribution, but tyrosinase (E) was localized mainly in the perinuclear area and was markedly absent from the cell periphery (arrowheads) (F = merged image).
Figure 4
Figure 4
TRP-1 is present within the melanosomes of HPS-2 melanocytes. Cultured HPS-2 (A), OCA-1 (B), and OCA-3 (C) melanocytes, containing predominantly hypomelanized melanosomes, were processed for the immunocytochemical localization of TRP-1 with the use (top row) or omission (bottom) of the MEL-5 primary antibody. Peroxidase reaction product occurred in melanosomes (arrows) of the HPS-2 melanocytes (A), null for β3A, and the OCA-1 melanocytes (B), null for tyrosinase. In contrast, peroxidase reaction product did not occur in melanosomes (arrowheads) of the OCA-3 melanocytes (C), null for TRP-1 and all melanocyte types not processed with Mel 5 primary antibody (bottom). Bar, 0.5 μm.
Figure 5
Figure 5
HPS-2 versus control melanocytes contain many melanosomes devoid of tyrosinase. Cultured HPS-2 (A) and control (B) melanocytes were processed for DOPA histochemistry to localize catalytically functional tyrosinase. Many of the melanosomes in the HPS-2 melanocytes remained amelanotic (arrowheads), indicating that they lacked tyrosinase, whereas those in the control melanocytes were heavily pigmented. However, in the HPS-2 melanocytes, reaction product and thus tyrosinase was apparent in some melanosomes (arrows) and in an occasional larger multivesiculated body/late endosome-like vesicles (open arrows). Bar, 2.0 μm.
Figure 6
Figure 6
HPS-2 melanocytes exhibit a normal tyrosinase profile in the TGN. Cultured control (A) and HPS-2 (B) melanocytes were processed for DOPA histochemistry. In both cell types, reaction product was similarly present in the trans-most cisternae of the Golgi apparatus (arrows) and in coated vesicles budding off (arrowheads) and free in the vicinity of the TGN. Bar, 0.5 μm.
Figure 7
Figure 7
Late endosome-like structures are prevalent in HPS-2 melanocytes. Cultured control (A) and HPS-2 (B and C) melanocytes were processed for DOPA histochemistry. (A) Multivesicular bodies with minimal (1) or no (2) reaction product were occasionally present in control melanocytes. (B) In contrast, multivesicular bodies with much reaction product (arrows) were abundant in HPS-2 melanocytes. (C) The HPS-2 multivesiculated bodies exhibited finger-like protrusions of their limiting membranes (1) and the DOPA positive reaction product appeared as aggregates (2), and/or within vesicles (3). Bars: A and B, 0.4 μm; C, 0.25 μm.
Figure 8
Figure 8
Tyrosinase localization on HPS-2 melanocytes transfected with the cDNA encoding the β subunit of AP-3. Cultured HPS-2 melanocytes were transfected with vector alone (A, C, and E) or the cDNA encoding the β subunit of AP-3 (B, D, and F). Selected transfectants were immunocytochemically processed for colocalization of the β3A subunit of AP-3 (red) and TRP-1 (green) (A and B); colocalization of the γ subunit of AP-1 (green) and tyrosinase (red) (C and D); and localization of the δ subunit of AP-3 (red) (E and F); G shows normal human melanocytes immunocytochemically processed for the localization of tyrosinase (red). Both the β3A (arrowheads in B) and the δ subunits of AP-3 (arrowheads in F) were up-regulated after transfection of the β3A subunit of AP-3. Concomitantly, expression of tyrosinase was normalized throughout the dendrites in the HPS-2 cells transfection with the β3A subunit of AP-3 (arrowheads in D) resembling tyrosinase expression in normal human melanocytes (G). Expression of both TRP-1 (green in A vs. B) and the γ subunit of AP-1 (green in C vs. D) were unaffected by transfection.
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