Fig 1.
The SLC45A2 transcript is highly expressed in human melanoma cells and primary melanocytes.
Expression of the SLC45A transcripts in various human melanoma cell lines (A), normal melanocytes from Caucasians and Asians (B), and human non-melanogenic skin cells (C) were examined by RT-qPCR. The data are representative of three independent experiments. (D) The expression of the MATP protein in human cells was analyzed by immunoblotting with an anti-MATP antibody. The arrowhead indicates the MATP protein. The data are representative of three independent experiments.
Fig 2.
The MATP protein is located in the melanosomes.
(A) MNT-1 cells were stained with anti-MATP antibody before (MATP) or after antigen pre-absorbance (Pre-absorbed) for 5 min prior to adding an anti-MATP antibody. (B) MNT-1 cells were stained with anti-TA99 or-HMB45 (a melanosomal protein) antibodies together with an anti-MATP antibody. The insets are magnified, and the co-localization of MATP and each melanosomal marker is indicated by arrowheads. Scale bars = 10 μm. (C) Cells were stained with anti-EEA1, anti-LAMP1, or anti-TNG46 antibodies to detect the endosomes, the lysosomes, or the Golgi apparatus, respectively, together with an anti-MATP antibody. These representative images were captured by confocal microscopy. Scale bars = 10 μm.
Fig 3.
The knockdown of MATP using siRNA reduces melanin production.
(A) The representative microscopic images after each siRNA treatment for 4 days. (B) The color of the cell lysates from 2 x 105 MNT-1 cells was monitored after each treatment, and the melanin contents were measured by measuring the absorbance at 450 nm. The data are representative of three independent experiments (*, P < 0.05). (C) The expression level of melanogenesis-related proteins in MNT-1 cells was analyzed by immunoblotting with the appropriate antibodies. TYR, tyrosinase; TYRP-1, tyrosinase related protein-1; PMEL17, premelanosome protein 17. (D) Electron microscopy analysis. The white and black arrowheads indicate mature (stage III or IV) and early (stage II) melanosomes, respectively. II, stage II melanosome; III, stage III melanosome; IV, stage IV melanosome.
Fig 4.
Copper treatment recovers the L-DOPA oxidase activity of tyrosinase in MATP-KD cells.
MNT-1 cells were treated with each siRNA for 2 days. The lysates were treated with or without 1 mM CuSO4 for 5 min before incubating with 2 mg/ml L-DOPA for 1 hour, after which the absorbance at 450 nm was measured. The data are representative of three independent experiments (***, P < 0.005).
Fig 5.
The knockdown of MATP acidifies the melanosomal pH.
(A) MNT-1 cells stably expressing scrambled or MATP shRNAs were treated with the pH indicator DAMP for 30 min. An anti-DNP antibody was used to detect DAMP by fluorescence microscopy. Scale bars = 200 μm. (B) The fluorescence intensity was calculated with ImageJ software (http://rsbweb.nih.gov/ij/download.html). The data are representative of three independent experiments (*, P < 0.05). (C) MNT-1 cells stably expressing scrambled or MATP shRNAs were co-stained with anti-DNP and anti-HMB45 antibodies after DAMP treatment for 30 min and then visualized by confocal microscopy. Each inset was magnified. Scale bars = 10 μm.
Fig 6.
Schematic models of MATP function as a putative transporter in regulating melanosomal pH in normal and OCA4 conditions.
Under normal conditions, MATP elevates the melanosomal pH by functioning as a transporter using a proton gradient. Under this condition, copper can bind to tyrosinase (Cu-Tyrosinase), resulting in active tyrosinase. In OCA4 melanosomes, MATP does not function properly and the melanosomal lumen becomes acidic. Under this condition, copper cannot incorporate into tyrosinase and tyrosinase activity is reduced. Apo-Tyrosinase, tyrosinase without copper; Cu-Tyrosinase, tyrosinase with copper.