Establishment of a synchronized tyrosinase transport system revealed a role of Tyrp1 in efficient melanogenesis by promoting tyrosinase targeting to melanosomes

Abstract

Tyrosinase (Tyr) is a key enzyme in the process of melanin synthesis that occurs exclusively within specialized organelles called melanosomes in melanocytes. Tyr is synthesized and post-translationally modified independently of the formation of melanosome precursors and then transported to immature melanosomes by a series of membrane trafficking events that includes endoplasmic reticulum (ER)-to-Golgi transport, post-Golgi trafficking, and endosomal transport. Although several important regulators of Tyr transport have been identified, their precise role in each Tyr transport event is not fully understood, because Tyr is present in several melanocyte organelles under steady-state conditions, thereby precluding the possibility of determining where Tyr is being transported at any given moment. In this study, we established a novel synchronized Tyr transport system in Tyr-knockout B16-F1 cells by using Tyr tagged with an artificial oligomerization domain FM4 (named Tyr-EGFP-FM4). Tyr-EGFP-FM4 was initially trapped at the ER under oligomerized conditions, but at 30 min after chemical dissociation into monomers, it was transported to the Golgi and at 9 h reached immature melanosomes. Melanin was then detected at 12 h after the ER exit of Tyr-EGFP-FM4. By using this synchronized Tyr transport system, we were able to demonstrate that Tyr-related protein 1 (Tyrp1), another melanogenic enzyme, is a positive regulator of efficient Tyr targeting to immature melanosomes. Thus, the synchronized Tyr transport system should serve as a useful tool for analyzing the molecular mechanism of each Tyr transport event in melanocytes as well as in the search for new drugs or cosmetics that artificially regulate Tyr transport.

Figure 1

EGFP-tagged Tyr was functional in Tyr-KO B16-F1 cells. (A) Schematic representation of mouse Tyr C-terminal tagged with FLAG (Tyr-FLAG) and EGFP (Tyr-EGFP). SS, signal sequence; TM, transmembrane domain. (B) Expression of Tyr-EGFP in Tyr-KO cells. After plasmid transfection, Tyr-KO B16-F1 cells expressing Tyr-EGFP or Tyr-FLAG were lysed at the times indicated and analyzed by immunoblotting with anti-Tyr and anti-β-actin antibodies. WT and Tyr-KO cells were used as a positive control and a negative control, respectively, for Tyr expression. (C) Representative images of Tyr-EGFP-expressing and Tyr-FLAG-expressing Tyr-KO cells after plasmid transfection. Scale bars, 20 μm. (D) The percentage of Tyr-KO cells expressing Tyr-EGFP or Tyr-FLAG and containing melanin shown in (C) was calculated at the times indicated after plasmid transfection. The error bars represent the means ± SEM of the data obtained in three independent experiments (n = 30 cells in each experiment). ∗ P < 0.05; NS, not significant (one-way ANOVA and Tukey’s test). Only the statistical significance between Tyr-EGFP and Tyr-FLAG at each time point was shown.

Figure 2

Establishment of a synchronized Tyr transport system by using the FM4-mediated oligomerization system. (A) Schematic representation of mouse Tyr C-terminal tagged with EGFP and four FM domains (Tyr-EGFP-FM4). FM is a FKBP12-derived artificial oligomerization domain. (B) Expression of Tyr-EGFP-FM4 in the presence and absence of D/D solubilizer. Tyr-KO B16-F1 cells expressing Tyr-EGFP-FM4 were treated or not treated with 500 nM D/D solubilizer (at time 0). The cells were then harvested at the times indicated and analyzed by immunoblotting with anti-Tyr and anti-β-actin antibodies. (C) Representative images of Tyr-EGFP-FM4-expressing Tyr-KO cells after treatment with D/D solubilizer or DMSO (–D/D solubilizer). Tyr-EGFP-FM4-expressing cells in the fluorescent images are outlined with broken white lines. Melanin-containing Tyr-EGFP-FM4-expressing cells and transparent Tyr-EGFP-FM4-expressing cells in the bright-field images are outlined with broken red lines and broken black lines, respectively. Scale bars, 20 μm. (D) The percentage of cells shown in (C) containing melanin was calculated after D/D solubilizer treatment (closed circles) or DMSO treatment (–D/D solubilizer; open circles). The error bars represent the means ± SEM of the data obtained in three independent experiments (n = 30 cells in each experiment). ∗ ∗ ∗ P < 0.001 (one-way ANOVA and Tukey’s test).

Figure 3

Colocalization of Tyr-EGFP-FM4 with GM130 at the Golgi and with Tyrp1 at melanosomes. (A) Tyr-KO B16-F1 cells expressing Tyr-EGFP-FM4 were treated with 500 nM D/D solubilizer or DMSO (–D/D solubilizer), fixed at the times indicated, and stained for GM130 (a Golgi marker). Scale bars, 20 μm. (B) Quantification of the colocalization ratio between Tyr and GM130 in the cells shown in (A) in the presence (black symbols) or absence of D/D solubilizer (red symbols). (C) Tyr-KO cells expressing Tyr-EGFP-FM4 were treated with 500 nM D/D solubilizer or DMSO (–D/D solubilizer), fixed at the times indicated, and stained for Tyrp1. Scale bars, 20 μm. (D) Quantification of the colocalization ratio between Tyr and Tyrp1 in the cells shown in (C) in the presence (black symbols) or absence of D/D solubilizer (red symbols). Pearson’s correlation coefficients in (B and D) were calculated (n = 10 cells), and the error bars represent the means ± SEM. ∗ P < 0.05; ∗ ∗ P < 0.01; ∗ ∗ ∗ P < 0.001; NS, not significant (one-way ANOVA and Tukey’s test). Only the statistical significance between the presence and absence of D/D solubilizer at each time point was shown.

Figure 4

Overexpression of Tyrp1 promoted Tyr-mediated melanin synthesis. (A) mRuby3-tagged Tyrp1 and Tyr-EGFP-FM4 were co-expressed in Tyr-KO B16-F1 cells, and the cells were treated with D/D solubilizer or DMSO (–D/D solubilizer) and fixed at the times indicated. Representative images are shown. Transparent Tyr-EGFP-FM4-expressing cells in the bright-field images are outlined with broken black lines. Scale bars, 20 μm. (B) The percentage of cells shown in (A) containing melanin after treatment with D/D solubilizer (closed circles) or DMSO (–D/D solubilizer; open circles) was calculated. Blue broken lines indicate the melanin-containing cells expressing Tyr-EGFP-FM4 alone (see Fig. 2D). The error bars represent the means ± SEM of the data obtained in three independent experiments (n = 10 cells in each experiment). ∗ ∗ ∗ P < 0.001 (one-way ANOVA and Tukey’s test). (C) Quantification of the colocalization ratio between Tyr-EGFP-FM4 and Tyrp1-mRuby3 in the cells shown in (A) in the presence (black symbols) or absence of D/D solubilizer (red symbols). Pearson’s correlation coefficients were calculated (n = 10 cells), and the error bars represent the means ± SEM. ∗ ∗ ∗ P < 0.001 (one-way ANOVA and Tukey’s test).

Figure 5

Knockdown of Tyrp1 delayed Tyr-mediated melanin synthesis. (A,B) Knockdown efficiency of Tyrp1 in Tyr-KO B16-F1 cells transfected with siRNA against Tyrp1 (siTyrp1) or control siRNA. The cells were analyzed by immunoblotting with anti-Tyrp1 and anti-β-actin antibodies (A) and by immunofluorescence using anti-Tyrp1 antibody (B). Scale bars, 20 μm. (C) Tyrp1-depleted (or control) Tyr-EGFP-FM4-expressing Tyr-KO cells were treated with D/D solubilizer and fixed at the times indicated. Melanin-containing Tyr-EGFP-FM4-expressing cells and transparent Tyr-EGFP-FM4-expressing cells in the bright-field images are outlined with broken red lines and broken black lines, respectively. Scale bars, 20 μm. (D) The percentage of cells shown in (C) containing melanin after treatment with D/D solubilizer was calculated. Tyrp1-depleted and controls cells are shown by closed circles and open circles, respectively. The error bars represent the means ± SEM of the data obtained in three independent experiments (n = 10 cells in each experiment). ∗ ∗ ∗ P < 0.001; NS, not significant (one-way ANOVA and Tukey’s test).