S-Palmitoylation of Tyrosinase at Cysteine⁵⁰⁰ Regulates Melanogenesis

Total RNA was isolated using the RNeasy Mini Kit (Qiagen,

Hilden, Germany). First-strand cDNA was synthesized from

1.0 mg of total RNA using a ReverTra Ace qPCR RT kit

(Toyobo). RT-qPCR was performed using THUNDERBIRD

SYBR qPCR Mix (Toyobo) and LightCycler 480 (Roche).

Tyrosinase mRNA levels were expressed relative to that of

GAPDH. The following primers (Sigma-Aldrich) were used:

Fukata Y, Iwanaga T, Fukata M. Systematic screening for palmitoyl transferase

activity of the DHHC protein family in mammalian cells. Methods

2006;40:177e82.

Screening for tyrosinase-specific DHHCs

Human embryonic kidney 293T cells (RRID:CVCL_0063;

2.5  105 cells/12-well plate) were cultured for 16e20 hours

in DMEM containing 10% FBS. HA-tagged tyrosinase and

DHHC1e23 plasmids (Fukata et al., 2006) were cotransfected. Metabolic labeling was performed using 0.2 mCi/ml

(3H)-palmitate (PerkinElmer, Branchburg, NJ) for 4 hours.

After incubation, cells were lysed with SDS-sample buffer

and separated using SDS-PAGE. After fixing the gels for 30

minutes in a fixing solution (isopropanol: water: acetic acid ¼

25: 65: 10), they were treated with amplification buffer (1 M

sodium salicylate/15% ethanol), dried under vacuum, and

exposed to an X-ray film. Radiolabeled bands were scanned

in the autoradiograph and analyzed using the National Institutes of Health (Bethesda, MD) software.

Statistical analysis

Korycka J, Łach A, Heger E, Bogusławska DM, Wolny M, Toporkiewicz M,

et al. Human DHHC proteins: a spotlight on the hidden player of palmitoylation. Eur J Cell Biol 2012;91:107e17.

Mizushima S, Nagata S. pEF-BOS, a powerful mammalian expression vector.

Nucleic Acids Res 1990;18:5322.

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Tyrosinase Palmitoylation in Melanogenesis

Supplementary Figure S1. Melanin synthesis pathway, the phylogenetic tree of the human DHHC protein family, and mRNA expression of DHHC2, DHHC3, DHHC7,

and DHHC15 in NHEMs and HM3KO cells. (a) Illustration of melanin (eumelanin and pheomelanin) synthesis pathway. (b) Phylogenetic tree of the human DHHC protein

family. DHHC2 and 15 and DHHC3 and 7 belong to the same subfamilies (Korycka et al., 2012). (c) Semiquantitative RT-PCR was performed using total RNA from NHEMs

and HM3KO cells; DHHC2-, DHHC3-, DHHC7-, and DHHC15-specific primers; and a one-step SuperScript RT-PCR kit. DHHC2, DHHC3, DHHC7, and DHHC15

mRNAs were expressed in both NHEMs and HM3KO cells. M denotes DNA ladder marker. DHHC, Asp-His-His-Cys; NHEM, normal human epidermal melanocyte.

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Supplementary Figure S2. Effects of DHHC2, DHHC3, DHHC7, DHHC15 siRNAs on mRNA expression levels of DHHC2, DHHC3, DHHC7, DHHC15, and

tyrosinase. HM3KO cells were transfected with either scr or DHHC2, DHHC3, DHHC7, or DHHC15 siRNA. Three days after siRNA transfection, mRNA

expression levels of (a) DHHC2, ****P < 0.0001 (DHHC2), **P ¼ 0.0089 (DHHC3), ***P ¼ 0.0003 (DHHC7), and **P ¼ 0.0032 (DHHC15); (b) DHHC3, ****P

< 0.0001; (c) DHHC7, ****P < 0.0001; (d) DHHC15, **P ¼ 0.0015 (DHHC3) and ****P < 0.0001 (DHHC15); and (e) tyrosinase (TYR) were analyzed using RTqPCR. The DHHC mRNA expression was normalized to that of GAPDH. n ¼ 3. Analysis was performed with one-way ANOVA with Tukey’s posthoc test.

DHHC, Asp-His-His-Cys; scr, scramble; siRNA, small interfering RNA.

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Tyrosinase Palmitoylation in Melanogenesis

Supplementary Figure S3. Intracellular localization of DHHCs in NHEMs. Magnified images of Figures 5 and 6a are shown. The fluorescence intensity profile

across the arrow for both green and red channels was analyzed using Zen software, 2010 (Carl Zeiss, Oberkochen, Germany) and shown in the graph. Bars ¼ 20

mm. DHHC, Asp-His-His-Cys; NHEM, normal human epidermal melanocyte.

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Supplementary Figure S4. A regulation model of melanin synthesis through palmitoylation-mediated tyrosinase degradation. Tyrosinase is synthesized in the

ER, matured in the Golgi apparatus, and trafficked to melanosomes through the endosomes. During this process, tyrosinase may be palmitoylated in the ER by

DHHC2 and DHHC3; in the Golgi apparatus by DHHC2, DHHC3, and DHHC15; and in melanosomes by DHHC2. Tyrosinase palmitoylation accelerates its

ubiquitination and degradation, thereby downregulating melanogenesis. Conversely, inhibition of tyrosinase palmitoylation increases tyrosinase stability and

melanogenesis. DHHC, Asp-His-His-Cys; ER, endoplasmic reticulum.

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