Tyrosinase and Tyrosinase-Related Protein 1 Require Rab7 for Their Intracellular Transport

Abstract

We have recently identified the association of Rab7 in melanosome biogenesis and proposed that Rab7 is involved in the transport of tyrosinase-related protein 1 from the trans-Golgi network to melanosomes, possibly passing through late-endosome-delineated compartments. In order to further investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and tyrosinase-related protein by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells (SK-mel-24) in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection of the recombinant adenoviruses carrying tyrosinase (Ad-HT) and TRP-1 (Ad-TRP-1) resulted in the enhancement of tyrosinase activity and melanin production compared to a single infection of Ad-HT. In the Ad-HT-infected SK-mel-24 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal lgp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization of tyrosinase and lgp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected SK-mel-24 cells, TRP-1, which is reported to be present exclusively in melanosomes, was detected throughout the cytoplasm, but not colocalized in prelysosomal (early endosomal) EEA-1 granules. In the presence of Rab7N125I, however, TRP-1 was retained in the EEA-1-positive granules. Our findings indicate that the dominant-negative mutant of Rab7 impairs vesicular transport of tyrosinase and TRP-1, suggesting that the transport of these melanogenic proteins from the trans-Golgi network to maturing melanosomes requires passage through endosome-delineated compartments.

Delta-Tocotrienol Causes Decrease of Melanin Content in Mouse Melanoma Cells

“…Tyrosinase activity was measured with a spectrophotometer at 475 nm. 18) Melanin Content --Cells dissolved in 1 ml of alkaline solution (1 N NaOH) and incubated at 80 • C for 2 hr were used to measure the protein concentration and melanin. The amount of melanin was measured with a spectrophotometer at 420 nm.…”

Efficacy of acetaminophen in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

“…These results also indicate that human melanoma cell lines were more susceptible to APAP than murine B16-F0 and B16-F10 melanoma cells. When tested in amelanotic SK-MEL-24 and C32 cells that do not express functional tyrosinase (23,24), APAP at 100 μM showed only a 17±8 and 12±2% antiproliferative effect 48 h after incubation (Fig. 1).…”

“…A similar approach was used to grow skin fibroblast BJ cells, prostate PC-3, osteosarcoma Saos-2, colorectal adenocarcinoma SW-620, amelanotic SK-MEL- 24 Cell viability assay. Cell viability was determined, as described previously (9)(10)(11).…”

Reduction of Mevalonate Pyrophosphate Decarboxylase in Mouse Melanoma Cells Treated with δ-Tocotrienol Is Not Associated with Reduction of Cholesterol Content or Release of Lysosomes and Melanosomes

“…Tyrosinase activity was measured with a spectrophotometer at 475 nm. 20) Melanin Content --Cells dissolved in 1 ml of alkaline solution (1 N NaOH) and incubated at 80 • C for 2 hr were used to measure levels of protein and melanin. The amount of melanin was measured with a spectrophotometer at 420 nm.…”