MOLECULAR BIOLOGY FOR SYNTHESIS OF GIANT PIGMENT GRANULES IN CONGENITAR PIGMENTARY DISEASES

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This study examines the target signals for the vesicular transport of tyrosinase and its related proteins (TRP-1,2) from Golgi (TGN) to melanosomes, in the hope to identify the biological and molecular mechanism involved in the synthesis of giant pigment granules in congenital pigmentary diseases. Specifically this study focused on characterization of the biological role of a low molecular weight GTP-binding protein, Rab7. To investigate the requirement of Rab7-containing compartments for vesicular transport of tyrosinase family proteins, we expressed tyrosinase and TRPs by recombinant adenovirus and analyzed their localization in human amelanotic melanoma cells in the presence or absence of a dominant-negative mutant of Rab7 (Rab7N125I). Co-infection (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 cells many of the newly synthesized tyrosinase proteins were colocalized in lysosomal Igp85-positive granules of the entire cytoplasm, whereas in the presence of Rab7N125I the colocalization tyrosinase and Igp85 proteins was decreased markedly in the distal area of the cytoplasm. In the Ad-TRP-1-infected cells, TRP-1 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.We are in the process of analyzing the functional domains of TRP-1 and tyrosinase in this vesicular transport of the melanosome biosynthesis.