Identification of rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein 1

The melanosome is a unique secretary granule of the melanocyte in which mel anin pigments are synthesized by tyrosinase gene family glycoproteins, Mela nogenesis is a highly regulated process because of its inherent toxicity. A n understanding of the various regulatory mechanisms is important in deline ating the pathophysiology involved in pigmentary disorders and melanoma, We have purified and analyzed the total melanosomal proteins from B16 mouse m elanoma tumors in order to identify new proteins that may be involved in th e control of the melanogenesis process, Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresi s, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised a nd digested with cyanogen bromide, and the fragments were sequenced. Synthe tic oligonucleotide primers were synthesized corresponding to the peptide s equences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out, Sequencing of one of the poly merase-chain-reaction-mediated clones demonstrated 80%-97% sequence homolog y of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated reg ion. GTP-binding assay on two-dimensional gels of melanosomal proteins show ed the presence of several (five to six) small GTP-binding proteins, sugges ting that small GTP-binding proteins are associated with the melanosome, Am ong the known GTP-binding proteins with similar molecular weight and isoele ctric point ranges, rab3, rab7, and rab8 were found to be present in the me lanosomal fraction by immunoblotting. Confocal immunofluorescence microscop y showed that rab7 is colocalized with the tyrosinase-related protein 1 aro und the perinuclear area as well as, in part, in the perikaryon, thereby su ggesting that rab7 might be involved in the intracellular transport of tyro sinase-related protein 1. Tyrosinase-related protein 1 transport was blocke d by the treatment of B16 cells with antisense oligonucleotide to rab7, We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosi nase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.