INTRACELLULAR SORTING AND TARGETING OF MELANOSOMAL MEMBRANE-PROTEINS - IDENTIFICATION OF SIGNALS FOR SORTING OF THE HUMAN BROWN LOCUS PROTEIN, GP75

The structural and functional integrity of cytoplasmic organelles is m aintained by intracellular mechanisms that sort and target newly synth esized proteins to their appropriate cellular locations. In melanocyti c cells, melanin pigment is synthesized in specialized organelles, mel anosomes. A family of melanocyte-specific proteins, known as tyrosinas e-related proteins that regulate melanin pigment synthesis, is localiz ed to the melanosomal membrane. The human brown locus protein, tyrosin ase-related protein-1 or gp75, is the most abundant glycoprotein in me lanocytic cells, and is a prototype for melanosomal membrane proteins. To investigate the signals that allow intracellular retention and sor ting of glycoprotein (gp)75, we constructed protein chimeras containin g the amino-terminal extracellular domain of the T lymphocyte surface protein CD8, and transmembrane and cytoplasmic domains of gp75. In fib roblast transfectants, chimeric CD8 molecules containing the 36-amino acid cytoplasmic domain of gp75 were retained in cytoplasmic organelle s. Signals in the gp75 cytoplasmic tail alone, were sufficient for int racellular retention and targeting of the chimeric proteins to the end osomal/lysosomal compartment. Analysis of subcellular localization of carboxyl-terminal deletion mutants of gp75 and the CD8/gp75 chimeras s howed that deletion of up to 20 amino acids from the gp75 carboxyl ter minus did not affect intracellular retention and sorting, whereas both gp75 and CD8/gp75 mutants lacking the carboxyl-terminal 27 amino acid s were transported to the cell surface. This region contains the amino acid sequence, asn-gln-pro-leu-leu-thr, and this hexapeptide is conse rved among other melanosomal proteins. Further evidence showed that th is hexapeptide sequence is necessary for intracellular sorting of gp75 in melanocytic cells, and suggested that a signal for sorting melanos omal proteins along the endosomal/lysosomal pathway lies within this s equence. These data provide evidence for common signals for intracellu lar sorting of melanosomal and lysosomal proteins, and support the not ion that lysosomes and melanosomes share a common endosomal pathway of biogenesis.