Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms mu 1A and mu 1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of mu 1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (mu 1A or mu 1B) showed two distinct distribution patterns that involved Pmel17, and only mu 1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking mu 1B expression. Finally, we established that expression of mu 1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.