Abnormal translocation of tyrosinase and tyrosinase-related protein 1 in cutaneous melanocytes of Hermansky-Pudlak syndrome and in melanoma cells transfected with anti-sense HPS1 cDNA

Hermansky-Pudlak syndrome is an autosomal recessive disorder characterized by oculocutaneous albinism, a bleeding disorder, and, in some patients, cer oid storage and progressive lung disease. Although Hermansky-Pudlak syndrom e exhibits locus heterogeneity, most patients have mutations in the HPS1 ge ne. Melanocytes in the basal epithelial layer of skin from patients with di fferent mutations in the HPS1 gene exhibited occasional large complexes con taining dihydroxyphenylalanine-positive cisterna and 50 nm vesicles. To cha racterize the role of the HPS1 protein in cells, human HPS1 cDNA was transf ected into pigmented SK-MEL-188 melanoma cells (M-188) in either the sense (S-188) or the antisense (A-188) orientation. Expression of the 79 kDa HPS1 protein (in M-188 and S-188 cells) or lack of expression (in A-188 cells) was confirmed by Western blotting using two HPS1-protein-specific polyclona l. antibodies. Significant reduction in expression of HPS1 protein in A-188 cells resulted in a significant decrease in tyrosinase activity and melani n content compared with M-188 and S-188 cells using an intact cell assay fo r tyrosinase. In contrast, tyrosinase activities in cell lysates of M-188, S-188, and A-188 cells were not significantly different. Knockout of HPS1 p rotein expression in A-188 cells caused both tyrosinase and tyrosinase-rela ted protein 1 to be localized to large granular complexes in the cell cytos ol and dendrites. Electron microscope analysis of the A-188 cells revealed that absence of HPS1 protein resulted in the deposition of dihydroxyphenyla lanine reaction products (i.e., tyrosinase) confined to large membrane-boun d structures with limiting membranes. We conclude that lack of HPS1 protein expression results in mistranslocation of tyrosinase and tyrosinase-relate d protein 1 to large granular complexes rather than melanosomes, compromisi ng melanin synthesis.