Pink-eyed dilution protein controls the processing of tyrosinase.
The processing of tyrosinase, which catalyzes the limiting reaction in melanin synthesis, was investigated in melan-p1 melanocytes, which are null at the p locus. Endoglycosidase H digestion showed that a significant fraction of tyrosinase was retained in the endoplasmic reticulum. This retention could be rescued either by transfection of melan-p1 cells with an epitope-tagged wild-type p transcript or by treatment with either bafilomycin A1 or ammonium chloride. We found that the endoplasmic reticulum contains a significant amount of p protein, thus supporting a role for p within this compartment. Using immunofluoresence, we showed that most mature full-length tyrosinase in melan-p1 cells was located in the perinuclear area near the Golgi, in contrast to its punctate melanosomal pattern in wild-type melanocytes. Expression of p in melan-p1 cells restored tyrosinase to melanosomes. Triton X-114 phase separation revealed that an increased amount of tyrosinase was proteolyzed in melan-p1 cells compared with wild-type melanocytes. The proteolyzed tyrosinase was no longer membrane bound, but remained enzymatically active and a large proportion was secreted into the culture medium of melan-p1 cells. We conclude that p regulates posttranslational processing of tyrosinase, and hypopigmentation in melan-p1 cells is the result of altered tyrosinase processing and trafficking.
Chen
Kun
K
The Ronald O. Perelman Department of Dermatology and The Department of Cell Biology, New York University School of Medicine, New York, New York 10016, USA.
Manga
Prashiela
P
Orlow
Seth J
SJ
D000818:Animals; D002352:Carrier Proteins; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000502:physiology / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000502:physiology / Q000648:ultrastructure; D008544:Melanocytes / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism; D010858:Pigmentation / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C076239:P protein, mouse; D014442:Monophenol Monooxygenase