The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells.
Tyrosinase-related protein-2 (TRP-2) is a DOPAchrome tautomerase catalyzing a distal step in the melanin synthesis pathway. Similar to the other two melanogenic enzymes belonging to the TRP gene family, tyrosinase and TRP-1, TRP-2 is expressed in melanocytes and melanoma cells. Despite the increasing evidence of its efficiency as a melanoma antigen, little is known about the maturation and intracellular trafficking of TRP-2. Here we show that TRP-2 is mainly distributed in the TGN of melanoma cells instead of being confined solely to melanosomes. This, together with the plasma membrane occasional localization observed by immunofluorescence, suggest the TRP-2 participation in a recycling pathway, which could include or not the melanosomes. Using pulse-chase experiments we show that the TRP-2 polypeptide folds in the endoplasmic reticulum (ER) in the presence of calnexin, until it reaches a dithiothreitol-resistant conformation enabling its ER exit to the Golgi. If N-glycosylation inhibitors prevent the association with calnexin, the TRP-2 nascent chain undergoes an accelerated degradation process. This process is delayed in the presence of proteasomal inhibitors, indicating that the misfolded chain is retro-translocated from the ER into the cytosol and degraded in proteasomes. This is a rare example in which calnexin although indispensable for the nascent chain folding is not required for its targeting to degradation. Therefore TRP-2 may prove to be a good model to document the calnexin-independent retro-translocation process of proteasomally degraded proteins. Clearly, TRP-2 has a distinct maturation pathway from tyrosinase and TRP-1 and possibly a second regulatory function within the cell.
Negroiu
Gabriela
G
Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania.
Dwek
Raymond A
RA
Petrescu
Stefana M
SM
D000818:Animals; D001693:Biological Transport, Active; D037281:Calnexin / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D008546:Melanoma, Experimental / Q000378:metabolism*; D020460:Melanosomes / Q000378:metabolism; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured; D021601:trans-Golgi Network / Q000378:metabolism
D037282:Calreticulin; D009097:Multienzyme Complexes; D011134:Polysaccharides; D037281:Calnexin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase