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J. Biol. Chem., Vol. 278, Issue 29, 27035-27042, July 18, 2003

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The Inhibition of Early N-Glycan Processing Targets TRP-2 to Degradation in B16 Melanoma Cells^*

Gabriela Negroiu , Raymond A. Dwek  and Stefana M. Petrescu  ¶

From the Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania and the Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom

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.

Received for publication, March 27, 2003 , and in revised form, April 25, 2003.

^* This work was supported by the Wellcome Trust Collaborative Research Initiative Grant 064227. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 40-21-223-9069; Fax: 40-21-223-9068; E-mail: Stefana.Petrescu{at}biochim.ro.

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