The Repeat Domain of the Melanosomal Matrix Protein PMEL17/GP100 Is Required for the Formation of Organellar Fibers

Toshihiko Hoashi,Jacqueline Muller,Wilfred D Vieira,Francois Rouzaud,Kanako Kikuchi,Kunihiko Tamaki,Vincent J Hearing

doi:10.1074/jbc.m601643200

Toshihiko Hoashi, Jacqueline Muller + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m601643200

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Abstract

Over 125 pigmentation-related genes have been identified to date. Of those, PMEL17/GP100 has been widely studied as a melanoma-specific antigen as well as a protein required for the formation of fibrils in melanosomes. PMEL17 is synthesized, glycosylated, processed, and delivered to melanosomes, allowing them to mature from amorphous round vesicles to elongated fibrillar structures. In contrast to other melanosomal proteins such as TYR and TYRP1, the processing and sorting of PMEL17 is highly complex. Monoclonal antibody HMB45 is commonly used for melanoma detection, but has the added advantage that it specifically reacts with sialylated PMEL17 in the fibrillar matrix in melanosomes. In this study, we generated mutant forms of PMEL17 to clarify the subdomain of PMEL17 required for formation of the fibrillar matrix, a process critical to pigmentation. The internal proline/serine/threonine-rich repeat domain (called the RPT domain) of PMEL17 undergoes variable proteolytic cleavage. Deletion of the RPT domain abolished its recognition by HMB45 and its capacity to form fibrils. Truncation of the C-terminal domain did not significantly affect the processing or trafficking of PMEL17, but, in contrast, deletion of the N-terminal domain abrogated both. We conclude that the RPT domain is essential for its function in generating the fibrillar matrix of melanosomes and that the luminal domain is necessary for its correct processing and trafficking to those organelles.

Highlights

Melanoma is one of the most notorious tumors because of its poor prognosis [1]
HMB45 Detects the M␣ Form of PMEL17 by Immunoblotting—Pigmented MNT-1 cells, unpigmented SK-MEL-28 cells, and HeLa cells transfected with empty vector, PMEL17-i, or PMEL17-is were analyzed by immunoblotting using HMB45 and ␣-PEP13h antibody
According to a previous report that the P2-to-M␣/M␤ cleavage occurs in post-Golgi and prelysosomal compartments [16], it is reasonable that M␤ was not detected in ⌬SIG. These results suggest that SIG in PMEL17 is essential for entering the secretory pathway, as expected. ⌬N-terminal domain (NTD) showed two bands recognized by HMB45 at relatively lower intensities, a major one that was smaller than M␣ and the other corresponding to M␣C

Summary

Introduction

Melanoma is one of the most notorious tumors because of its poor prognosis [1]. Several groups have raised monoclonal antibodies that recognize melanoma cells, with HMB45 being the most popular and frequently used in clinics for the specific detection of melanocytic tumors [2, 3]. These results suggest that N-terminal domains (NTD, PKD, GAP1, and GAP2) are required for the correct trafficking of PMEL17 in these transfected cells. The RPT Domain Is Required for the Proper Maturation of Fibrillar Structure—Previous reports by our group [14] and others [13] have shown that HMB45 recognizes melanosomal matrix fibers.

Results

Conclusion