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J. Biol. Chem., Vol. 283, Issue 4, 2307-2322, January 25, 2008
Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes*![]() ![]() 1![]() 2
From the
Melanin pigments are synthesized within specialized organelles called melanosomes and polymerize on intraluminal fibrils that form within melanosome precursors. The fibrils consist of proteolytic fragments derived from Pmel17, a pigment cell-specific integral membrane protein. The intracellular pathways by which Pmel17 accesses melanosome precursors and the identity of the Pmel17 derivatives within fibrillar melanosomes have been a matter of debate. We show here that antibodies that detect Pmel17 within fibrillar melanosomes recognize only the luminal products of proprotein convertase cleavage and not the remaining products linked to the transmembrane domain. Moreover, antibodies to the N and C termini detect only Pmel17 isoforms present in early biosynthetic compartments, which constitute a large fraction of detectable steady state Pmel17 in cell lysates because of slow early biosynthetic transport and rapid consumption by fibril formation. Using an antibody to a luminal epitope that is destroyed upon modification by O-linked oligosaccharides, we show that all post-endoplasmic reticulum Pmel17 isoforms are modified by Golgi-associated oligosaccharide transferases, and that only processed forms contribute to melanosome biogenesis. These data indicate that Pmel17 follows a single biosynthetic route from the endoplasmic reticulum through the Golgi complex and endosomes to melanosomes, and that only fragments encompassing previously described functional luminal determinants are present within the fibrils. These data have important implications for the site and mechanism of fibril formation.
Received for publication, September 25, 2007 , and in revised form, November 7, 2007. * This work was supported in part by National Institutes of Health Grant R01 AR048155, The Institut Curie, and CNRS. 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. 1 Supported in part by NCI Training Grant T32-CA-009140 from the National Institutes of Health. 2 To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania, 513 Stellar-Chance Laboratories, 422 Curie Blvd., Philadelphia, PA 19104-6100. Tel.: 215-898-3204; Fax: 215-573-4345; E-mail: marksm{at}mail.med.upenn.edu.
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