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W2034456929 abstract "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. 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. Melanin pigments function in photoprotection in the skin and ocular development and visual acuity in the eye. They are synthesized and stored within specialized lysosome-related organelles of melanocytes and ocular pigment epithelia called melanosomes (1Marks M.S. Seabra M.C. Nat. Rev. Mol. Cell Biol. 2001; 2: 738-748Crossref PubMed Scopus (347) Google Scholar, 2Hearing V.J. Pigm. Cell Res. 2000; 13: 23-34Crossref PubMed Scopus (105) Google Scholar). Melanosomes bearing brown and black melanins, or eumelanins, develop within melanocytes through four morphologically distinct stages. Stage I and II melanosomes lack melanins and are characterized by the progressive development of intraluminal fibrillar striations, upon which melanins are deposited as they are synthesized in stages III and IV. The fibrils likely serve to detoxify oxidative melanin intermediates and concentrate them for storage (in ocular pigment cells) or for transfer to keratinocytes (in epidermal melanocytes) (3Theos A.C. Truschel S.T. Raposo G. Marks M.S. Pigm. Cell Res. 2005; 18: 322-336Crossref PubMed Scopus (189) Google Scholar). The fibrils resemble amyloid both morphologically and structurally (4Fowler D.M. Koulov A.V. Alory-Jost C. Marks M.S. Balch W.E. Kelly J.W. Plos Biol. 2006; 4: e6Crossref PubMed Scopus (658) Google Scholar). Thus, understanding the nature of their formation may help to decipher mechanisms controlling pathological amyloid biogenesis. The major biogenetic component of the melanosome fibrils is the pigment cell-specific protein, Pmel17 (also known as gp100 or SILV; referred to here as Pmel). 3The abbreviations used are: PmelPmel17CSproprotein convertase cleavage siteEndoHendoglycosidase HERendoplasmic reticulumIFMimmunofluorescence microscopyKLDKringle-like domainNTRN-terminal regionPEphycoerythrinPKDpolycystic kidney disease-1 repeat-like regionPNGase Fprotein N-glycanase FRPTregion of internal repeatsWTwild-typeCHOCHO-K1 cellsFACSfluorescence-activated cell sorterPBSphosphate-buffered salinemAbmonoclonal antibodyERGICER-Golgi intermediate compartment. 3The abbreviations used are: PmelPmel17CSproprotein convertase cleavage siteEndoHendoglycosidase HERendoplasmic reticulumIFMimmunofluorescence microscopyKLDKringle-like domainNTRN-terminal regionPEphycoerythrinPKDpolycystic kidney disease-1 repeat-like regionPNGase Fprotein N-glycanase FRPTregion of internal repeatsWTwild-typeCHOCHO-K1 cellsFACSfluorescence-activated cell sorterPBSphosphate-buffered salinemAbmonoclonal antibodyERGICER-Golgi intermediate compartment. Pmel is the only pigment cell-specific protein required for fibril formation, as its ectopic expression in non-pigment cells is sufficient to induce the formation of melanosome-like fibrils (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar). Conversely, Pmel gene mutations are associated with hypopigmentation in several animal models (6Brunberg E. Andersson L. Cothran G. Sandberg K. Mikko S. Lindgren G. BMC Genet. 2006; 7: 46Crossref PubMed Scopus (134) Google Scholar, 7Clark L.A. Wahl J.M. Rees C.A. Murphy K.E. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 1376-1381Crossref PubMed Scopus (188) Google Scholar, 8Kerje S. Sharma P. Gunnarsson U. Kim H. Bagchi S. Fredriksson R. Schutz K. Jensen P. von Heijne G. Okimoto R. Andersson L. Genetics. 2004; 168: 1507-1518Crossref PubMed Scopus (170) Google Scholar, 9Martínez-Esparza M. Jiménez-Cervantes C. Bennett D.C. Lozano J.A. Solano F. García-Borrón J.C. Mamm. Genome. 1999; 10: 1168-1171Crossref PubMed Scopus (46) Google Scholar, 10Reissmann M. Bierwolf J. Brockmann G.A. Anim. Genet. 2007; 38: 1-6Crossref PubMed Scopus (33) Google Scholar, 11Schonthaler H.B. Lampert J.M. von Lintig J. Schwarz H. Geisler R. Neuhauss S.C. Dev. Biol. 2005; 284: 421-436Crossref PubMed Scopus (83) Google Scholar), including silver mice (9Martínez-Esparza M. Jiménez-Cervantes C. Bennett D.C. Lozano J.A. Solano F. García-Borrón J.C. Mamm. Genome. 1999; 10: 1168-1171Crossref PubMed Scopus (46) Google Scholar) in which eumelanosomes are depleted of fibrils and altered in morphology (12Theos A.C. Berson J.F. Theos S.C. Herman K.E. Harper D.C. Tenza D. Sviderskaya E.V. Lamoreux M.L. Bennett D.C. Raposo G. Marks M.S. Mol. Biol. Cell. 2006; 17: 3598-3612Crossref PubMed Scopus (74) Google Scholar). Pmel immunoreactivity is detected on fibrils in stage II melanosomes (13Lee Z.H. Hou L. Moellmann G. Kuklinska E. Antol K. Fraser M. Halaban R. Kwon B.S. J. Investig. Dermatol. 1996; 106: 605-610Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 14Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. J. Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (350) Google Scholar), and Pmel fragments copurify with fibrils (15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar) or stage II melanosomes (16Kushimoto T. Basrur V. Valencia J. Matsunaga J. Vieira W.D. Ferrans V.J. Muller J. Appella E. Hearing V.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10698-10703Crossref PubMed Scopus (188) Google Scholar, 17Basrur V. Yang F. Kushimoto T. Higashimoto Y. Yasumoto K. Valencia J. Muller J. Vieira W.D. Watabe H. Shabanowitz J. Hearing V.J. Hunt D.F. Appella E. J. Proteome Res. 2003; 2: 69-79Crossref PubMed Scopus (131) Google Scholar) by subcellular fractionation. Finally, purified recombinant Pmel fragments produced in bacteria adopt a fibrillar conformation in vitro (4Fowler D.M. Koulov A.V. Alory-Jost C. Marks M.S. Balch W.E. Kelly J.W. Plos Biol. 2006; 4: e6Crossref PubMed Scopus (658) Google Scholar), suggesting that Pmel may be the sole component of the melanosome fibrils. Pmel17 proprotein convertase cleavage site endoglycosidase H endoplasmic reticulum immunofluorescence microscopy Kringle-like domain N-terminal region phycoerythrin polycystic kidney disease-1 repeat-like region protein N-glycanase F region of internal repeats wild-type CHO-K1 cells fluorescence-activated cell sorter phosphate-buffered saline monoclonal antibody ER-Golgi intermediate compartment. Pmel17 proprotein convertase cleavage site endoglycosidase H endoplasmic reticulum immunofluorescence microscopy Kringle-like domain N-terminal region phycoerythrin polycystic kidney disease-1 repeat-like region protein N-glycanase F region of internal repeats wild-type CHO-K1 cells fluorescence-activated cell sorter phosphate-buffered saline monoclonal antibody ER-Golgi intermediate compartment. Although Pmel is clearly a critical component of melanosome fibrils, the mechanism by which Pmel adopts a fibrillar conformation in vivo remains unknown. To define this mechanism, it is critical to clearly understand Pmel biosynthetic trafficking within melanocytes to compartments in which fibrils form. Human Pmel is synthesized as a type I integral membrane protein with an N-terminal signal sequence, a large luminal domain, a single 24-residue membrane-spanning domain, and a 45-residue cytoplasmic domain (18Kwon B.S. Halaban R. Kim G.S. Usack L. Pomerantz S. Haq A.K. Mol. Biol. Med. 1987; 4: 339-355PubMed Google Scholar, 19Maresh G.A. Marken J.S. Neubauer M. Aruffo A. Hellström I. Hellström K.E. Marquardt H. DNA Cell Biol. 1994; 13: 87-95Crossref PubMed Scopus (27) Google Scholar, 20Adema G.J. de Boer A.J. Vogel A.M. Loenen W.A.M. Figdor C.G. J. Biol. Chem. 1994; 269: 20126-20133Abstract Full Text PDF PubMed Google Scholar). Four Pmel products with luminal domains of 525, 532, 567, and 574 residues result from alternatively spliced mRNAs (21Bailin T. Lee S.T. Spritz R.A. J. Investig. Dermatol. 1996; 106: 24-27Abstract Full Text PDF PubMed Scopus (17) Google Scholar, 22Kim K.K. Youn B.S. Heng H.H. Shi X.M. Tsui L.C. Lee Z.H. Pickard R.T. Kwon B.S. Pigm. Cell Res. 1996; 9: 42-48Crossref PubMed Scopus (9) Google Scholar, 23Nichols S.E. Harper D.C. Berson J.F. Marks M.S. J. Investig. Dermatol. 2003; 121: 821-830Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), with the 567-residue form predominating in most melanocytic cells. As for other type I integral membrane proteins, the signal sequence is cleaved, and at least four core N-linked oligosaccharides are added to consensus attachment sites, presumably cotranslationally, in the endoplasmic reticulum (ER) (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 24Maresh G.A. Wang W-C. Beam K.S. Malacko A.R. Hellström I. Hellström K.E. Marquardt H. Arch. Biochem. Biophys. 1994; 311: 95-102Crossref PubMed Scopus (37) Google Scholar). At least a fraction of Pmel traverses the Golgi complex, where some of the N-linked oligosaccharides are modified by resident mannosidases and glysosyltransferases to a complex form that is resistant to digestion in vitro by endoglycosidase H (EndoH) (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 24Maresh G.A. Wang W-C. Beam K.S. Malacko A.R. Hellström I. Hellström K.E. Marquardt H. Arch. Biochem. Biophys. 1994; 311: 95-102Crossref PubMed Scopus (37) Google Scholar) and where O-linked oligosaccharides are added, elaborated, and modified by sialic acid (25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). At least a fraction of the mature form is cleaved into two fragments, referred to here as Mα and Mβ, in a post-Golgi compartment (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar) (likely endosomes; see Ref. 26Theos A.C. Truschel S.T. Tenza D. Hurbain I. Harper D.C. Berson J.F. Thomas P.C. Raposo G. Marks M.S. Dev. Cell. 2006; 10: 343-354Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) by a proprotein convertase (15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar). A small fraction of the resulting luminal fragment is secreted (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 24Maresh G.A. Wang W-C. Beam K.S. Malacko A.R. Hellström I. Hellström K.E. Marquardt H. Arch. Biochem. Biophys. 1994; 311: 95-102Crossref PubMed Scopus (37) Google Scholar). Pmel that accumulates in fibrillar stage II melanosomes is reactive with three commonly used antibodies, HMB45, HMB50, and NKI-beteb, all developed initially as melanoma markers (27Adema G.J. de Boer A.J. van't Hullenaar R. Denijn M. Ruiter D.J. Vogel A.M. Figdor C.G. Am. J. Pathol. 1993; 143: 1579-1585PubMed Google Scholar). By immunoblotting, HMB45 recognizes predominantly proteolytic products that are derived from Mα (25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Yasumoto K. Watabe H. Valencia J.C. Kushimoto T. Kobayashi T. Appella E. Hearing V.J. J. Biol. Chem. 2004; 279: 28330-28338Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and that harbor sialic acid-containing oligosaccharides (25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 29Chiamenti A.M. Vella F. Bonetti F. Pea M. Ferrari S. Martignoni G. Benedetti A. Suzuki H. Melanoma Res. 1996; 6: 291-298Crossref PubMed Scopus (35) Google Scholar, 30Kapur R.P. Bigler S.A. Skelly M. Gown A.M. J. Histochem. Cytochem. 1992; 40: 207-212Crossref PubMed Scopus (100) Google Scholar), suggesting that at least some Pmel17 within melanosomes is processed in the Golgi. Although these aspects of Pmel biosynthesis are well accepted, the proposed fate of the majority of Pmel has remained controversial (3Theos A.C. Truschel S.T. Raposo G. Marks M.S. Pigm. Cell Res. 2005; 18: 322-336Crossref PubMed Scopus (189) Google Scholar, 31Marks M.S. Theos A.C. Raposo G. Truschel S.T. Pigm. Cell Res. 2006; 19: 253-256Crossref Scopus (1) Google Scholar, 32Valencia J.C. Hoashi T. Pawelek J.M. Solano F. Hearing V.J. Pigm. Cell Res. 2006; 19: 250-252Crossref PubMed Scopus (11) Google Scholar). Two different fates have been proposed. One model posits that fibril formation initiates on the internal membranes of multivesicular endosomes, subsequent to Pmel traversing the secretory pathway, including the Golgi complex, and accessing the early endosomal system (3Theos A.C. Truschel S.T. Raposo G. Marks M.S. Pigm. Cell Res. 2005; 18: 322-336Crossref PubMed Scopus (189) Google Scholar, 26Theos A.C. Truschel S.T. Tenza D. Hurbain I. Harper D.C. Berson J.F. Thomas P.C. Raposo G. Marks M.S. Dev. Cell. 2006; 10: 343-354Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). This model assumes that all Pmel isoforms that exit the ER are processed by Golgi enzymes and cleaved by a proprotein convertase, such that only processed forms of the luminal Mα fragment accumulate in fibril-containing subcellular compartments. Although full-length, immature Pmel, unprocessed by the Golgi, is the predominant form detected in whole cell lysates by immunoblotting, and Golgi-modified and proteolytically processed forms of Pmel are not recovered stoichiometrically by various methods, these features can be explained as follows: (i) a slow rate of folding (and hence slow ER release to downstream secretory compartments) balanced by (ii) a rapid loss of extractable Golgi-processed Pmel fragments to insoluble fibrils (15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar), rendered even more insoluble by deposition of melanin (33Donatien P.D. Orlow S.J. Eur. J. Biochem. 1995; 232: 159-164Crossref PubMed Scopus (51) Google Scholar). The model is supported by quantitative immunoelectron microscopy analyses, in which uncleaved Pmel forms are detected throughout the Golgi and endosomes, but not in stage II melanosomes (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 14Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. J. Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (350) Google Scholar, 15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar, 34Theos A.C. Tenza D. Martina J.A. Hurbain I. Peden A.A. Sviderskaya E.V. Stewart A. Robinson M.S. Bennett D.C. Cutler D.F. Bonifacino J.S. Marks M.S. Raposo G. Mol. Biol. Cell. 2005; 16: 5356-5372Crossref PubMed Scopus (191) Google Scholar), and by studies showing that pharmacologic or mutagenic inhibition of acidification, cleavage, or endosomal sorting interfere with fibril formation (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar, 26Theos A.C. Truschel S.T. Tenza D. Hurbain I. Harper D.C. Berson J.F. Thomas P.C. Raposo G. Marks M.S. Dev. Cell. 2006; 10: 343-354Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The model is also consistent with an early ultrastructural study by Maul (35Maul G.G. J. Ultrasctruct. Res. 1969; 26: 163-176Crossref PubMed Scopus (95) Google Scholar) showing melanosome fibrils forming in compartments contiguous with smooth tubular membranes in chick feather melanocytes. Although Maul (35Maul G.G. J. Ultrasctruct. Res. 1969; 26: 163-176Crossref PubMed Scopus (95) Google Scholar) interpreted these membranes at the time to correspond to “smooth endoplasmic reticulum,” the study predated a morphologic characterization of the endocytic pathway and the trans Golgi network, and the results would be more appropriately interpreted today to support either of these membrane systems as precursors to premelanosomes. The second model posits that the majority of Pmel is targeted directly from the ER to immature stage I melanosomes, bypassing the Golgi complex (16Kushimoto T. Basrur V. Valencia J. Matsunaga J. Vieira W.D. Ferrans V.J. Muller J. Appella E. Hearing V.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10698-10703Crossref PubMed Scopus (188) Google Scholar). This model, stemming initially from a literal interpretation of the 1969 conclusion by Maul (35Maul G.G. J. Ultrasctruct. Res. 1969; 26: 163-176Crossref PubMed Scopus (95) Google Scholar), was based on subcellular fractionation results in which full-length Pmel, bearing EndoH-sensitive core N-linked oligosaccharides, was identified in fractions enriched in fibrillar stage II melanosomes (16Kushimoto T. Basrur V. Valencia J. Matsunaga J. Vieira W.D. Ferrans V.J. Muller J. Appella E. Hearing V.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10698-10703Crossref PubMed Scopus (188) Google Scholar, 17Basrur V. Yang F. Kushimoto T. Higashimoto Y. Yasumoto K. Valencia J. Muller J. Vieira W.D. Watabe H. Shabanowitz J. Hearing V.J. Hunt D.F. Appella E. J. Proteome Res. 2003; 2: 69-79Crossref PubMed Scopus (131) Google Scholar). Epitope mapping studies by Yasumoto et al. (28Yasumoto K. Watabe H. Valencia J.C. Kushimoto T. Kobayashi T. Appella E. Hearing V.J. J. Biol. Chem. 2004; 279: 28330-28338Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) assigned antibody reactivity to monoclonal antibodies HMB50 and NKI-beteb, both well established to identify stage II melanosomes by immunoelectron microscopy (14Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. J. Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (350) Google Scholar, 15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar, 36Vennegoor C. Hageman P. van Nouhuijs H. Ruiter D.J. Calafat J. Ringens P.J. Rümke P. Am. J. Pathol. 1988; 130: 179-192PubMed Google Scholar), to membrane-proximal regions of the luminal domain that should be absent in Mα. A recent study showed that Pmel is modified by O-linked oligosaccharides, a modification that occurs in the Golgi complex (37Van den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-208Crossref PubMed Scopus (602) Google Scholar), but the results were interpreted to conclude that an alternative post-ER form of Pmel lacking O-linked oligosaccharides is found in melanosomes (25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Because immature forms of Pmel also copurified by subcellular fractionation with clathrin-associated adaptor complexes AP-1 and AP-2 (38Valencia J.C. Watabe H. Chi A. Rouzaud F. Chen K.G. Vieira W.D. Takahashi K. Yamaguchi Y. Berens W. Nagashima K. Shabanowitz J. Hunt D.F. Appella E. Hearing V.J. J. Cell Sci. 2006; 119: 1080-1091Crossref PubMed Scopus (44) Google Scholar), which are known to facilitate endosomal protein sorting from the trans-Golgi network, endosomes, and the plasma membrane (39Robinson M.S. Bonifacino J.S. Curr. Opin. Cell Biol. 2001; 13: 444-453Crossref PubMed Scopus (434) Google Scholar), it was concluded that Pmel reaches endocytic compartments without passing through the Golgi complex (25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 38Valencia J.C. Watabe H. Chi A. Rouzaud F. Chen K.G. Vieira W.D. Takahashi K. Yamaguchi Y. Berens W. Nagashima K. Shabanowitz J. Hunt D.F. Appella E. Hearing V.J. J. Cell Sci. 2006; 119: 1080-1091Crossref PubMed Scopus (44) Google Scholar). These conclusions were supported by immunofluorescence microscopy (IFM) analyses in which labeling by antibodies that react with immature forms of Pmel, but not with Mα, overlapped with labeling by HMB45/HMB50/NKI-beteb and by adaptor complexes, respectively (16Kushimoto T. Basrur V. Valencia J. Matsunaga J. Vieira W.D. Ferrans V.J. Muller J. Appella E. Hearing V.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10698-10703Crossref PubMed Scopus (188) Google Scholar, 25Valencia J.C. Rouzaud F. Julien S. Chen K.G. Passeron T. Yamaguchi Y. Abu-Asab M. Tsokos M. Costin G.E. Yamaguchi H. Miller Jenkins L.M. Nagashima K. Appella E. Hearing V.J. J. Biol. Chem. 2007; 282: 11266-11280Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 28Yasumoto K. Watabe H. Valencia J.C. Kushimoto T. Kobayashi T. Appella E. Hearing V.J. J. Biol. Chem. 2004; 279: 28330-28338Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 38Valencia J.C. Watabe H. Chi A. Rouzaud F. Chen K.G. Vieira W.D. Takahashi K. Yamaguchi Y. Berens W. Nagashima K. Shabanowitz J. Hunt D.F. Appella E. Hearing V.J. J. Cell Sci. 2006; 119: 1080-1091Crossref PubMed Scopus (44) Google Scholar). Many of the data supporting the second model are based on subcellular fractionation, the results of which might reflect impurities in isolated fractions. Several conclusions, however, seem incompatible with the first model, including the following: (i) the epitope reactivity of several of the antibodies; (ii) the inferred detection of Pmel isoforms modified by some Golgi oligosaccharide transferases but not others; and (iii) the overlap of epitopes for immature Pmel isoforms with stage II melanosomes by IFM. Here we present data that counter these conclusions and suggest that the data supporting them might warrant reevaluation. We show that immature forms of Pmel are found only in pre-Golgi compartments of melanocytic cells and that stage II melanosomes harbor only Golgi-modified Pmel fragments that are derived from Mα and that bear sialylated O-linked oligosaccharides. The results do not support a model in which a distinct cohort of Pmel accesses melanosomes either directly from the ER or with unmodified oligosaccharides. Nevertheless, we show that neither N- nor O-linked glycans are required for trafficking of Pmel through the conventional biosynthetic pathway to melanosome precursor compartments, and we discuss the potential roles of different types of glycosylation in fibrillogenesis. Chemicals and Antibodies—All chemicals were obtained from Sigma or Thermo Fisher Scientific (Fremont, CA) unless stated otherwise. The following mAbs were used: HMB45, HMB50, and NKI-beteb to human Pmel were purchased from LabVision/Thermo Fisher Scientific (Fremont, CA); XD5.A11 to human leukocyte antigen class II β chains (40Radka S.F. Machamer C.E. Cresswell P. Hum. Immunol. 1984; 10: 177-188Crossref PubMed Scopus (49) Google Scholar) and TA99 (also called mel-5) anti-Tyrp1 (41Thomson T.M. Mattes M.J. Roux L. Old L.J. Lloyd K.O. J. Investig. Dermatol. 1985; 85: 169-174Abstract Full Text PDF PubMed Scopus (105) Google Scholar) were produced in-house from hybridomas originally purchased from American Type Culture Collection (Manassas, VA); mAb G1/93 to ERGIC-53 (42Schweizer A. Fransen J.A. Bachi T. Ginsel L. Hauri H.P. J. Cell Biol. 1988; 107: 1643-1653Crossref PubMed Scopus (373) Google Scholar) was from Axxora (San Diego) or a kind gift of H. P. Hauri (University of Basel, Basel, Switzerland); mAb 3126 to calnexin was from Chemicon (Temecula, CA); and anti-tubulin was from Sigma. The following polyclonal anti-peptide antibodies were prepared in-house and affinity-purified as described previously: αPep13h to the C-terminal 14 residues of human Pmel (14Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. J. Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (350) Google Scholar) (identical to αPep13h generated by Kushimoto et al. (16Kushimoto T. Basrur V. Valencia J. Matsunaga J. Vieira W.D. Ferrans V.J. Muller J. Appella E. Hearing V.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10698-10703Crossref PubMed Scopus (188) Google Scholar)); αPmel-N to the N-terminal 17 residues of human Pmel (15Berson J.F. Theos A.C. Harper D.C. Tenza D. Raposo G. Marks M.S. J. Cell Biol. 2003; 161: 521-533Crossref PubMed Scopus (220) Google Scholar, 23Nichols S.E. Harper D.C. Berson J.F. Marks M.S. J. Investig. Dermatol. 2003; 121: 821-830Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar); and αPmel-I to residues 326-344 of human Pmel (23Nichols S.E. Harper D.C. Berson J.F. Marks M.S. J. Investig. Dermatol. 2003; 121: 821-830Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Rabbit anti-Tac to human interleukin-2 receptor α chain (43Santini F. Marks M.S. Keen J.H. Mol. Biol. Cell. 1998; 9: 1177-1194Crossref PubMed Scopus (40) Google Scholar) or pre-immune serum was used as a negative control. Rabbit anti-LAMP-1 was purchased from Affinity BioReagents (Golden, CO). Cell Culture and Transfections—MNT-1 human melanoma cells (44Cuomo M. Nicotra M.R. Apollonj C. Fraioli R. Giacomini P. Natali P.G. J. Investig. Dermatol. 1991; 96: 446-451Abstract Full Text PDF PubMed Scopus (28) Google Scholar) and HeLa cells were cultured as described previously (5Berson J.F. Harper D. Tenza D. Raposo G. Marks M.S. Mol. Biol. Cell. 2001; 12: 3451-3464Crossref PubMed Scopus (254) Google Scholar, 14Raposo G. Tenza D. Murphy D.M. Berson J.F. Marks M.S. J. Cell Biol. 2001; 152: 809-823Crossref PubMed Scopus (350) Google Scholar). CHO-K1 and ldlD14 cells (45Kingsley D.M. Kozarsky K.F. Hobbie L. Krieger M. Cell. 1986; 44: 749-759Abstract Full Text PDF PubMed Scopus (235) Google Scholar) were obtained with the kind permission of Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge) from American Type Culture Collection and were cultured in Ham's F-12 medium (Invitrogen) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT) and penicillin/streptomycin (Invitrogen). HeLa cells were transfected using FuGENE 6 reagent (Roche Diagnostics) according to manufa" @default.
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W2034456929 title "Premelanosome Amyloid-like Fibrils Are Composed of Only Golgi-processed Forms of Pmel17 That Have Been Proteolytically Processed in Endosomes" @default.
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W2034456929 doi "https://doi.org/10.1074/jbc.m708007200" @default.
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