Abstract
Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.
Highlights
Following co-translational translocation into the endoplasmic reticulum (ER)[2] membrane as a so-called P1 precursor form (100 kDa), Pmel[17] gets transported through the Golgi apparatus where it becomes a substrate for sialyltransferases (6)
Because this sorting defect has so far only been described in transient overexpression systems employing the cervical carcinoma cell line HeLa, we first decided to examine whether it would be observed in melanoma cells
Expression was confirmed by Western blot using antibody Pep13h (Fig. 1B, left panel), which recognizes newly synthesized Pmel[17], but not mature fibrils (19, 30). ⌬NTR appeared to undergo proprotein convertase (pPC)-mediated cleavage as judged by the detection of M (Fig. 1B, left panel) and M␣ (Fig. 1B, right panel) fragments, but did not give rise to the set of fibrillogenic fragments reactive with antibody HMB45 (Fig. 1B, right panel), suggesting that it represents a loss-of-function mutant
Summary
Following co-translational translocation into the endoplasmic reticulum (ER)[2] membrane as a so-called P1 precursor form (100 kDa), Pmel[17] gets transported through the Golgi apparatus where it becomes a substrate for sialyltransferases (6). In line with this data and consistent with previous reports (10, 13) we did not observe co-localization of ⌬NTR with the lysosomal/melanosomal marker LAMP1 (Fig. 1F), indicating that the NTR is essential to route the protein to the appropriate compartments within the cell.
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