Characterization and Subcellular Localization of Human Pmel 17/silver, a 100-kDa (Pre)Melanosomal Membrane Protein Associated With 5,6,-Dihydroxyindole-2-Carboxylic Acid (DHICA) Converting Activity

Abstract

Pmel 17 is preferentially expressed in pigment cells in a manner suggestive of involvement in melanin biosynthesis. The gene is identical to the silver (si) pigmentation locus in mice. We now produced a recombinant glutathione-S-transferase-human Pmel 17 infusion protein and raised polyclonal antibodies against it to confirm the ultrastructural location and presumed site of action predicted by the deduced primary structure of Pmel 17/silver, and to authenticate the specificity of the DHICA converting function as inherent to the silver-locus protein. Full-length Pmel 17 cDNA also produced in insect cells in a baculovirus expression vector to ensure that activity did not originate from a co-precipitated protein. Natural hPmel 17 from human melanoma cells has an approximate molecular size of 100 kDa. By immunoperoxidase electron microscopic cytochemistry, the antigen was localized to the limiting membranes of premelanosomes and presumed premelanogenic cytosolic vesicles and, to a minor extent, in the premelanosomal matrix. In an in vitro assay, both the natural and the recombinant Pmel 17 accelerated the conversion of DHICA to melanin. This activity was inhibited by the anti-Pmel 17 polyclonal antibodies, indicating that the acceleration of DHICA conversion by natural protein is genuine and cannot be due to contaminating complexed proteins. We suggest that in situ Pmel 17/silver is a component of a postulated premelanosomal/melanosomal complex of membrane-bound melanogenic oxidoreductive enzymes and cofactors, in analogy to the electron transfer chain in mitochondria.

Light- or Dark-Colored, L-DOPA-Based Melanins

“…1,[33][34] Similarly, the amyloid-like protein Pmel17/Silver/gp100 is capable of generating MN-like pigments in vitro or in vivo. 6,[35][36] Pmel17/Silver/gp100 is a melanocyte-specific protein within the matrix of the melanocytes upon which the MN pigment is deposited and organized. [37][38][39] It is worth noting that in the case of lignins, a plant-based polymeric substance derived from phenolic precursors, extracellular proteins containing so-called dirigent domains are involved in the deposition and organization of this pigment.…”

Retrotransposon insertion in SILV is responsible for merle patterning of the domestic dog

“…The SILV protein appears to be necessary for the formation of the fibril matrix upon which melanin intermediates are deposited late in melanosome maturation (24). Other studies have shown that SILV may also participate in melanin biosynthesis by accelerating the conversion of 5,6-dihydroxyindole-2-carboxylic acid to melanin (34,35). The mutant phenotype of SILV in the human is unknown (28).…”

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation

“…The amyloid sheets are critical determinants of the ellipsoid melanosome shape (12,13), which is required for proper melanosome motility into the apical processes of retinal pigment epithelial cells (14). The amyloid sheets have also been proposed to accelerate melanin polymerization (10,15,16), and organisms that lack PMEL or carry mutations in the PMEL gene are characterized by various degrees of hypopigmentation (13,(17)(18)(19)(20)(21). Because PMEL is synthesized in the endoplasmic reticulum (ER) as a type I transmembrane protein (22)(23)(24) but only initiates amyloid fibril formation within the lumen of endosomal membrane compartments (25)(26)(27), PMEL must navigate the secretory pathway from the ER to endosomes in a non-amyloid form.…”