Characterization and subcellular localization of human Pmel 17/silver, a 110-kDa (pre)melanosomal membrane protein associated with 5,6,-dihydroxyindole-2-carboxylic acid (DHICA) converting activity.
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.
Lee
Z H
ZH
Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, USA.
Hou
L
L
Moellmann
G
G
Kuklinska
E
E
Antol
K
K
Fraser
M
M
Halaban
R
R
Kwon
B S
BS
D000818:Animals; D006801:Humans; D007211:Indoles / Q000378:metabolism*; D008543:Melanins / Q000378:metabolism*; D008544:Melanocytes / Q000737:chemistry* / Q000648:ultrastructure; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D009154:Mutation; D010088:Oxidoreductases; D010858:Pigmentation / Q000235:genetics; D011506:Proteins / Q000032:analysis* / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D058951:gp100 Melanoma Antigen
D007211:Indoles; D008543:Melanins; D008562:Membrane Glycoproteins; C548637:PMEL protein, human; C548642:Pmel protein, mouse; D011506:Proteins; D058951:gp100 Melanoma Antigen; C030692:5,6-dihydroxy-2-indolylcarboxylic acid; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1