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The Pmel 17/silver locus protein. Characterization and investigation of its melanogenic function
T Kobayashi1, K Urabe, S J Orlow
1Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
The silver mutation in mice causes progressive graying of hair due to the loss of functional follicular melanocytes. Recently the silver locus gene (called Pmel 17) has been cloned; its encoded product shares homology with a chick melanosomal matrix protein and a bovine retinal pigment epithelial protein. Although the sequence of the silver gene and the correlation of its expression with pigment production have been reported, its function in melanogenesis is still unknown. In an effort to characterize that function, we have synthesized the predicted carboxyl-terminal peptide of the mouse Pmel 17 protein and generated a rabbit polyclonal antibody (alpha PEP13) to it; that antibody recognized the silver protein specifically. The immunoaffinity-purified silver protein lacked all of the known melanogenic catalytic activities which other tyrosinase-related proteins (TRP) have, nor did it appear to modulate any of those TRP activities. Metabolic labeling experiments demonstrated that the silver protein disappears in vivo within a few hours, indicating that it is rapidly degraded, or quickly processed to lose its carboxyl terminus. Cross-reactivity experiments showed that a recently reported anti-melanosomal matrix protein antibody (alpha MX) also recognizes the silver protein, although at a different epitope from that of alpha PEP13. Using Western immunoblotting, we analyzed subcellular fractions isolated from B16 F10 melanoma cells and found that the silver protein was rich in the melanosome fraction but was absent from coated vesicles which deliver TRPs to melanosomes. These results suggest that the silver locus product is a melanosomal matrix protein which may contribute to melanogenesis as a structural protein, although the possibility remains that it also has a novel catalytic function in melanogenesis.
Insights
The silver gene (Pmel 17) product, linked to graying hair, is a melanosomal matrix protein. It lacks known catalytic activity, suggesting a structural role in melanogenesis, not enzymatic.
Area of Science:
- Cell Biology
- Genetics
- Biochemistry
Background:
- The silver mutation in mice leads to progressive graying hair due to loss of functional follicular melanocytes.
- The silver locus gene, Pmel 17, has been identified, with its product showing homology to melanosomal matrix and retinal pigment epithelial proteins.
- The precise function of the Pmel 17 protein in melanogenesis remains unknown despite gene identification and expression correlation.
Purpose of the Study:
- To characterize the function of the mouse Pmel 17 protein in melanogenesis.
- To investigate the protein's localization and enzymatic activity within melanocytes.
Main Methods:
- Synthesis of the predicted carboxyl-terminal peptide of mouse Pmel 17 and generation of a specific polyclonal antibody (alpha PEP13).
- Immunoaffinity purification and testing for melanogenic catalytic activities.
- Metabolic labeling experiments to assess protein stability in vivo.
- Western immunoblotting analysis of subcellular fractions from B16 F10 melanoma cells using alpha PEP13 and an anti-melanosomal matrix protein antibody (alpha MX).
Main Results:
- The silver protein, recognized by alpha PEP13, lacks known melanogenic catalytic activities and does not modulate other tyrosinase-related proteins (TRPs).
- Metabolic labeling indicates rapid in vivo degradation or processing of the silver protein.
- The silver protein is localized to the melanosome fraction but absent from coated vesicles, and recognized by both alpha PEP13 and alpha MX antibodies at different epitopes.
- Absence from coated vesicles suggests a role after TRP delivery to melanosomes.
Conclusions:
- The Pmel 17 protein is a melanosomal matrix protein.
- Its function in melanogenesis is likely structural, contributing to the melanosome, rather than catalytic.
- A novel catalytic function cannot be entirely excluded, but its primary role appears structural.
