Proteomic and Bioinformatic Characterization of the Biogenesis and Function of Melanosomes
Abstract
Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.
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Fractionation of NHM extracts revealed NCKX5, dark pigment, and TGN46 were enriched in fractions enriched for melanosomal marker TYRP1.
“Fractionation of our NHM extracts using a sucrose density gradient similar to that used in the proteomic study (18) revealed that NCKX5, dark pigment, and TGN46 were all enriched in the fractions enriched for melanosomal marker TYRP1.”
From SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis (2008) · cites this paper as [18]
NCKX5 was detected in melanosome-enriched fractions in a proteomic analysis.
“In a proteomic analysis of fractionated melanocytes, NCKX5 was detected in melanosomeenriched fractions (18), although we have not detected any colocalization between NCKX5 and melanosomal markers by immunofluorescence microscopy with our antisera (Fig. 3D).”
From SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis (2008) · cites this paper as [18]
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- Authors
- Chi, A., Valencia, J. C., Hu, Z.-Z., Watabe, H., Yamaguchi, H., Mangini, N. J., Huang, H., Canfield, V. A., Cheng, K. C., Yang, F., Abe, R., Yamagishi, S., Shabanowitz, J., Hearing, V. J., Wu, C., Appella, E., & Hunt, D. F.
- Affiliations
- National Institutes of Health (US)
- Department of Chemistry, University of Virginia, Charlottesville, Virginia, 22904, Laboratory of Cell Biology, National Institutes of Health, Bethesda, Maryland, 20892, Protein Information Resource, Georgetown University Medical Center, Washington, DC, Indiana University School of MedicineNorthwest, Gary, Indiana, Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, Jake Gittlen Cancer Research Foundation, Pennsylvania State University College of Medicine,...
- ORCID
- Venue
- Journal of Proteome Research
- Publisher
- American Chemical Society (ACS)
- Published
- 2006-10-04
- Volume
- 5
- Issue
- 11
- Pages
- 3135-3144
- ISSN
- 1535-3893 (ISSN-L)
- 1535-3907
- OpenAlex
- W2134091914
- Cited by
- 199
- References
- 50
- Citations by year
- 2026: 7 · 2025: 2 · 2024: 6 · 2023: 10 · 2022: 6 · 2021: 8 · 2020: 8 · 2019: 6 · 2018: 12 · 2017: 8 · 2016: 6 · 2015: 8 · 2014: 10 · 2013: 14 · 2012: 10
- FWCI
- 7.18
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Proteome Research (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (5r01hd040179-05)
- National Human Genome Research Institute (U01HG02712, U01HG002712)
- National Institute of General Medical Sciences (GM37537, R01GM037537)
- National Center for Research Resources (RR01744)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (HD40179)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › melanin and skin pigmentation
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Melanoma and MAPK Pathways
- Keywords
- melanosomes, melanin, melanosome biogenesis, melanocyte development, coat color genes, lysosome-related organelles, pigment epithelium-derived factor, SLC24A5, melanogenesis, organelle evolution
- MeSH terms
- Animals, Organelle Biogenesis, Chromatography, Affinity, Eye Color, Hair Color, Humans, Melanoma, Neoplasm Proteins, Peptide Fragments, Trypsin, Reproducibility of Results, Computational Biology, Melanosomes, Gene Expression Profiling, Proteomics, Cell Line, Tumor, Mice
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