title: 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.
authors.affiliation: Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
authors.forename: An
authors.initials: A
authors.lastname: Chi
authors.forename: Julio C
authors.initials: JC
authors.lastname: Valencia
authors.forename: Zhang-Zhi
authors.initials: ZZ
authors.lastname: Hu
authors.forename: Hidenori
authors.initials: H
authors.lastname: Watabe
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamaguchi
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Mangini
authors.forename: Hongzhan
authors.initials: H
authors.lastname: Huang
authors.forename: Victor A
authors.initials: VA
authors.lastname: Canfield
authors.forename: Keith C
authors.initials: KC
authors.lastname: Cheng
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Riichiro
authors.initials: R
authors.lastname: Abe
authors.forename: Shoichi
authors.initials: S
authors.lastname: Yamagishi
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Hearing
authors.forename: Cathy
authors.initials: C
authors.lastname: Wu
authors.forename: Ettore
authors.initials: E
authors.lastname: Appella
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D019295:Computational Biology / Q000379:methods; D005127:Eye Color; D020869:Gene Expression Profiling; D006200:Hair Color; D006801:Humans; D008545:Melanoma; D020460:Melanosomes / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001678:Organelle Biogenesis; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D014357:Trypsin
chemical_list: D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014357:Trypsin