Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.
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.
Chi
An
A
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Valencia
Julio C
JC
Hu
Zhang-Zhi
ZZ
Watabe
Hidenori
H
Yamaguchi
Hiroshi
H
Mangini
Nancy J
NJ
Huang
Hongzhan
H
Canfield
Victor A
VA
Cheng
Keith C
KC
Yang
Feng
F
Abe
Riichiro
R
Yamagishi
Shoichi
S
Shabanowitz
Jeffrey
J
Hearing
Vincent J
VJ
Wu
Cathy
C
Appella
Ettore
E
Hunt
Donald F
DF
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
D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014357:Trypsin