Prev. Article Next Article Table of Contents

Article

Proteomic and Bioinformatic Characterization of the Biogenesis and Function of Melanosomes

Supporting Info

An Chi,^†^# Julio C. Valencia,^‡^# Zhang-Zhi Hu,^§^# Hidenori Watabe,^‡ Hiroshi Yamaguchi,^‡ Nancy J. Mangini, Hongzhan Huang,^§ Victor A. Canfield, Keith C. Cheng, Feng Yang,^† Riichiro Abe, Shoichi Yamagishi,⁺ Jeffrey Shabanowitz,^† Vincent J. Hearing,^‡ Cathy Wu,^§ Ettore Appella,*^‡ and Donald F. Hunt*^†^¶

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, Hershey, Pennsylvania, Department of Dermatology, Hokkaido University Graduate School, Sapporo, Japan, Kurume University School of Medicine, Kurume, Japan, and Department of Pathology, University of Virginia, Charlottesville, Virginia, 22908

J. Proteome Res., 2006, 5 (11), pp 3135–3144

DOI: 10.1021/pr060363j

Publication Date (Web): October 4, 2006

†

Department of Chemistry, University of Virginia.

These authors contributed equally to this research.

‡

Laboratory of Cell Biology, National Institutes of Health.

Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center.

Indiana University School of MedicinesNorthwest.

Department of Pharmacology, Pennsylvania State University College of Medicine.

Jake Gittlen Cancer Research Foundation, Pennsylvania State University College of Medicine.

Hokkaido University Graduate School.

Kurume University School of Medicine.

Authors for correspondence. For Dr. Ettore Appella: Building 37, Room 2140, National Institutes of Health, Bethesda, MD 20892; e-mail, appellae@ nih.gov. For Dr. Donald F. Hunt: Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA 22904, E-mail: dfh@virginia.edu.

Department of Pathology, University of Virginia.

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 1500 proteins in melanosomes of all stages, with 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.