Isolation of Intact and Functional Melanosomes from the Retinal Pigment Epithelium

Melanosomes of retinal pigment epithelium (RPE) have many vision supporting functions. Melanosome research would benefit from a method to isolate pure and characterized melanosomes. Sucrose gradient centrifugation is the most commonly used method for isolation of RPE melanosomes, but the isolated products are insufficiently characterized and their quality is unclear. Here we introduce a new gentle method for fractionation of porcine RPE that produces intact functional melanosomes with minimal cross-contamination from other cell organelles. The characterization of isolated organelles was conducted with several methods confirming the purity of the isolated melanosomal fraction (transmission electron microscopy, immunoblotting) and presence of the melanosomal membrane (fluorescence staining of melanosomal membrane, zeta potential measurement). We demonstrate that our isolation method produces RPE melanosomes with the ability to generate free phosphate (P(i)) from ATP thereby proving that many membrane proteins remain functional after isolation. The isolated porcine RPE melanosomes represented V-type H(+)ATPase activity that was demonstrated with bafilomycin A1, a specific V-ATPase inhibitor. We anticipate that the isolation method described here can easily be optimized for the isolation of stage IV melanosomes from other pigmented cell types and tissues.