Coated vesicles: characterization, selective dissociation, and reassembly.

Abstract

Sodium dodecyl sulfate/polyacrylamide gels of coated vesicles from porcine brain (mean 76% coated vesicles) show 3 major proteins (180,000, 125,000 and 55,000 daltons) that account for 73% of the total protein. Preparations consisting predominantly of coats (65%) have less of the 55,000 dalton protein. Clathrin (180,000 daltons) comprises 40% of the protein of a coated vesicle. Conditions of 2 M urea, 0.25 M MgCl2, or pH 7.5 disrupt the coat and solubilize clathrin. Solubulized clathrin reforms coat structures after dilution of urea or MgCl2. High-pH-solubilized clathrin reassembles after dialysis against buffer at pH 6.5 containing dithiothreitol (5 mM). Reassembled coats are predominantly clathrin.