Abstract
The multimeric clathrin assembly proteins AP-1 and AP-2 with molecular masses of approximately 270 kDa and the monomeric clathrin assembly proteins AP180 and auxilin with molecular masses of approximately 90 kDa catalyze the assembly of clathrin into artificial clathrin baskets under physiological conditions. We have now identified a much smaller approximately 20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0, extracts of bovine-brain coated vesicles and purified it to near homogeneity. A polyclonal antibody against this protein did not cross-react with any of the other assembly proteins, and sequencing data suggest that this new protein is similar or identical to myelin basic protein (MBP). At a molar ratio of 3 molecules per clathrin triskelion, MBP catalyzes polymerization of clathrin into artificial baskets that appear structurally similar to the baskets assembled by the other assembly proteins. In addition, like the other baskets, the clathrin-MBP baskets are uncoated by hsp70. MBP represents a significant fraction of the total assembly protein activity present in 0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not clear if it acts as an assembly protein in vivo, but because it is well characterized and easily available, MBP will be a useful protein to investigate the mechanism of clathrin assembly and disassembly in vitro.
Highlights
From the Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, ‡Clinical Neurosciences Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-0301
We report the purification of this 20-kDa protein and identify it as myelin basic protein (MBP)
Like the previously discovered higher molecular weight clathrin assembly proteins, AP-1, AP-2, AP180, and auxilin, MBP copurifies with coated vesicles and interacts with clathrin in vitro under physiological conditions inducing it to polymerize into baskets
Summary
Materials—Superose-6 was obtained from Pharmacia Biotech Inc. Hydroxylapatite was from Calbiochem. Assembly proteins, and 100-kDa cofactor protein were obtained from the coated vesicles as reported previously [20]. The amount of clathrin assembled was quantified by SDS-gel electrophoretic analysis of the solutions before and after centrifugation. Uncoating Assay—Uncoating assays were performed as described previously [17, 18, 20, 21] The dependence of this uncoating reaction on cofactor concentration was determined [20, 21]. The pure protein was obtained from the Coomassie-stained protein bands of the SDS gels. Amino Acid Sequence Determination—The 20-kDa protein was digested with Lys-C endoproteinase overnight at 37 °C at a enzyme to substrate ratio of 1 to 40. Concentrations of the hsp, clathrin, and assembly proteins were determined as described by Prasad et al [20]
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