Identification of three coated vesicle components as alpha- and beta-tubulin linked to a phosphorylated 50,000-dalton polypeptide.

Abstract

Coated vesicles are involved in the intracellular transport of membrane proteins between a variety of membrane compartments. The coats of bovine brain coated vesicles contain at least 6 polypeptides in addition to an 180,000-dalton polypeptide called clathrin. The 54,000- and 56,000-dalton coated vesicle polypeptides are .alpha.- and .beta.-tubulin, determined by immunoblotting and 2-dimensional gel electrophoresis. An affinity-purified tubulin antiserum can precipitate coated vesicles. The tubulin polypeptides are tightly associated with a 50,000-dalton coated vesicle polypeptide, which is phosphorylated. The phosphorylated 50,000-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinity-purified antiserum directed against these proteins. Gel filtration experiments indicate that at least a fraction of the 50,000-dalton polypeptide may associate with the 100,000-dalton coated vesicle polypeptide. Since brain is a tissue rich in tubulins, liver coated vesicles were analyzed for the presence of .alpha.- and .beta.-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same MW and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and .alpha.- and .beta.-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton.