Identification of Three Coated Vesicle Components as and /3-Tubulin Linked to a Phosphorylated 50,000-dalton Polypeptide

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 six polypeptides in addition to an 180,000-dalton polypeptide called clathrin. In this report we show that the 54,000and 56,000-dalton coated vesicle polypeptides are aand B-tubulin, determined by immunoblotting and two-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,O00-dalton polypeptide appears to be related to brain microtubule-associated tau proteins since it can be specifically immunoprecipitated by an affinitypurified antiserum directed against these proteins. In addition, gel filtration experiments indicate that at least a fraction of the 50,O00-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 c~and ~-tubulin. Like brain coated vesicles, liver coated vesicles also contain an endogenous kinase activity, which phosphorylates polypeptides of the same molecular weights and isoelectric points as the brain coated vesicle 50,000-dalton, tau-like polypeptide, and aand ~-tubulin. The phosphorylated 50,000-dalton polypeptide may link the membrane and contents of coated vesicles with components of the cytoskeleton. The mechanisms by which membrane and secreted proteins are sorted and targeted to specific intracellular destinations are for the most part not understood. Coated vesicles are thought to mediate the transport of membrane proteins between various membrane compartments within the cell, for example between the endoplasmic reticulum and the Golgi apparatus and between the Golgi apparatus and the plasma membrane. They are also involved in the internalization of selected plasma membrane components during receptor-mediated endocytosis (12, 30). Isolated bovine brain coated vesicles contain ~ 100 copies (10) of a 180,000-dalton polypeptide called clathrin (28). Two proteins of 33,000 and 36,000 daltons are tightly bound to clathrin (16, 29) and comprise an assembly unit of the coat structure ("triskelion") (l 7, 40). Several other polypeptides have been shown to copurify with clathrin during various separation procedures (31, 34). The properties and precise functions of each of the coated vesicle polypeptides are only beginning to be understood. A 50,000-dalton polypeptide appears to be phosphorylated by a cAMPand Ca++-independent protein kinase activity associated with coated vesicles (14, 26). Little is known about the 54,000and 56,000-dalton polypeptides or about a series of polypeptides between 100,000 and 150,000 daltons, which are present in coated vesicle preparations at stoichiometries comparable with those of the 33,000and 36,000-dalton polypeptides (3 l). In this report we identify the 54,000and 56,000-dalton coated vesicle polypeptides as aand/3-tubulin. As would be expected, these two polypeptides are not unique to coated vesicles (31), yet they maintain a constant molar ratio relative to clathrin through several purification procedures and require harsh treatments for their dissociation from the coated vesicle structure. We have confirmed the report of Pauloin et al. (26) and Kadota et al. (14) that a 50,000-dalton coated vesicle polypeptide can be phosphorylated by a kinase activity present in THE JOURNAL OF CELL BIOLOGY VOLUME 97 JULY 1983 40-47 4 0 © The Rockefeller University Press . 0021-9525/97/07/0040/08 $I .00 on M ay 0, 2017 D ow nladed fom Published July 1, 1983