THE ROLE OF COATED VESICLES IN INTRACELLULAR TRANSPORT

Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification I have made use of the relatively uniform diameter of coated vesicles to fractionate conventionally purified brain coated vesicles according to size on glass bead columns. Permeation chromatography can remove larger membrane contaminants, and gel electrophoretic analysis shows that only three major polypeptide chains and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin and are unique to fractions containing coated vesicles. Polypeptides of 54,000 and 56,000 molecular weight appear to be components of brain coated vesicles yet are also present in other membrane fractions.

I have identified the 54,000 and 56,000 dalton brain coated vesicle polypeptides as (alpha)- and (beta)-tubulin using immunoblotting and two-dimensional gel electrophoresis techniques. The tubulin polypeptides associate with coated vesicles by interacting with a 50,000 dalton, phosphorylated coated vesicle polypeptide. The 50,000 dalton polypeptide is antigenically related but distinct from microtubule associated tau proteins. At least a fraction of the 50,000 dalton polypeptide may also associate with the 100,000 dalton coated vesicle polypeptide. The 50,000 dalton polypeptide and (alpha)- and (beta)-tubulin are each phosphorylated by an endogenous coated vesicle kinase activity. An analogous endogenous kinase activity phosphorylates polypeptides of identical molecular weights and isoelectric points in a preparation of liver coated vesicles. The 50,000 dalton polypeptide may be a common coated vesicle constituent which links the membrane and contents of some types of coated vesicles with components of the cytoskeleton.

I have identified the presence of a cytoplasmically-oriented synaptic vesicle membrane component in brain coated vesicles and have shown that a monoclonal antibody directed against this polypeptide can penetrate the clathrin coat and recognize this polypeptide on the outer membrane surface of intact coated vesicles. An effective purification procedure for the isolation of coated vesicles from the electric organ of the marine ray Torpedo californica is also presented.