FUNCTION ANALYSIS OF COATOMER IN VESICLE TRANSPORT

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The cDNA encoding epsilon-COP,the 36-kD subunit of coatomer, was cloned form a bovine liver cDNA library and sequenced. Immunoblotting with an anti-epsilon-COP antibody showed that epsilon-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA,recombinant His_6-tagged epsilon-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the His_6 tag. Radiolabeled coatomer was employed to establish that all the subunits of the enter coated vesicles as an intact unit.cDNA encoding the 20-kD subunit of coatomer, zeta-COP,predicts a protein of 177-amino acid residues, similar in sequence to AP17 and AP19, subunits of the clathrin adaptor complexes. Polyclonal antibody directed to zeta-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (beta-, beta'-, gamma-, and epsilon-COP), zeta-COP exists in both coatomer bound and free pools.