1995 Fiscal Year Final Research Report Summary

FUNCTION ANALYSIS OF COATOMER IN VESICLE TRANSPORT

Research Project

Project/Area Number	06680596
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Structural biochemistry
Research Institution	SASAKI INSTITUTE
Principal Investigator	KUGE Sayuri SASAKI INSTITUTE BIOCHEMISTRY RESEARCHER, 生化学部, 研究員 (50260104)
Project Period (FY)	1994 – 1995
Keywords	Vesicle Transport / Coatomer / epsilon-COP
Research Abstract	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.