Electron Microscopy to Examine the Effect of the a Anti-~-COP Antibody on Coated Vesicle Assembly

cDNA encoding the 20-kD subunit of coatomer, ~'-COE 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 ~'-COP blocks the binding of coatomer to Golgi membranes and prevents the assembly of COP-coated vesicles on Golgi cisternae. Unlike other coatomer subunits (/3-,/Y-, 3% and e-COP), ~'-COP exists in both coatomer bound and free pools. p ROTEIN transport between membrane-bound compartments of eukaryotic cells is carried out by the budding and fusion of transport vesicles (Palade, 1975). IntraGolgi transport appears to be mediated by COP-coated vesicles that have been isolated and shown to contain a set of coat proteins of apparent molecular mass 160 (a-COP), 110 (/3-COP), 110 (B'-COP), 98 (3"-COP), and 61 kD (&COP), and several smaller subunits (Orei et al., 1986; Malhotra et al., 1989; Serafini et al., 1991; Stenbeck et al., 1993). Purification of cytosolic B-COP from bovine brain has revealed that it exists as a 650-700-kD complex named coatomer, which contains five peptides with molecular mass identical to o~-, ~-, B'-, 3'-, and g-COP, and three other peptides with molecular mass 36, 35, and 20 kD, respectively (Waters et al., 1991; Stenbeck et al., 1993). Peptide sequence comparisons have shown that at least three polypeptides (/~-, ~'-, and 3'-COP) are components of both COP-coated vesicles and coatomer (Duden et al., 1991; Serafini et al., 1991; Stenbeck et al., 1992, 1993), and recent studies (Hara-Kuge, S., O. Kuge, L. Orci, M. Arnherdt, E T. Wieland, and J. E. Rothman, manuscript submitted for publication) have directly confirmed that coatomer enters in coated vesicles as an intact unit. Recently, we have shown that coated vesicle formation and vesicular transport in a cell-free system are dependent on coatomer (Elazar, Z., L. Orci, J. Ostermann, M. Amherdt, G. Tanigawa, and J. E. Rothman, manuscript submitted for publication; Orci et al., 1993). In addition, antibody to B-COP microinjected into cells inhibits transport to the trans-Golgi network (Pepperkok et al., 1993), and 3'-COP is related to Sec2 lp, a protein encoded by a yeast gene reAddress all correspondence to James E. Rothman, Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. quired for ER to Golgi transport (Hosobuchi et al., 1992; Stenbeck et al., 1992). Thus, coatomer is required both for intra-Golgi transport and for transport from ER to Golgi, both in vivo and in vitro. In this study, we define a new coat protein ~'-COP, previously known as p20, the 20-kD subunit of coatomer. We show that this protein is present in coated vesicles, and is required for coatomer binding to the Golgi membranes and for coated-vesicle assembly. While anti-~'-COP antibody completely inhibits coated vesicle formation, it only partially inhibits the assay signal of intra-Golgi transport reconstituted in a cell-free system. This evidence confirms the dual role of coat assembly in budding and in coupling fusion to budding (Elazar, Z., L. Orci, J. Ostermann, M. Amherdt, G. Tanigawa, and J. E. Rothman, manuscript submitted for publication). Materials and Methods