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Abstract. The cDNA encoding e-COP, the 36-kD subunit of coatomer, was cloned from a bovine liver cDNA library and sequenced. Immunoblotting with an anti-e-COP antibody showed that e-COP exists in COP-coated vesicles as well as in the cytosolic coatomer. Using the cloned cDNA, recombinant Hisstagged e-COP was overexpressed in cultured Chinese hamster ovary (CHO) cells, from which metabolically radiolabeled coatomer was purified by taking advantage of the Hiss tag. Radiolabeled coatomer was employed to establish that all the subunits of the coatomer enter coated vesicles as an intact unit.I NTRACELLULAR traffic between the membrane compartments of eukaryotic cells relies on the movement of vesicular carriers (Jamieson and Palade, 1967;Palade, 1975; Rothman et al., 1984a,b). Golgi-derived (non-clathrin or COP) coated vesicles can be produced in a cell-free system that reconstitutes intercisternal protein transport (Balch et al., 1983(Balch et al., , 1984Balch and Rothman, 1985;Orci et al., 1986Orci et al., , 1989 and purified (Malhotra et al., 1989;. This led to the identification of four of the subunits of the coat, or COPs, termed or-COP (160 kD),/3-COP (110 kD), -y-COP (98 kD), and 8-COP (61 kD).Independently, a cytosollc complex that acts as the coat protomer (termed 'coatomer') containing the same four coat proteins was purified (Waters et al., 1991). The coatomer also contains polypeptides of Mr 35-36 kD (a doublet) and 20 kD (~'-COP: Kuge et al., 1993). The coatomer is required to form Golgi-derived coated vesicles and these contain at least the/3-COP subunit. But there has not been direct proof that the entire coatomer is incorporated en bloc into the coat.We cloned the cDNA encoding the 36-kD subunit of coatomer (which we now term e-COP) and used it to express 36-kD protein containing six histidine residues in mammalian cells. These transfectants enabled us to develop a simple method to purify radiolabeled coatomer using the NiAddress all correspondence to Dr. J. E. Rothman, Program of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York 10021.Dr. Hara-Kuge's current address is Department of Biochemistry, Sasaki Institute, Kanda-Surugadai, Chiyoda-ku, Tokyo 101, Japan.Dr. Kuge's current address is Department of Biochemistry and Cell Biology, National Institute of Heaith, Toyama, Shinjuku-ku, Tokyo 162, Japan.NTA affinity resin. Using this radiolabeled coatomer, we could follow coat assembly on Golgi membranes, and also determine the stoichiometry of each subunit at every stage. Materials and Methods Materials DNA ManipulationsDNA manipulations, including restriction enzyme digestion, ligation, plasmid isolation, subcloning, E. coli transformation, 32p-labeling of DNA, and oligonucleotide probes for filter hybridization, were carried out by the standard methods, unless otherwise stated. DNA nucleotide sequences were determined by the dideoxy chain termination methods with Sequenase, using walking primers. Purification of e-COPCoatomer was prepared from...

VIP36, an intracellular lectin that recognizes high mannose-type glycans (Hara-Kuge, S., Ohkura, T., Seko, A., and Yamashita, K. (1999) Glycobiology 9, 833-839), was shown to localize not only to the early secretory pathway but also to the plasma membrane of MadinDarby canine kidney (MDCK) cells. In the plasma membrane, VIP36 exhibited an apical-predominant distribution, the apical/basolateral ratio being ϳ2. Like VIP36, plasma membrane glycoproteins recognized by VIP36 were found in the apical and basolateral membranes in the ratio of ϳ2 to 1. In addition, secretory glycoproteins recognized by VIP36 were secreted ϳ2-fold more efficiently from the apical membrane than from the basolateral membrane. Thus, the apical/basolateral ratio of the transport of VIP36-recognized glycoproteins was correlated with that of VIP36 in MDCK cells. Upon overproduction of VIP36 in MDCK cells, the apical/basolateral ratios of both VIP36 and VIP36-recognized glycoproteins were changed from ϳ2 to ϳ4, and the secretion of VIP36-recognized glycoproteins was greatly stimulated. In contrast to the overproduction of VIP36, that of a mutant version of VIP36, which has no lectin activity, was of no effect on the distribution of glycoproteins to apical and basolateral membranes and inhibited the secretion of VIP36-recognized glycoproteins. Furthermore, the overproduction of VIP36 greatly stimulated the secretion of a major apical secretory glycoprotein of MDCK cells, clusterin, which was found to carry at least one high mannose-type glycan and to be recognized by VIP36. In contrast to the secretion of clusterin, that of a non-glycosylated apical-secretion protein, galectin-3, was not stimulated through the overproduction of VIP36. These results indicated that VIP36 was involved in the transport and sorting of glycoproteins carrying high mannose-type glycan(s).Newly synthesized secretory and membrane proteins exit from the ER 1 in transport vesicles targeted to the Golgi apparatus. Vesicular transport through the Golgi is often accompanied by post-translational modifications, such as glycosylation of cargo proteins until they have reached the trans-Golgi Network. In the trans-Golgi Network, proteins are sorted into vesicles bound for different destinations including the plasma membrane, the endosome/lysosome, and secretory granules. The protein sorting has been one of the most interesting issues in the study of vesicular protein traffic processes, but its molecular mechanisms remain largely unresolved.It has been recently demonstrated that intracellular lectins play important roles in vesicular transport: for example, mannose-6-phosphate receptor (1) as a receptor recognizing the marker for lysosomal enzymes, calnexin (2, 3) and calreticulin (4) as molecular chaperones, and ERGIC-53 (5) possibly as a transport cargo receptor. ERGIC-53 is an intermediate compartment marker (6), and it is identical to MR60, a mannosespecific membrane lectin (7) with a carbohydrate-binding domain homologous to that of lectins of leguminous plants (8). The N-term...

Abstract. 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.