Transcobalamin II-receptor (TC II-R)1 is a single chain glycoprotein with a molecular mass of 62 kDa (1) containing about 27% carbohydrate. It promotes plasma transport of cobalamin (Cbl; vitamin B 12 ) bound to transcobalamin II (TC II) (2). TC II-R functions as a noncovalent homodimer with a molecular mass of 124 kDa in tissue membranes (1), and its dimerization occurs not in the ER, where it is a monomer, but rather in the plasma membrane (3). In addition, the dimerization of TC II-R is a membrane fluidity-driven event requiring a highly ordered rigid bilayer (4). In polarized epithelial Caco-2 cells, TC II-R is predominantly present in basolateral membranes (5), where it functions in the delivery of Cbl to be utilized as coenzymes (6). Treatment of the basolateral surface of Caco-2 cells with TC II-R antiserum in vitro or circulating TC II-R antibody in vivo results in a failure of TC II-Cbl uptake, causing intracellular Cbl deficiency (6). These studies have suggested that plasma membrane delivery of TC II-R is extremely important for cells to obtain their Cbl from the circulation to maintain their normal metabolic, differentiation, and proliferation status. Despite the importance of plasma membrane expression of TC II-R in the tissue transport of Cbl, no information is available regarding its intracellular trafficking and plasma membrane expression.Recent studies have shown that formation of intracellular inter-or intrachain disulfide bonds and their final rearrangement by thiol-disulfide exchange reaction is one of the co/posttranslational modifications of some proteins (7,8). The correct formation of intrachain and, in the case of oligomeric proteins, interchain disulfide bonds is crucial for the proper assembly of secretory and plasma membrane proteins, which in turn determines their stability, intracellular transport, maturation, and function (9 -11). Inhibition of disulfide bond formation by DTT treatment has been shown to affect secretion of albumin and H1 subunit of asialoglycoprotein receptor from fibroblasts (12) and of gp80 (clusterin, apolipoprotein J) in polarized MadinDarby canine kidney cells (13). However, the secretion of nondisulfide bond-containing proteins such as ⣠1 -antitrypsin (14) in HepG2 cells or of a 20-kDa peptide derived from osteoponin in Madin-Darby canine kidney cells (13) is not affected. These studies have proposed that the retention of proteins in the ER following treatment with DTT is due to a failure of these proteins to form disulfide bonds, which then fold improperly. As a consequence, these misfolded proteins fail to achieve a conformation essential for their function as well as their exit from the ER for further processing and targeting (15).Human TC II-R contains 10 half-cysteines (16), and recent immunoblot studies of placental plasma membrane proteins under nonreducing and reducing conditions (1) have shown that TC II-R dimers contain intramolecular disulfide bonds that, upon reduction, increase the apparent molecular mass of the dimer by 20 kDa. Taken together, ...