Monoclonal antibodies as markers of the endocytic and secretory pathways

A galactosyltransferase-rich subcellular fraction and wheat germ agglutinin(WGA)-binding microsomal proteins from rat myeloma cells have been used to immunize BALB/c mice. Fusion of the corresponding spleen cells with the Sp2/0 mouse myeloma has lead to the production of hybridomas secreting monoclonal antibodies directed against four proteins of the Golgi complex (GC) and other smooth membranes (SM). Subcellular fractionation of myeloma cells and rat liver, Triton X-114 partitioning, protease treatment and lectin binding studies have permitted us to identify--by immunoblotting--the molecular weight of the proteins involved, their topology and their mode of association with membranes. Morphological analysis has been performed by immunocytochemistry at the light and electron microscopic level. Judging by these criteria, the GCII antigen is a protein of 44 kDa which is loosely associated with the endodomain of Golgi cisternae. GCIII is a detergent-binding glycoprotein of 130 kDa whose epitope is on the endodomain of Golgi cisternae. SMI is a detergent-binding glycoprotein of 58 to 90 kDa found at several stations along the endocytic path: in coated pits, coated vesicles, endocytic vesicles, but not in lysosomes. The epitope recognized by the corresponding antibody faces the ectodomain. When this antibody is added to living cells in culture, it is rapidly internalized. SMII is a detergent-binding glycoprotein of 140 kDa. The epitope recognized is restricted to membranes of Golgi complex cisternae and multivesicular bodies. These reagents should be useful for dissection and perturbation of vesicular traffic.