Characterization of molecules involved in protein translocation using a specific antibody.

Abstract

The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum requires a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of MW 60,000. Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule in Madin-Darby canine kidney (MDCK) cells. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of MW 72,000, representing the membrane-bound protein from which the MW 60,000 peptide was proteolytically derived. The antibody could also be used to remove exclusively the MW 60,000 peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Immunoprecipitation of elastase extracts alkylated with [14C]N-ethylmaleimide selected a single species of MW 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the MW 72,000 membrane protein irrespective of the duration of labeling or if followed by a chase. Subsequent treatment with protease generated the MW 60,000 fragment. In addition, the antibody could be used to visualize reticular structures in intact cells which correspond to endoplasmic reticulum at the ultrastructural level. It is thus clear that one membrane component required in the vectorial translocation of nascent secretory (and membrane) proteins is a peptide of MW 72,000.