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Characterization of Molecules Involved in Protein Translocation Using a Specific Antibody

Meyer, David I. ; Louvard, Daniel ; Dobberstein, Bernhard

In: The Journal of Cell Biology, 92 (1982), pp. 579-583

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The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol. 87:503-508). Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived . The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system. Moreover,immunoprecipitation of elastase extracts alkylated with [t4C] N-ethylmaleimide selected a single species of mol wt 60,000. Immunoprecipitation of in vivo radiolabeled proteins from the appropriate cell type yielded the 72,000-mol wt membrane protein irrespective of the duration of labeling, or if followed by a chase. Subsequent treatment with protease generated the 60,000-mol wt 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 mol wt 72,000.

Item Type: Article
Journal or Publication Title: The Journal of Cell Biology
Volume: 92
Date Deposited: 15 Jul 2008 17:26
Date: 1982
Page Range: pp. 579-583
Faculties / Institutes: Service facilities > Center for Molecular Biology Heidelberg
Subjects: 570 Life sciences
Schriftenreihe ID: Works by Bernhard Dobberstein
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