A major glycoprotein of the nuclear pore complex is a membrane-spanning polypeptide with a large lumenal domain and a small cytoplasmic tail.
One of a small number of polypeptides of the nuclear pore complex that have been identified is a major glycoprotein called gp210. Since it is very resistant to chemical extractions from membranes, gp210 was suggested to be integrated into nuclear membranes. In this study we have determined the membrane topology of this protein by biochemical and immunological approaches. We found that limited proteolysis of isolated nuclear envelopes with papain released a 200 kd water-soluble fragment of gp210 containing concanavalin A-reactive carbohydrate. Immunogold electron microscopy with a monoclonal antibody showed that this domain is localized on the lumenal side of nuclear membranes at pore complexes. Anti-peptide antibodies against two sequences near the C-terminus of gp210 were used to map possible membrane spanning and cytoplasmically disposed regions of this protein. From analysis of the protease sensitivity of these epitopes in sealed membrane vesicles, we determined that gp210 contains a small cytoplasmic tail and only a single membrane-spanning region. Thus, gp210 is a transmembrane protein with most of its mass, including the carbohydrate, located in the perinuclear space. This topology suggests that gp210 is involved primarily in structural organization of the pore complex, for which it may provide a membrane attachment site.
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
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Greber
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D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D003593:Cytoplasm / Q000032:analysis; D005455:Fluorescent Antibody Technique; D008562:Membrane Glycoproteins / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology*; D008854:Microscopy, Electron; D009685:Nuclear Envelope / Q000502:physiology*; D009687:Nuclear Proteins / Q000502:physiology*; D010447:Peptide Hydrolases; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000906:Antibodies; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D010447:Peptide Hydrolases