Molecular and cellular studies of the human homolog of the 160-kD alpha-subunit of the coatomer protein complex.
The traffic of proteins through the eukaryotic secretory pathway is achieved in part by nonclathrin-coated vesicles mediating transport between the Golgi network and the endoplasmic reticulum. These transit vesicles are coated with coat proteins (COP), which assemble to form a complex of seven polypeptides known as coatomer. From the Hep3B human hepatocellular carcinoma cell line, we have previously isolated and sequenced the cDNA of a novel gene, HEP-COP, whose predicted amino acid sequence, calculated relative molecular mass, and hydrophilicity are strikingly similar to the 160-kD alpha-subunit of the coatomer complex in yeast. Four synthetic peptides were designed for immunizing pairs of rabbits to generate polyclonal antisera. In Western blot experiments, these antibodies could specifically recognize protein bands of 160 kD, which were absent when control preimmune sera were used. Immunoblotting of subcellular components of Hep3B cells probed with one of the antisera revealed 160-kD protein bands predominantly in the microsomal and cytosolic fractions, but virtually none in the nuclear compartment. Indirect immunofluorescence of Hep3B cells using the same antibody exhibited fluorescent staining chiefly in the cytoplasm. Taken together with the cDNA data, the results of this immunological analysis of the putative HEP-COP protein support the suggestion that the latter is the human homolog of alpha-COP.
Quek
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HH
Department of Microbiology, Faculty of Medcine, National University of Singapore, Republic of Singapore.
Chow
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VT
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020756:Coatomer Protein; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D015151:Immunoblotting; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011817:Rabbits; D014407:Tumor Cells, Cultured
D020756:Coatomer Protein; D018076:DNA, Complementary; D008565:Membrane Proteins