The Cytoplasmic Coatomer Protein COPI

• Published in: The Journal of biological chemistry 1999-10, Vol.274 (44), p.31135-31138
• Main Authors: de la Vega, Luis A., Stockert, Richard J.
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.44.31135

Expression of the asialoglycoprotein receptor (ASGR) by the human hepatocellular carcinoma cell lines HepG2 and HuH-7 in response to intracellular cGMP concentrations was previously shown to be regulated at the translational level (1). Stable transfection of COS-7 cells with deletion constructs encoding the asialoglycoprotein receptor H2b subunit localized the cGMP-responsive cis-acting element to the mRNA 5′-untranslated region. Resolution by anion exchange chromatography of an S-100 isolated from human liver resulted in the partial purification of an RNA-binding protein specific to thiscis-acting element. Northwestern analysis using the 5′-untranslated region as probe indicated that a 140-kDa protein was the potential RNA-binding protein. Sequence of tryptic peptides suggested that the 140-kDa protein was the α-COP subunit of coatomer protein COPI, usually associated with trans-Golgi network membrane traffic. Immunoblot analysis confirmed the presence of α-COP in the Mono-Q fraction as well as that of a second coatomer subunit, β-COP. Antibody induced gel retardation supershift confirmed the identification of the RNA-binding proteins as α- and β-COP. Although the RNA recognition motif appears to reside solely in α-COP, antibody-induced supershift strongly indicated that the entire coatomer complex was the trans-acting factor. Depletion of S-100 with the antibody to β-COP confirmed that the coatomer was the sole protein binding to the ASGR mRNA 5′-untranslated region in liver cytosol and responsible for inhibition of in vitrotranslation of the asialoglycoprotein receptor.