trans-Golgi retention of a plasma membrane protein: mutations in the cytoplasmic domain of the asialoglycoprotein receptor subunit H1 result in trans-Golgi retention

Preparation of the stable MDCK cell lines M1, M1A (= Ml[A4-33A]), M1 rs, M2, M12 (= M[Y+f]) has been described previously Geffen et al., 1993; Leitinger et al., 1994; Fuhrer et al., 1994) . To produce stable cell lines, the eDNA of Hl(A4-33A) TM was cloned into pK21 (with a eytomegalovirus promoter; provided by R. Gentz, Hoffmann-La Roche, Basel), and transfected into MDCK cells together with one tenth the amount of the resistance plasmid pSVneo using polybrene and dimethyl sulfoxide according to Kawai and Nishizawa (1984) . Clonal cell lines resistant to 0.5 mg/ml G418-sulfate were isolated and screened for expression by immunofluorescence and immunoblot analysis using a polyclonal antiserum directed against the ASGP receptor. The eDNA of Hl(A4-33A) was subcloned into pBamHis (with a murine leukemia virus promoter and the Salmonella his gene as a resistance marker; from R. Mulligan, Whitehead Institute, Cambridge, MA) and transfected into the cell line M2, an MDCK-derived cell line expressing the second ASGP receptor subunit H2 and G418 resistance (Fuhrer et al., 1994) . Clonal cell lines were selected in the presence of 2 mg/ml histidinol. The resulting cell lines were named M1A Ts and M2/1A, respectively.