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

Mutagenesis of the cDNAs encoding the mutants H1(A4-33A), HI(A4-11), and HI(A12-33) have been described previously (Beltzer et al., 1991; Geffen et al., 1993) . Hl(A12-33/5A) was constructed using the mutagenesis kit by Amersham Corp. (Arlington Heights, IL), the eDNA of HI(A12-33) subcloned into M13mp19 as the template, and the anti-sense oligonucleotide GGTCTTGAG..~CCTCCTTG as the primer (the mismatched nucleotides are underlined). To generate the cDNA of H1(A4-33m), two complementary oligonucleotides were synthesized, CCAGGAACAAAA-Gq'TGATrTCTGAAGAAGACITGGCTGCA and GCCAAGTCITCT-TCAGAAATCAACITITGTTC, which encode the e-myc epitope EQK-LISEEDLA recognized by the monoclonal antibody 9E10 (Evan et al., 1985) and produce "sticky ends" for in-frame insertion between the StyI site at codons 2/3 and the PstI site at codons 32/33 of the H1 cDNA.