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

The other eDNA constructs were made by polymerase chain reaction (PCR) using the wild-type eDNA of H1 (Spiess et al., 1985; Hind III-EcoRI subcloned into pSP64 or pGEM3) as the template, a mutagenic primer and a second primer corresponding to a flanking sequence in the plasmid vector. For Hl(~Pro), a 5' eDNA fragment containing codons 1-24 and 31-33 was amplified using the oligonucleotide CGTCCTGCAG-GAGCCCTITrCTGAGCTG as the mutagenic primer. Codons 32 and 33 correspond to a PstI restriction site that was used to ligate this amplified 5' segment to the 3' rest of the H1 eDNA starting from this PstI site. The HI(A2-37) construct was made using the mutagenic primer CCTAC-CATGGGACCTCGCCTCCT to amplify an amino terminally truncated eDNA with an ATG and an NdeI restriction site for subcloning. The cDNAs of HI(A2-19), HI(A2-24), HI(A2-28), HI(A2-33), and HI(A26-40) were constructed by PCR and splicing by overlap extension according to Ho et al. (1989a) , thereby retaining the original 5' untranslated sequence and ATG.