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

To construct the eDNA of AN2 and AN2A a BamHI site was introduced into the aminopeptidase N eDNA at codons 33/34 corresponding to the exoplasmic end of the transmembrane domain using the mutagenic anti-sense primer TCCTGGGATCCCACCACTG and the Amersham mutagenesis kit. The eDNA fragment 3' of this BamHI site was ligated to the 5' t-IindIfI-BamHI fragment of either H1 or Hl(A4-33A) eDNA. From the plasmid 19L, a gift from G. von Heijne (Karolinska Institute, Huddinge, Sweden) a sequence encoding 19 leucines, preceded by a KpnI site and the codons for Met-Gly-Pro-Arg, and followed by a BamHI site was amplified by PCR and ligated in front of the BamHI-EcoRI fragment of H1 eDNA to obtain the construct for H1ALeu19. The construct for H1Leu19 was made by ligating the HindlII-NruI fragment of plasmid pSAll/3 (Spiess and Handschin, 1987) to the H1ALeu~9 eDNA cut with KpnI and blunted.