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 test whether Hl(A4-33A) reached the trans-Golgi, we analyzed the incorporation of sialic acid and sulfation of tyrosine residues, two trans-Gol~-specific modifications (Roth et al., 1985; Baeuerle and Huttner, 1987) . Upon neuraminidase digestion of M1 and M1A cell lysates, the endo H-resistant forms of wild-type H1 and of Hl(A4-33A) were both slightly shifted on immunoblots, indicative of desialylation (Fig. 4, lanes 6-9) . Subunit H1 is not naturally sulfated. However, we have recently shown that addi-tion of a 9--residue tyrosine sulfation peptide to the exoplasmic carboxy terminus of the protein allows efficient labeling with laSS]sulfate in a stable-transfected MDCK cell line (Leitinger et al., 1994) . The tyrosine sulfation tag was introduced into the mutant Hl(A4-33A) and stable expressing MDCK cell lines, named M1A rs, were generated. The tagged protein Hl(A4-33A) rs accumulated intracellularly like Hl(A4-33A), as judged by immunofluorescence microscopy and proteinase K digestion (not shown). Upon labeling with [35S]sulfate, radioactivity was incorporated into the endo H-resistant form of Hl(A4-33A) rs with similar efficiency as into H1 xs, the tagged version of H1 (Fig. 4, lanes 10--12) . These results indicate that Hl(A4-33A) reaches the trans-GolgitTGN.