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

In the mutant protein H1 (A4-33A) amino acids 4-33 of the 40--residue cytoplasmic domain have been replaced by an alanine (Fig. 1) . In transfected fibroblast and MDCK cells, this mutant protein was efficiently expressed but could not be detected at the plasma membrane, Fig. 2 (A-D) shows immunofluorescence analysis of wild-type H1 and Hl(A4-33A) expressed by the two stable-transfected MDCK cell lines M1 and MIA, respectively, using an antiserum directed against the carboxy-terminal sequence of H1. The wild-type protein was stained on the surface of nonpermeabilized M1 cells (Fig. 2 A) . Upon permeabilization the staining reflects the distribution of H1 throughout the secretory pathway, in endosomes and on the cell surface ( ing pattern could be observed for H1(A4-33A) expressed by a stable NIH/3T3 fibroblast cell line (E) and by transiently transfected BHK-21 (F), COS-7, CaCo-2, and HeLa cells (not shown). In 5-10% of the transiently transfected cells, but not in the stable cell lines, the mutant protein could be detected also on the cell surface in addition to the typical intracellular structures. Since surface appearance of Hl(A4-33A) was also observed in a small fraction of mock-transfected M1A cells, this phenomenon is probably caused by the transfection conditions. The absence of Hl(A4-33A) from the cell surface was confirmed biochemically by immunoblot analysis of proteinase K digested M1A and M1 cells (Fig. 3) . In untreated cells, two immunoreactive forms of 40 kD and 45 kD for H1, and 35 kD and 40 kD for Hl(A4-33A) were detected. The two forms of each protein correspond to the highmannose glycosylated precursor which is sensitive to endoglycosidase H (endo H) digestion and to the complex glycosylated, endo H-resistant form (Fig. 4, lanes 1-5) .