DAD1 IS REQUIRED FOR THE FUNCTION AND THE STRUCTURAL INTEGRITY OF THEOLIGOSACCHARYLTRANSFERASE COMPLEX

Asparagine-linked glycosylation is a highly conserved protein modifica tion reaction that occurs in all eukaryotic organisms. The oligosaccha ryltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of pre assembled high mannose oligosaccharides onto certain asparagine residu es of nascent polypeptides. The mammalian OST complex was initially th ought to be composed of three transmembrane proteins, ribophorin I (RI ), ribophorin II (RII), and OST48, Most recently, a small integral mem brane protein of 12 kDa called DAD1 has been identified as an addition al member of the mammalian OST complex. A point mutation in the DAD1 g ene is responsible for the temperature-sensitive phenotype of a baby h amster kidney-derived cell line (tsBN7) that undergoes apoptosis at th e non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non- permissive temperature. This temperature-sensitive cell Line offered u nique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylatio n, Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins wer e reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protei n) and the Sec61 complex, which are components of the translocation ap paratus, are not affected by the instability of the OST subunits, Furt hermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temp erature caused also the underglycosylation of a secretory glycoprotein , We concluded that degradation of DAD1 at the non-permissive temperat ure not only affects the stability of OST48 and the ribophorins but al so results in the functional inactivation of the OST complex.