Biosynthesis and processing of ribophorins in the endoplasmic reticulum

Analysis of the products of in vitro translation of poly A ÷ mRNAs extracted from free (Fig. 3, lanes a and c) and membrane-bound polysomes (lanes b and d) isolated from cl-9 cells showed that both ribophorin mRNAs are found only in polysomes bound to ER membranes and therefore indicated that in intact cells the polypeptides must be co-translationally inserted into the ER membranes. The in vitro synthesized ribophorin polypeptides had the same electrophoretic mobility as the corresponding proteins found in cl-9 cells (Fig. 4, compare lanes a and c for ribophorin I) . Because both ribophorins are glycoproteins (45, 46) , the size of the primary translation products were also compared with that of the respective unglycosylated polypeptides synthesized in cells treated with tunicamycin, an inhibitor of N-linked glycosylation (30) (Fig. 4, lane b) . It was observed that each of the unglycosylated ribophorins synthesized in vivo was ~2,000 mol wt smaller (ribophorin I, Mr = 63,000; ribophorin II, Mr = 61,000, not shown) than the products of in vitro translation. These observations strongly suggest that both ribophorins contain amino-terminal signal sequences which are removed during co-translational insertion of the polypeptides into the ER membrane. Co-translational core glycosylation must also take place, however, so that no net change in the apparent molecular weight of the ribophorins is observed after their insertion into the ER membrane is completed. These presumptions were confirmed when the co-translational insertion of ribophorin I into the ER membrane was carried out in a cell-free translation system supplemented with dog pancreas microsomes (Fig. 5) . The presence of membranes during