Biosynthesis and processing of ribophorins in the endoplasmic reticulum

THE JOURNAL OF CELL BIOLOGY " VOLUME 99, 1984 FIGURE 3 Site of synthesis of ribophorins. Messenger RNA isolated from free (lanes a and c) or membrane-bound polysomes (lanes b and d) prepared from rat hepatocyte cultures (cl-9) was used to program a wheat germ cell-free translation system (12 A26o of mRNA/ml). Samples (0.2 ml) containing ~107 cpm of [3SS]methionine-incorporated radioactivity were used for immuneprecipitation of in vitro synthesized polypeptides with antibodies against ribophorin I or ribophorin II (300 ~,g/ml IgG for each). Immuneprecipitates were analyzed by SDS PAGE (10%) followed by fluorography (72 h). translation (Fig. 5, lane a) , but not their addition after translation was completed (Fig. 5, lane c) , rendered the product (Mr = 65,000) sensitive to endoglycosidase H treatment, which resulted in a decrease in the apparent molecular weight to Mr = 63,000 (Fig. 5, lane b) , the size of the polypeptide found in tunicamycin-treated cells (Fig. 4, lane b) . After their insertion in the ER, newly synthesized ribophorins did not appear to undergo major posttranslational modifications, since the electrophoretic mobility of the labeled proteins did not change when incubation of cl-9 cells with [35S]methionine was extended for various periods, from 6 min to 15 h (for ribophorin I see Fig. 6 ). Thus, it appeared unlikely that the asparagine-linked oligosaccharides were modified to the complex forms characteristic of many glycoproteins (21, 25) , or that O-linked sugars are added to the polypeptides (9, 24) . Indeed, the effect of endoglycosidases on mature ribophorins demonstrated the presence of high mannose oligosaccharide chains in these proteins. ~25I-labeled ribophorins were resistant to endoglycosidase D (Fig. 7 , lane b for ribophorin I) and sensitive to endoglycosidase H treatment (Fig. 8, lanes b and d) . Moreover, treatment with endoglycosidase H released >95% of the [3H]mannose radioactivity in ribophorins obtained from cells labeled for 48 h with [3H]mannose (data not shown). It can therefore be concluded that all mannosecontaining oligosaccharides in ribophorin I are of the high mannose type.