Biosynthesis and processing of ribophorins in the endoplasmic reticulum

The extent to which the high mannose oligosaccharide side chains in ribophorin I undergo removal of peripheral mannose residues was assessed by a kinetic analysis of the distribution of radioactive mannose in ribophorin oligosaccharides obtained from cells labeled with 2[3H]mannose for l, 7, and 24 h. The oligosaccharides obtained from immuneprecipitated ribophorin I by pronase digestion and endoglycosidase H treatment were analyzed by high resolution Biogel P-4 gel filtration chromatography (13) . After l h of labeling, the major radioactive peaks corresponded to Man9GlcNAc and MansGlcNAc, and a small shoulder to GlcMan9GlcNAc (Fig. 9 C) . After 7 h of labeling ( Fig. 9 B) , peaks corresponding to these species were well resolved and Man7GlcNAc, ManrGlcNAc, and MansGlcNAc species could also be identified. A steady state seemed to be reached after 24 h (Fig. 9A) , when the most prominent peaks corresponded to MansGlcNAc and ManrGlcNAc, but all species above MansGlcNAc were represented. The preceding observations demonstrate that ribophorins are synthesized in membrane-bound ribosomes and that during their co-translational insertion into the ER membrane the nascent polypeptides undergo cleavage of an amino-terminal insertion signal, as well as core glycosylation of asparagine residues. Aside from trimming of the oligosaccharide chains, no major posttranslational modifications such as addition of terminal sugars, O-glycosylation, or further proteolytic cleavage of the polypeptide seemed to affect the ribophorins. A kinetic analysis indicated that after rapid removal of glucose residues, further trimming of mannoses from the core oligosaccharide is a slow process: a steady-state distribution of labeled mannoses in the different stages is attained only after 24 h. Several lines of reasoning indicate that this trimming takes place in the ER itself. If the slow trimming of ribophorin oligosaccharides were carried out by the Golgi mannosidases which process other glycoproteins (55) (56) (57) , ribophorins would be expected to reside for a rather long period of time in Golgi membranes, where their presence cannot be detected by immuneprecipitation (unpublished observations). The fact that no ribophorin molecules containing oligosaccharides shorter than MansGlcNAc could be detected in cl-9 cells, even after 72 h of labeling (not shown), also suggests that the proteins do not reach the cis portion of the Golgi apparatus where the enzyme transferase I, the action of which is required for the subsequent removal of mannoses from Man5GlcNAc, is thought to be located (54) .