CYTOSOLIC DEGLYCOSYLATION PROCESS OF NEWLY SYNTHESIZED GLYCOPROTEINS GENERATES OLIGOMANNOSIDES POSSESSING ONE GLCNAC RESIDUE AT THE REDUCING END

Recent studies on the mechanism of degradation of newly synthesized gl ycoproteins suggest the involvement of a retrotranslocation of the gly coprotein from the lumen of the rough endoplasmic reticulum into the c ytosol, where a deglycosylation process takes place. In the studies re ported here, we used a glycosylation mutant of Chinese hamster ovary c ells that does not synthesize mannosylphosphoryldolichol and has an in creased level of soluble oligomannosides originating from glycoprotein degradation. In the presence of anisomycin, an inhibitor of protein s ynthesis, we observed an accumulation of glucosylated oligosaccharide- lipid donors (Glc(3)Man(5)GlcNAc(2-)PP-Dol), which are the precursors of the soluble neutral oligosaccharide material. Inhibition of rough e ndoplasmic reticulum glucosidase(s) by castanospermine led to the form ation of Glc(3)Man(5)GlcNAc(2)(OSGn2) (in which OSGn2 is an oligomanno side possessing two GlcNAc residues at its reducing end), which was th en retained in the lumen of intracellular vesicles. Thus they were pro tected during an 8 h chase period from the action of cytosolic chitobi ase, which is responsible for the conversion of OSGn2 to oligomannosid es possessing one GlcNAc residue at the reducing end (OSGn1). In contr ast, when protein synthesis was maintained in the presence of castanos permine, glucosylated oligomannosides (Glc(1-3)Man(5)GlcNAc(1)) were r ecovered in cytosol. Except for monoglucosylated Man(5) species, which are potential substrates for luminal calnexin and calreticulin, the p attern of oligomannosides was similar to that observed on glycoprotein s. The occurrence in the cytosol of glucosylated species with one GlcN Ac residue at the reducing end implies that the deglycosylation proces s that generates glucosylated OSGn1 from glycoproteins occurs in the c ytosol.