Prompted by previous observations which suggested that the release of
polymannose oligosaccharides shortly after the cotranslational N-glycosylation of
proteins is a function of the ER-associated quality control system (Moore and Spiro (1994) J. Biol. Chem., 269, 12715-12721), we evaluated the effect which
proteasome inhibitors have on the appearance of these free saccharide components. Employing as a model system
castanospermine-treated BW5147 mouse T-
lymphoma cells in which accelerated degradation of the
T-cell receptor (TCR) alpha subunit takes place (Kearse et al. (1994) EMBO J., 13, 3678-3686), we noted that both
lactacystin and N-acetyl-L-leucyl-L-leucyl-L-norleucinal, but not
leupeptin, brought about a rapid and substantial reduction in the release of free
polymannose oligosaccharides into the cytosol during pulse-chase studies, while the
oligosaccharides in the intravesicular compartment remained unchanged, as measured by
streptolysin O permeabilization. This inhibition was furthermore selective in that it affected solely the components terminating in a single
N-acetylglucosamine residue (OS-GlcNAc(1)) and not the
oligosaccharides terminating in a
di-N-acetylchitobiose sequence (OS-GlcNAc(2)), which reside primarily in the intravesicular compartment. Despite the quantitative effect of the
proteasome inhibitors on the cytosolic
oligosaccharides, the molar distribution of the triglucosyl OS-GlcNAc(1) species was unaffected. The decrease in cytosolic
oligosaccharides brought about by
proteasome inhibition was reflected in a pronounced increase in the stability of the TCRalpha subunit. Our findings suggest that the N-deglycosylation and
proteasome mediated degradation are coupled events. On the basis of our data and those of others we propose that the quality control mechanism involves proteasomes associated with the cytosolic side of the endoplasmic reticulum acting in concert with a membrane situated
N-glycanase. Such a complex by removing the
carbohydrate units could facilitate the retrograde ER to cytosol translocation of
glycoproteins.