Glycobiology, 2000, Vol. 10, No. 7 727-735
© 2000 Oxford University Press
Effect of proteasome inhibitors on the release into the cytosol of free polymannose oligosaccharides from glycoproteins
Departments of Biological Chemistry and Medicine, Harvard Medical School, and the Joslin Diabetes Center, Boston, MA 02215, USA
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) 
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-GlcNAc1) and not the oligosaccharides terminating in a di-N-acetylchitobiose sequence (OS-GlcNAc2), 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-GlcNAc1 species was unaffected. The decrease in cytosolic oligosaccharides brought about by proteasome inhibition was reflected in a pronounced increase in the stability of the TCR 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.
1 Present address: Department of Chemistry, East Carolina University, NC 27858
2 To whom correspondence should be addressed at: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215
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