Release of polymannose oligosaccharides from vesicular stomatitis virus G protein during endoplasmic reticulum-associated degradation

To further explore the localization of the N-deglycosylation involved in th e endoplasmic reticulum (ER)-associated quality control system we studied H epG2 cells infected with vesicular stomatitis virus (VSV) and its ts045 mut ant, as in this system oligosaccharide release can be attributed solely to the VSV glycoprotein (G protein). We utilized the restricted intracellular migration of the mutant protein as well as dithiothreitol (DTT), low temper ature, and a castanospermine (CST)-imposed glucosidase blockade to determin e in which intracellular compartment deglycosylation takes place. Degradati on of the VSV ts045 G protein was considerably greater at the nonpermissive than at the permissive temperature; this was reflected by a substantial in crease in polymannose oligosaccharide release. Under both conditions these oligosaccharides were predominantly in the characteristic cytosolic form, w hich terminates in a single N-acetylglucosamine (OS-GlcNAc(1)); this was al so the case in the presence of DTT, which retains the G protein completely in the ER. However when cells infected with the VSV mutant were examined at 15 degreesC or exposed to CST, both of which represent conditions that imp air ER-to-cytosol transport, the released oligosaccharides were almost excl usively (>95%) in the vesicular OS-GlcNAC(2) form; glucosidase blockade had a similar effect on the wld-type virus. Addition of puromycin to glucosida se-inhibited cells resulted in a pronounced reduction (>90%) in oligosaccha ride release, which reflected a comparable impairment in glycoprotein biosy nthesis and indicated that the OS-GlcNAc(2) components originated from prot ein degradation rather than hydrolysis of oligosaccharide lipids. Our findi ngs are consistent with N-deglycosylation of the VSV G protein in the ER an d the subsequent transport of the released oligosaccharides to the cytosol where OS-GlcNAc(2) to OS-GlcNAc(1) conversion by an endo-beta -N-acetylgluc osaminidase takes place. Studies with the ts045 G protein at the nonpermiss ive temperature permitted us to determine that it can be processed by Golgi endomannosidase although remaining endo H sensitive, supporting the concep t that it recycles between the ER and cis-Golgi compartments.