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

Abstract

To further explore the localization of the N-deglycosylation involved in the endoplasmic reticulum (ER)-associated quality control system we studied HepG2 cells infected with vesicular stomatitis virus (VSV) and its ts045 mutant, 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 temperature, and a castanospermine (CST)-imposed glucosidase blockade to determine in which intracellular compartment deglycosylation takes place. Degradation of the VSV ts045 G protein was considerably greater at the nonpermissive than at the permissive temperature; this was reflected by a substantial increase in polymannose oligosaccharide release. Under both conditions these oligosaccharides were predominantly in the characteristic cytosolic form, which terminates in a single N-acetylglucosamine (OS-GlcNAc(1)); this was also 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 degrees C or exposed to CST, both of which represent conditions that impair ER-to-cytosol transport, the released oligosaccharides were almost exclusively (> 95%) in the vesicular OS-GlcNAc(2) form; glucosidase blockade had a similar effect on the wild-type virus. Addition of puromycin to glucosidase-inhibited cells resulted in a pronounced reduction (> 90%) in oligosaccharide release, which reflected a comparable impairment in glycoprotein biosynthesis and indicated that the OS-GlcNAc(2) components originated from protein degradation rather than hydrolysis of oligosaccharide lipids. Our findings are consistent with N-deglycosylation of the VSV G protein in the ER and the subsequent transport of the released oligosaccharides to the cytosol where OS-GlcNAc(2) to OS-GlcNAc(1) conversion by an endo-beta-N-acetylglucosaminidase takes place. Studies with the ts045 G protein at the nonpermissive temperature permitted us to determine that it can be processed by Golgi endomannosidase although remaining endo H sensitive, supporting the concept that it recycles between the ER and cis-Golgi compartments.