The occurrence of non‐glycosylated forms of N‐glycoprotein upon proteasome inhibition does not confirm cytosolic deglycosylation

N-Glycosylated substrates that undergo ER-associated degradation (ERAD) are often deglycosylated by the cytosolic peptide:N-glycanase (Ngly1) during their proteasomal degradation in the cytosol. Consequently, the presence of non- or deglycosylated forms of such proteins after treatment with proteasome inhibitors is widely used as evidence for cytosolic deglycosylation by Ngly1. However, in this study, the accumulation of nonglycosylated RTAm, a model ERAD substrate, was still observed in mouse cells lacking cytosolic de-N-glycosylating enzymes, when treated with proteasome inhibitors. It was found that RTAm is normally partially N-glycosylated, while the nonglycosylated form is rapidly degraded by proteasomes in the cytosol. Our results suggest that the occurrence of 'nonglycosylated' ERAD substrates upon treatment with proteasome inhibitors is not necessarily a clue for cytosolic deglycosylation.